Sample records for adenoviral-mediated gene delivery

  1. Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

    PubMed Central

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting

  2. Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

    PubMed

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

  3. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    PubMed Central

    2011-01-01

    Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

  4. Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

    PubMed

    Clausen, Björn E; Brand, Anna; Karram, Khalad

    2015-06-01

    Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine

    PubMed Central

    Lee, Cody S.; Bishop, Elliot S.; Zhang, Ruyi; Yu, Xinyi; Farina, Evan M.; Yan, Shujuan; Zhao, Chen; Zheng, Zongyue; Shu, Yi; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; Ho, Sherwin; Athiviraham, Aravind; Lee, Michael J.; Wolf, Jennifer Moriatis; Reid, Russell R.; He, Tong-Chuan

    2017-01-01

    With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2,000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and pre-clinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine. PMID:28944281

  6. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  7. Targeted adenoviral vectors

    NASA Astrophysics Data System (ADS)

    Douglas, Joanne T.

    The practical implementation of gene therapy in the clinical setting mandates gene delivery vehicles, or vectors, capable of efficient gene delivery selectively to the target disease cells. The utility of adenoviral vectors for gene therapy is restricted by their dependence on the native adenoviral primary cellular receptor for cell entry. Therefore, a number of strategies have been developed to allow CAR-independent infection of specific cell types, including the use of bispecific conjugates and genetic modifications to the adenoviral capsid proteins, in particular the fibre protein. These targeted adenoviral vectors have demonstrated efficient gene transfer in vitro , correlating with a therapeutic benefit in preclinical animal models. Such vectors are predicted to possess enhanced efficacy in human clinical studies, although anatomical barriers to their use must be circumvented.

  8. Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

    PubMed Central

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

    2006-01-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

  9. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    PubMed

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  10. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  11. Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

    PubMed Central

    Weiskirchen, Ralf; Kneifel, Jens; Weiskirchen, Sabine; van de Leur, Eddy; Kunz, Dagmar; Gressner, Axel M

    2000-01-01

    Background The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. Results With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. Conclusions Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible

  12. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

    PubMed Central

    Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

    1999-01-01

    The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

  13. A Novel Nonviral Gene Delivery System: Multifunctional Envelope-Type Nano Device

    NASA Astrophysics Data System (ADS)

    Hatakeyama, Hiroto; Akita, Hidetaka; Kogure, Kentaro; Harashima, Hideyoshi

    In this review we introduce a new concept for developing a nonviral gene delivery system which we call "Programmed Packaging." Based on this concept, we succeeded in developing a multifunctional envelope-type nano device (MEND), which exerts high transfection activities equivalent to those of an adenovirus in a dividing cell. The use of MEND has been extended to in vivo applications. PEG/peptide/DOPE ternary conjugate (PPD)-MEND, a new in vivo gene delivery system for the targeting of tumor cells that dissociates surface-modified PEG in tumor tissue by matrix metalloproteinase (MMP) and exerts significant transfection activities, was developed. In parallel with the development of MEND, a quantitative gene delivery system, Confocal Image-assisted 3-dimensionally integrated quantification (CIDIQ), also was developed. This method identified the rate-limiting step of the nonviral gene delivery system by comparing it with adenoviral-mediated gene delivery. The results of this analysis provide a new direction for the development of rational nonviral gene delivery systems.

  14. Nanoparticle-mediated gene delivery.

    PubMed

    Jin, Sha; Leach, John C; Ye, Kaiming

    2009-01-01

    Nonviral gene delivery has been gaining considerable attention recently. Although the efficacy of DNA transfection, which is a major concern, is low in nonviral vector-mediated gene transfer compared with viral ones, nonviral vectors are relatively easy to prepare, less immunogenic and oncogenic, and have no potential of virus recombination and no limitation on the size of a transferred gene. The ability to incorporate genetic materials such as plasmid DNA, RNA, and siRNA into functionalized nanoparticles with little toxicity demonstrates a new era in pharmacotherapy for delivering genes selectively to tissues and cells. In this chapter, we highlight the basic concepts and applications of nonviral gene delivery using super paramagnetic iron oxide nanoparticles and functionalized silica nanoparticles. The experimental protocols related to these topics are described in the chapter.

  15. Improved exercise capacity and reduced systemic inflammation after adenoviral-mediated SERCA-2a gene transfer.

    PubMed

    Gupta, Dipin; Palma, Jon; Molina, Ezequiel; Gaughan, John P; Long, Walter; Houser, Steven; Macha, Mahender

    2008-04-01

    We hypothesized that sarcoplasmic reticulum Ca2+ ATPase pump (SERCA-2a) gene delivery would have beneficial effects upon exercise capacity and markers of inflammation in the setting of heart failure. A pressure-overload model of experimental heart failure was used in rats. Following a decrease in fractional shortening of >or=25%, animals underwent intracoronary adenoviral-mediated gene transfection using SERCA-2a. Heart failure animals were randomized to receive the SERCA-2a gene, the beta galactosidase (control) gene, or followed without any further intervention. Exercise and hemodynamic testing were performed, and myocardial and systemic markers of inflammation were assayed after 7 and 21 d. Animals receiving Ad.SERCA-2a showed an increase in exercise tolerance (499.0 +/- 14.9 versus 312.8 +/- 10.5 s, P < 0.0001) relative to Ad.Gal group. Groups treated with Ad.SERCA-2a had significantly decreased serum levels of the inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor-alpha compared with Ad.Gal-treated animals. Serum levels of atrial natriuretic peptide were decreased in animals receiving Ad.SERCA-2a compared with animals receiving Ad.Gal at day 7 (0.35 +/- 0.03 versus 0.52 +/- 0.11 pg/mL, P = 0.001). Myocardial levels of the proapoptotic protein bax were reduced in Ad.SERCA-2a -treated animals compared with those receiving Ad.Gal at day 7 (protein level/actin: 0.24 +/- 0.05 versus 0.33 +/- 0.04, P = 0.04) and day 21 (protein level/actin: 0.61 +/- 0.04 versus 0.69 +/- 0.01, P = 0.001). Genetic modulation of heart failure using the SERCA-2a gene was associated with improvement in cardiac function and exercise capacity as well as improvements in heart-failure associated inflammatory markers.

  16. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors

    PubMed Central

    Chorny, Michael; Fishbein, Ilia; Tengood, Jillian E.; Adamo, Richard F.; Alferiev, Ivan S.; Levy, Robert J.

    2013-01-01

    Gene therapeutic strategies have shown promise in treating vascular disease. However, their translation into clinical use requires pharmaceutical carriers enabling effective, site-specific delivery as well as providing sustained transgene expression in blood vessels. While replication-deficient adenovirus (Ad) offers several important advantages as a vector for vascular gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal transduction efficiency in vascular cells, and serious systemic adverse effects. We hypothesized that novel zinc oleate-based magnetic nanoparticles (MNPs) loaded with Ad would enable effective arterial cell transduction by shifting vector processing to an alternative pathway, protect Ad from inactivation by neutralizing factors, and allow site-specific gene transfer to arteries treated with stent angioplasty using a 2-source magnetic guidance strategy. Ad-loaded MNPs effectively transduced cultured endothelial and smooth muscle cells under magnetic conditions compared to controls and retained capacity for gene transfer after exposure to neutralizing antibodies and lithium iodide, a lytic agent causing disruption of free Ad. Localized arterial gene expression significantly stronger than in control animal groups was demonstrated after magnetically guided MNP delivery in a rat stenting model 2 and 9 d post-treatment, confirming feasibility of using Ad-loaded MNPs to achieve site-specific transduction in stented blood vessels. In conclusion, Ad-loaded MNPs formed by controlled precipitation of zinc oleate represent a novel delivery system, well-suited for efficient, magnetically targeted vascular gene transfer.—Chorny, M., Fishbein, I., Tengood, J. E., Adamo, R. F., Alferiev, I. S., Levy, R. J. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors. PMID:23407712

  17. Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

    PubMed

    Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

    2000-02-01

    Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44

  18. Regulated Expression of Adenoviral Vectors-Based Gene Therapies

    PubMed Central

    Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

    2008-01-01

    Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

  19. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  20. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

  1. Adenoviral Mediated Gene Transfer of IGF-1 Enhances Wound Healing and Induces Angiogenesis

    PubMed Central

    Balaji, S.; LeSaint, M.; Bhattacharya, S. S.; Moles, C.; Dhamija, Y.; Kidd, M.; Le, L.D.; King, A.; Shaaban, A.; Crombleholme, T. M.; Bollyky, P.; Keswani, S. G.

    2014-01-01

    to control treatments. Conclusions In two different models, our data demonstrates that adenoviral mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization. PMID:24725678

  2. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  3. Helper-Dependent Adenoviral Vectors.

    PubMed

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2011-10-29

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology.

  4. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  5. Field distribution and DNA transport in solid tumors during electric field-mediated gene delivery.

    PubMed

    Henshaw, Joshua W; Yuan, Fan

    2008-02-01

    Gene therapy has a great potential in cancer treatment. However, the efficacy of cancer gene therapy is currently limited by the lack of a safe and efficient means to deliver therapeutic genes into the nucleus of tumor cells. One method under investigation for improving local gene delivery is based on the use of pulsed electric field. Despite repeated demonstration of its effectiveness in vivo, the underlying mechanisms behind electric field-mediated gene delivery remain largely unknown. Without a thorough understanding of these mechanisms, it will be difficult to further advance the gene delivery. In this review, the electric field-mediated gene delivery in solid tumors will be examined by following individual transport processes that must occur in vivo for a successful gene transfer. The topics of examination include: (i) major barriers for gene delivery in the body, (ii) distribution of electric fields at both cell and tissue levels during the application of external fields, and (iii) electric field-induced transport of genes across each of the barriers. Through this approach, the review summarizes what is known about the mechanisms behind electric field-mediated gene delivery and what require further investigations in future studies.

  6. Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

    PubMed Central

    Karandashova, Sophia; Florova, Galina; Azghani, Ali O.; Komissarov, Andrey A.; Koenig, Kathy; Tucker, Torry A.; Allen, Timothy C.; Stewart, Kris; Tvinnereim, Amy

    2013-01-01

    Elevated concentrations of plasminogen activator inhibitor–1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, β-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40–80 and 200–400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10–15 nM in control animals to 20–40 nM in hPAI-1–overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment. PMID:23002099

  7. Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

    PubMed

    Cao, Huibi; Ouyang, Hong; Grasemann, Hartmut; Bartlett, Claire; Du, Kai; Duan, Rongqi; Shi, Fushan; Estrada, Marvin; Seigel, Kyle E; Coates, Allan L; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Moraes, Theo J; Hu, Jim

    2018-06-01

    A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

  8. High-capacity 'gutless' adenoviral vectors.

    PubMed

    Kochanek, S; Schiedner, G; Volpers, C

    2001-10-01

    Adenoviral vectors are promising gene transfer vehicles for different gene therapy applications. High-capacity adenoviral (HC-Ad) vectors address some of the problems that have been observed with replication-defective, E1-deleted first-generation adenoviral vectors: toxicity and immunogenicity due to viral gene expression and 7 to 8 kb capacity limit for the transport of therapeutic DNA. This review summarizes HC-Ad vector-related publications from the past 18 months that are mainly concerned with vector design/production and in vivo applications in different murine models.

  9. Suppression of adenoviral gene expression in the liver: role of innate vs adaptive immunity and their cell lysis mechanisms.

    PubMed

    Minagawa, Masahiro; Kawamura, Hiroki; Liu, Zhangxu; Govindarajan, Sugantha; Dennert, Gunther

    2005-06-01

    Injection of adenoviral constructs causes liver infection prompting immunity, which suppress viral gene expression. Innate and adaptive immunity mediate these processes raising the question which pathways are the most prominent. Adenovirus expressing the beta-galactosidase (beta-gal) gene was injected into normal and immunodeficient mice. Elimination of beta-gal-expressing hepatocytes and increases in liver enzymes were assayed. Major histocompatibility complex (MHC) class I densities, perforin channel insertion and apoptosis by Fas and tumor necrosis factor (TNF)-alpha were assayed. At high virus doses, suppression of viral gene expression was as efficient in immunodeficient as in normal mice, while at low doses effects of cytotoxic T lymphocytes (CTL) were demonstrable. Despite CTL priming and elimination of infected hepatocytes no liver injury is detected. Hepatocyte MHC I densities were able to trigger CTL granule exocytosis and perforin lysis in vitro but not in vivo. This is we show is because of decreased sensitivity of hepatocytes from infected mice to perforin and increased sensitivity to Fas and TNF-alpha lysis. Effector cells of the innate immune system are exceedingly effective in suppressing adenoviral gene expression. Perforin-independent pathways, those mediated by TNF-alpha and Fas are very efficient in hepatocytes from virus-infected livers.

  10. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

    PubMed Central

    Zhou, Qiu-Lan; Chen, Zhi-Yi; Yang, Feng

    2014-01-01

    With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation) on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo), including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers. PMID:25202710

  11. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  12. High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

    PubMed

    Gwiazda, Kamila S; Grier, Alexandra E; Sahni, Jaya; Burleigh, Stephen M; Martin, Unja; Yang, Julia G; Popp, Nicholas A; Krutein, Michelle C; Khan, Iram F; Jacoby, Kyle; Jensen, Michael C; Rawlings, David J; Scharenberg, Andrew M

    2016-09-29

    Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

  13. Pancreatic Transduction by Helper-Dependent Adenoviral Vectors via Intraductal Delivery

    PubMed Central

    Morró, Meritxell; Teichenne, Joan; Jimenez, Veronica; Kratzer, Ramona; Marletta, Serena; Maggioni, Luca; Mallol, Cristina; Ruberte, Jesus; Kochanek, Stefan; Bosch, Fatima

    2014-01-01

    Abstract Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue. PMID:25046147

  14. Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats.

    PubMed

    Badie, B; Drazan, K E; Kramar, M H; Shaked, A; Black, K L

    1995-06-01

    Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.

  15. Electroporation-mediated Delivery of Genes in Rodent Models of Lung Contusion

    PubMed Central

    Machado-Aranda, David; Raghavendran, Krishnan

    2015-01-01

    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated gene delivery to the lung. Electroporation is a highly efficient and easily reproducible technique that allows circumvention of several of lung gene delivery challenges and safety issues present with other forms of lung gene therapy. PMID:24510825

  16. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    PubMed

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  17. Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice - role of regulatory T cells.

    PubMed

    Singpiel, Alena; Kramer, Julia; Maus, Regina; Stolper, Jennifer; Bittersohl, Lara Friederike; Gauldie, Jack; Kolb, Martin; Welte, Tobias; Sparwasser, Tim; Maus, Ulrich A

    2018-03-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγ pos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery.

    PubMed

    Sun, Ryan R; Noble, Misty L; Sun, Samuel S; Song, Shuxian; Miao, Carol H

    2014-05-28

    Ultrasound (US)-mediated gene delivery has emerged as a promising non-viral method for safe and selective gene delivery. When enhanced by the cavitation of microbubbles (MBs), US exposure can induce sonoporation that transiently increases cell membrane permeability for localized delivery of DNA. The present study explores the effect of generalizable MB customizations on MB facilitation of gene transfer compared to Definity®, a clinically available contrast agent. These modifications are 1) increased MB shell acyl chain length (RN18) for elevated stability and 2) addition of positive charge on MB (RC5K) for greater DNA associability. The MB types were compared in their ability to facilitate transfection of luciferase and GFP reporter plasmid DNA in vitro and in vivo under various conditions of US intensity, MB dosage, and pretreatment MB-DNA incubation. The results indicated that both RN18 and RC5K were more efficient than Definity®, and that the cationic RC5K can induce even greater transgene expression by increasing payload capacity with prior DNA incubation without compromising cell viability. These findings could be applied to enhance MB functions in a wide range of therapeutic US/MB gene and drug delivery approach. With further designs, MB customizations have the potential to advance this technology closer to clinical application. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.

    PubMed

    Guan, Yuting; Ma, Yanlin; Li, Qi; Sun, Zhenliang; Ma, Lie; Wu, Lijuan; Wang, Liren; Zeng, Li; Shao, Yanjiao; Chen, Yuting; Ma, Ning; Lu, Wenqing; Hu, Kewen; Han, Honghui; Yu, Yanhong; Huang, Yuanhua; Liu, Mingyao; Li, Dali

    2016-05-01

    The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

    PubMed

    Broaddus, W C; Liu, Y; Steele, L L; Gillies, G T; Lin, P S; Loudon, W G; Valerie, K; Schmidt-Ullrich, R K; Fillmore, H L

    1999-12-01

    The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy

  1. MicroRNA Silencing Improves the Tumor Specificity of Adenoviral Transgene Expression

    PubMed Central

    Card, Paul B.; Hogg, Richard T.; del Alcazar, Carlos Gil

    2012-01-01

    Adenoviral technology has been thoroughly evaluated for delivering genetic material to tumor tissue and the surrounding microenvironment. Almost any gene can be cloned into an adenovirus (Ad) vector, which when combined with strong, constitutively active promoters permit up to a million-fold amplification of the transgene in a single adenoviral particle, thus facilitating their use in cancer therapy and imaging. However, widespread infection of the liver and other non-targeted tissues by Ad vectors is a substantial problem that often results in significant liver inflammation and hepatotoxicity at doses required to achieve efficient tumor transduction. miR-122 is a highly expressed liver-specific microRNA that provides a unique opportunity for down-regulating adenoviral transgene expression in liver tissue. The binding of endogenous miRNAs to complementary miRNA targeting elements (miRTs) incorporated into the 3′ untranslated region of adenoviral transgenes interferes with message stability and/or protein translation, and miRT elements against miR-122 (miRT-122) can selectively reduce adenoviral transgene expression in the liver. Previous studies using miR-122-based regulation, with and without other types of transcriptional targeting, have yielded promising preliminary results. However, investigations to date evaluating miRT-122 elements for improving tumor specificity have used either non-tumor bearing animals or direct intratumoral injection as the mode of delivery. In the present study, we confirmed the ability of miRT-122 sequences to selectively down-regulate adenoviral luciferase expression in the liver in vitro and in vivo, and show that this strategy can improve tumor specific transgene expression in a HT1080 human fibrosarcoma model. Rapid growth and the inefficient flow of blood through tumor neovasculature often results in profound hypoxia, which provides additional opportunities for targeting solid tumors and their microenvironment using vectors

  2. Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors.

    PubMed

    Camargo, F D; Huey-Louie, D A; Finn, A V; Sassani, A B; Cozen, A E; Moriwaki, H; Schneider, D B; Agah, R; Dichek, D A

    2000-11-01

    The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens.

  3. Cardiac Gene Therapy: Optimization of Gene Delivery Techniques In Vivo

    PubMed Central

    Katz, Michael G.; Swain, JaBaris D.; White, Jennifer D.; Low, David; Stedman, Hansell

    2010-01-01

    Abstract Vector-mediated cardiac gene therapy holds tremendous promise as a translatable platform technology for treating many cardiovascular diseases. The ideal technique is one that is efficient and practical, allowing for global cardiac gene expression, while minimizing collateral expression in other organs. Here we survey the available in vivo vector-mediated cardiac gene delivery methods—including transcutaneous, intravascular, intramuscular, and cardiopulmonary bypass techniques—with consideration of the relative merits and deficiencies of each. Review of available techniques suggests that an optimal method for vector-mediated gene delivery to the large animal myocardium would ideally employ retrograde and/or anterograde transcoronary gene delivery,extended vector residence time in the coronary circulation, an increased myocardial transcapillary gradient using physical methods, increased endothelial permeability with pharmacological agents, minimal collateral gene expression by isolation of the cardiac circulation from the systemic, and have low immunogenicity. PMID:19947886

  4. Improvement in adenoviral gene transfer efficiency after preincubation at +37 degrees C in vitro and in vivo.

    PubMed

    Kossila, Maija; Jauhiainen, Suvi; Laukkanen, Mikko O; Lehtolainen, Pauliina; Jääskeläinen, Maiju; Turunen, Päivi; Loimas, Sami; Wahlfors, Jarmo; Ylä-Herttuala, Seppo

    2002-01-01

    Adenovirus is a widely used vector in gene transfer experiments because it produces high transduction efficiency in vitro and in vivo by means of the coxsackie-adenovirus receptor (CAR) and major histocompatibility complex (MHC) class I alpha-2 domain. Adenoviral gene transfer efficiency has been reported to correlate with cellular CAR expression. We report here a simple method to increase adenoviral gene transfer efficiency in cells that do not express high levels of CAR: preincubation of adenovirus for 30-40 minutes at +37 degrees C significantly increased the transduction efficiency in vitro in CHO and BALB/3T3 cells, in which CAR is expressed at very low levels. Increased transduction efficiency of preincubated adenovirus was also detected in vivo in rat brain tissue. In addition, we found that adenoviruses were rapidly inactivated in human serum in a complement-independent manner, whereas fetal bovine serum (FBS) had hardly any effects on the viral infectivity. We conclude that preincubation of adenoviral vectors at +37 degrees C may substantially increase gene transfer efficiency in applications in which target cells do not express high levels of CAR.

  5. PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review

    NASA Astrophysics Data System (ADS)

    Bakar, L. M.; Abdullah, M. Z.; Doolaanea, A. A.; Ichwan, S. J. A.

    2017-08-01

    Cancer becomes a serious issue on society with increasing of their growth and proliferation, either in well economic developed countries or not. Recent years, oral cancer is one of the most threatening diseases impairing the quality of life of the patient. Scientists have emphasised on application of gene therapy for oral cancer by using nanoparticle as transportation vectors as a new alternative platform in order to overcome the limitations of conventional approaches. In modern medicine, nanotechnologies’ application, such as nanoparticles-mediated gene delivery, is one of promising tool for therapeutic devices. The objective of this article is to present a brief review summarizes on the current progress of nanotechnology-based gene delivery treatment system targeted for oral cancer.

  6. Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

    PubMed Central

    Salido, Eduardo C.; Li, Xiao M.; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J.; Roy-Chowdhury, Jayanta

    2006-01-01

    Mutations in the alanine–glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt−/− mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

  7. Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

    PubMed

    Kraljević Pavelić, Sandra; Marjanović, Marko; Poznić, Miroslav; Kralj, Marijeta

    2009-12-01

    p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and

  8. Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder.

    PubMed

    Buscarini, Maurizio; Quek, Marcus L; Gilliam-Hegarich, Susan; Kasahara, Nori; Bochner, Bernard

    2007-01-01

    observed that both alpha(v)beta3 and alpha(v)beta5 are strongly expressed in muscle cells surrounding the neoplastic cells, as well as within the peritumoral connective tissue. In cases of invasive bladder cancer that have lost CAR expression, an adenoviral vector may still be utilized through the less efficient interactions with the integrins. Bladder tumor tissue may be less susceptible to an adenoviral-mediated gene therapy approach in which a significant percentage of tumor cells require transduction. Adenoviral uptake by tumor or peritumoral cells with subsequent gene transfer could be predicted by the level of CAR and alpha(v)-based integrin expression. This would enhance our ability to identify those patients whose tumors would be more susceptible to Ad-mediated gene delivery as part of an antitumor treatment. 2007 S. Karger AG, Basel

  9. Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel J; Kalle, Wouter H J

    2005-06-01

    Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

  10. In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

    PubMed

    Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C; Da, Xiaoyu; David, Shiran Ben; Gavrity, Jayne; Awad, Hani A; Bae, Hyun W; Ley, Eric J; Kremen, Thomas J; Gazit, Zulma; Ferrara, Katherine W; Pelled, Gadi; Gazit, Dan

    2017-05-17

    More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 ( BMP - 6 ) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. Copyright © 2017, American Association for the Advancement of Science.

  11. In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

    PubMed Central

    Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C.; Da, Xiaoyu; Ben David, Shiran; Gavrity, Jayne; Awad, Hani A.; Bae, Hyun W.; Ley, Eric J.; Kremen, Thomas J.; Gazit, Zulma; Ferrara, Katherine W.; Pelled, Gadi; Gazit, Dan

    2017-01-01

    More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. PMID:28515335

  12. Preliminary studies of particle-mediated gene delivery to the joints of dogs.

    PubMed

    Campbell, S E; Nasir, L; Gault, E A; Argyle, D J; Bennett, D

    2007-04-07

    This paper describes a preliminary evaluation of particle-mediated bombardment via the Helios gene gun for the delivery of therapeutic genes to synovial cells in culture. A reporter gene, enhanced green fluorescent protein, was delivered to rabbit synovial fibroblasts (HIG-82) using gold particle (1.0 microm) bombardment to evaluate transfection efficiency at helium pressures of 100 and 150 psi. Transfection of cells occurred at these pressures despite some cell death. The in vitro delivery of gold particles to samples of synovial membrane and articular cartilage from a freshly euthanased dog was also studied to examine depth of penetration of gold particles (1.0 microm) at helium pressures of 250 and 500 psi. Light microscopical examination of histological sections of the synovial membrane showed that particles of gold had penetrated the lining cells of the synovium. However, no gold particles had penetrated the articular cartilage even at 500 psi.

  13. Adenoviral vector tethering to metal surfaces via hydrolysable cross-linkers for the modulation of vector release and transduction

    PubMed Central

    Fishbein, Ilia; Forbes, Scott P.; Chorny, Michael; Connolly, Jeanne M.; Adamo, Richard F.; Corrales, Ricardo; Alferiev, Ivan S.; Levy, Robert J.

    2013-01-01

    The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolysable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37°C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolysable cross-linkers with structure-specific release kinetics. PMID:23777912

  14. Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections.

    PubMed

    Sanches, Pedro Gomes; Mühlmeister, Mareike; Seip, Ralf; Kaijzel, Eric; Löwik, Clemens; Böhmer, Marcel; Tiemann, Klaus; Grüll, Holger

    2014-12-10

    Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200 μg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimb skeletal muscles were exposed to ultrasound from a non-focused transducer (1 MHz, 1.25 MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated/control muscles of up to 101× were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. 3D Porous Chitosan-Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer.

    PubMed

    Wang, Kui; Kievit, Forrest M; Florczyk, Stephen J; Stephen, Zachary R; Zhang, Miqin

    2015-10-12

    Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.

  16. Helper-dependent adenoviral vectors for liver-directed gene therapy

    PubMed Central

    Brunetti-Pierri, Nicola; Ng, Philip

    2011-01-01

    Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration resulting in acute toxicity, the severity of which is dependent on vector dose. Intense efforts have been focused on elucidating the factors involved in this acute response and various strategies have been investigated to improve the therapeutic index of HDAd vectors. These strategies have yielded encouraging results with the potential for clinical translation. PMID:21470977

  17. Gene delivery systems by the combination of lipid bubbles and ultrasound.

    PubMed

    Negishi, Yoichi; Endo-Takahashi, Yoko; Maruyama, Kazuo

    2016-11-28

    Gene therapy is promising for the treatment of many diseases including cancers and genetic diseases. From the viewpoint of safety, ultrasound (US)-mediated gene delivery with nano/ microbubbles was recently developed as a novel non-viral vector system. US-mediated gene delivery using nano/microbubbles are able to produce transient changes in the permeability of the cell membrane after US-induced cavitation while reducing cellular damage and enables the tissue-specific or the site-specific intracellular delivery of gene both in vitro and in vivo. We have recently developed novel lipid nanobubbles (Lipid Bubbles). These nanobubbles can also be used to enhance the efficacy of the US-mediated genes (plasmid DNA, siRNA, and miRNA etc.) delivery. In this review, we describe US-mediated delivery systems combined with nano/microbubbles and discuss their feasibility as non-viral vector systems.

  18. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

    PubMed Central

    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  19. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    NASA Astrophysics Data System (ADS)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  20. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

    PubMed

    Campos, Samuel K; Barry, Michael A

    2004-11-01

    There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

  1. Chromosomal integration of adenoviral vector DNA in vivo.

    PubMed

    Stephen, Sam Laurel; Montini, Eugenio; Sivanandam, Vijayshankar Ganesh; Al-Dhalimy, Muhseen; Kestler, Hans A; Finegold, Milton; Grompe, Markus; Kochanek, Stefan

    2010-10-01

    So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH(Deltaexon5) mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 x 10(-5) per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 x 10(-7). This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.

  2. Adenoviral beta-adrenergic receptor kinase inhibitor gene transfer improves exercise capacity, cardiac contractility, and systemic inflammation in a model of pressure overload hypertrophy.

    PubMed

    Gupta, Dipin; Molina, Ezequiel J; Palma, Jon; Gaughan, John P; Long, Walter; Macha, Mahender

    2008-10-01

    We hypothesized that intracoronary adenoviral-mediated delivery of betaARKct would improve heart failure associated pathophysiologic abnormalities related to exercise capacity, cardiac contractility, systemic inflammation and volume overload. After aortic banding, a cohort of Sprague-Dawley rats was followed by echocardiography. When an absolute decline of 25% in fractional shortening was detected, animals were randomized to intracoronary delivery of Ad.ssARKct (n=14), Ad.beta-Gal (n=13), or followed without any other further intervention (n=18). Assessment of exercise tolerance and hemodynamic profile and measurement of markers of systemic inflammation and volume overload was performed at 7, 14, and 21 days after gene delivery. Data were analyzed using ANOVA. Animals receiving Ad.ssARKct showed improved exercise tolerance compared to Ad.Gal-treated animals at 14 days (507+/-26 s vs. 408+/-19 s, P=0.01) and 21 days (526+/-55 s vs. 323+/-19 s, P<0.001) following injection. Animals receiving Ad.ssARKct demonstrated improved +dP/dtmax (mean+/-SD, 5,581+/-960 mmHg/s vs. 3,134+/-438 mmHg/s, P<0.01) and -dP/dtmax (mean+/-SD, -3,494+/-1,269 mmHg/s vs. -1,925+/-638 mmHg/s, P<0.01) compared to Ad.Gal-treated animals at 7 days. These differences were observed up to 21 days following injection. Serum levels of IL-1, IL-6, and TNF-alpha, as well as ANP were also decreased in animals receiving Ad.betaARKct. Genetic modulation of heart failure using the betaARKct gene was associated with improved exercise capacity and cardiac function as well as amelioration in heart failure-associated profiles of systemic inflammation and volume overload.

  3. Stent-mediated gene and drug delivery for cardiovascular disease and cancer: A brief insight.

    PubMed

    Krishnagopal, Akshaya; Reddy, Aakash; Sen, Dwaipayan

    2017-05-01

    This review concisely recapitulates the different existing modes of stent-mediated gene/drug delivery, their considerable advancement in clinical trials and a rationale for other merging new technologies such as nanotechnology and microRNA-based therapeutics, in addition to addressing the limitations in each of these perpetual stent platforms. Over the past decade, stent-mediated gene/drug delivery has materialized as a hopeful alternative for cardiovascular disease and cancer in contrast to routine conventional treatment modalities. Regardless of the phenomenal recent developments achieved by coronary interventions and cancer therapies that employ gene and drug-eluting stents, practical hurdles still remain a challenge. The present review highlights the limitations that each of the existing stent-based gene/drug delivery system encompasses and therefore provides a vision for the future with respect to discovering an ideal stent therapeutic platform that would circumvent all the practical hurdles witnessed with the existing technology. Further study of the improvisation of next-generation drug-eluting stents has helped to overcome the issue of restenosis to some extent. However, current stent formulations fall short of the anticipated clinically meaningful outcomes and there is an explicit need for more randomized trials aiming to further evaluate stent platforms in favour of enhanced safety and clinical value. Gene-eluting stents may hold promise in contributing new ideas for stent-based prevention of in-stent restenosis through genetic interventions by capitalizing on a wide variety of molecular targets. Therefore, the central consideration directs us toward finding an ideal stent therapeutic platform that would tackle all of the gaps in the existing technology. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

    PubMed

    Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

    2012-03-01

    The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

  5. Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

    PubMed

    Penny, Gervette M; Cochran, Rebecca B; Pihlajoki, Marjut; Kyrönlahti, Antti; Schrade, Anja; Häkkinen, Merja; Toppari, Jorma; Heikinheimo, Markku; Wilson, David B

    2017-10-01

    Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6 , two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4 flox/flox ; Gata6 flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes ( Hsd3b1 , Cyp17a1 and Hsd17b3 ) was reduced, whereas expression of another Leydig cell marker, Insl3 , was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2. © 2017 Society for Reproduction and Fertility.

  6. In vivo, cardiac-specific knockdown of target protein, Malic Enzyme-1, in rat via adenoviral delivery of DNA for non-native miRNA

    PubMed Central

    O'Donnell, J. Michael; Kalichira, Asha; Bi, Jian; Lewandowski, E. Douglas

    2013-01-01

    This study examines the feasibility of using the adenoviral delivery of DNA for a non-native microRNA to suppress expression of a target protein (cytosolic NADP+-dependent malic-enzyme 1, ME1) in whole heart in vivo, via an isolated-heart coronary perfusion approach. Complementary DNA constructs for ME1 microRNA were inserted into adenoviral vectors. Viral gene transfer to neonatal rat cardiomyocytes yielded 65% suppression of ME1 protein. This viral package was delivered to rat hearts in vivo (Adv.miR_ME1, 1013 vp/ml PBS) via coronary perfusion, using a cardiac-specific isolation technique. ME1 mRNA was reduced by 73% at 2-6 days post-surgery in heart receiving the Adv.miR_ME1. Importantly, ME1 protein was reduced by 66% (p<0.0002) at 5-6 days relative to sham-operated control hearts. Non-target protein expression for GAPDH, calsequestrin, and mitochondrial malic enzyme, ME3, were all unchanged. The non-target isoform, ME2, was unchanged at 2-5 days and reduced at day 6. This new approach demonstrates for the first time significant and acute silencing of target RNA translation and protein content in whole heart, in vivo, via non-native microRNA expression. PMID:22974418

  7. Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

    PubMed

    Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2016-08-01

    Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

  8. Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma

    PubMed Central

    Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J.; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2016-01-01

    Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of 111In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies. PMID:27357626

  9. Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

    PubMed

    Rouet, Romain; Thuma, Benjamin A; Roy, Marc D; Lintner, Nathanael G; Rubitski, David M; Finley, James E; Wisniewska, Hanna M; Mendonsa, Rima; Hirsh, Ariana; de Oñate, Lorena; Compte Barrón, Joan; McLellan, Thomas J; Bellenger, Justin; Feng, Xidong; Varghese, Alison; Chrunyk, Boris A; Borzilleri, Kris; Hesp, Kevin D; Zhou, Kaihong; Ma, Nannan; Tu, Meihua; Dullea, Robert; McClure, Kim F; Wilson, Ross C; Liras, Spiros; Mascitti, Vincent; Doudna, Jennifer A

    2018-05-30

    CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

  10. Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

    PubMed

    Sheikh, Muhammad Abid; Malik, Yousra Saeed; Xing, Zhenkai; Guo, Zhaopei; Tian, Huayu; Zhu, Xiaojuan; Chen, Xuesi

    2017-05-01

    Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD. For cell culture experiments, we utilized 6-hydroxydopamine (6-OHDA) mediated cell death model of MN9D cells following transfection with either a control plasmid or VEGF expressing plasmid. As compared to control transfected cells, PEI-PLL mediated VEGF gene delivery to MN9D cells resulted in increased cell viability, increase in the number of Tyrosine hydroxylase (TH) positive cells and decreased apoptosis following 6-OHDA insult. Next, we studied the therapeutic potential of PEI-PLL mediated VEGF gene delivery in SNPc by using unilateral 6-OHDA Medial forebrain bundle (MFB) lesion model of PD in rats. VEGF administration prevented the loss of motor functions induced by 6-OHDA as determined by behavior analysis. Similarly, VEGF inhibited the 6-OHDA mediated loss of DA neurons in Substantia Nigra Pars Compacta (SNPc) as well as DA nerve fibers in striatum as determined by TH immunostaining. In addition, PEI-PLL mediated VEGF gene delivery also prevented apoptosis and microglial activation in PD rat models. Together, these results clearly demonstrated the beneficial effects of PEI-PLL mediated VEGF gene delivery on dopaminergic system in both cell culture and animal models of PD. In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene

  11. Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

    PubMed

    Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

    2016-05-01

    Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

  12. Adenoviral vector gene delivery via the round window membrane in guinea pigs.

    PubMed

    Suzuki, Mitsuya; Yamasoba, Tatsuya; Suzukawa, Keigo; Kaga, Kimitaka

    2003-10-27

    We have found that damage from a local anesthetic solution containing phenol permitted beta-galactosidase (beta-gal) gene delivery to the guinea pig inner ear via the round window membrane (RWM). RWM damage was evident as degeneration of the outer epithelium. After adenovirus lacZ vector was applied to the damaged RWM, immunohistochemistry showed strong beta-gal expression in the RWM, mesothelial cells, organ of Corti, spiral limbus, spiral ligament and spiral ganglion. In the vestibular labyrinth, expression was seen in the sensory and supporting cells, transitional cells, and the dark-cell area. Thus, adenovirus can transfect a variety of inner ear cells in the guinea pig through a damaged RWM.

  13. Phage-Mediated Gene Therapy.

    PubMed

    Hosseinidoust, Zeinab

    2017-01-01

    Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Optimization and physicochemical characterization of a cationic lipid-phosphatidylcholine mixed emulsion formulated as a highly efficient vehicle that facilitates adenoviral gene transfer.

    PubMed

    Kim, Soo-Yeon; Lee, Sang-Jin; Kim, Jin-Ki; Choi, Han-Gon; Lim, Soo-Jeong

    2017-01-01

    Cationic lipid-based nanoparticles enhance viral gene transfer by forming electrostatic complexes with adenoviral vectors. We recently demonstrated the superior complexation capabilities of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) emulsion compared with a liposomal counterpart but the cytotoxicity of DOTAP emulsions remained a challenge. The present study is aimed at formulating an emulsion capable of acting as a highly effective viral gene transfer vehicle with reduced cytotoxicity and to physicochemically characterize the structures of virus-emulsion complexes in comparison with virus-liposome complexes when the only difference between emulsions and liposomes was the presence or absence of inner oil core. The emulsion formulation was performed by 1) reducing the content of DOTAP while increasing the content of zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2) optimizing the oil content. The complexation capability of formulated DOTAP:DMPC mixed emulsions was similar to those of emulsions containing DOTAP alone while displaying significantly lower cytotoxicity. The complexation capabilities of the DOTAP:DMPC mixed emulsion were serum-compatible and were monitored in a variety of cell types, whereas its liposomal counterpart was totally ineffective. Characterization by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering studies indicated that the optimized emulsions spontaneously surrounded the virus particles to generate emulsions that encapsulated the viral particles, whereas viral particles merely attached to the surfaces of the counterpart liposomes to form multiviral aggregates. Overall, these studies demonstrated that optimized DOTAP:DMPC mixed emulsions are potentially useful for adenoviral gene delivery due to less cytotoxicity and the unique ability to encapsulate the viral particle, highlighting the importance of nanoparticle formulation.

  15. Optimization and physicochemical characterization of a cationic lipid-phosphatidylcholine mixed emulsion formulated as a highly efficient vehicle that facilitates adenoviral gene transfer

    PubMed Central

    Kim, Soo-Yeon; Lee, Sang-Jin; Kim, Jin-Ki; Choi, Han-Gon; Lim, Soo-Jeong

    2017-01-01

    Cationic lipid-based nanoparticles enhance viral gene transfer by forming electrostatic complexes with adenoviral vectors. We recently demonstrated the superior complexation capabilities of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) emulsion compared with a liposomal counterpart but the cytotoxicity of DOTAP emulsions remained a challenge. The present study is aimed at formulating an emulsion capable of acting as a highly effective viral gene transfer vehicle with reduced cytotoxicity and to physicochemically characterize the structures of virus-emulsion complexes in comparison with virus–liposome complexes when the only difference between emulsions and liposomes was the presence or absence of inner oil core. The emulsion formulation was performed by 1) reducing the content of DOTAP while increasing the content of zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2) optimizing the oil content. The complexation capability of formulated DOTAP:DMPC mixed emulsions was similar to those of emulsions containing DOTAP alone while displaying significantly lower cytotoxicity. The complexation capabilities of the DOTAP:DMPC mixed emulsion were serum-compatible and were monitored in a variety of cell types, whereas its liposomal counterpart was totally ineffective. Characterization by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering studies indicated that the optimized emulsions spontaneously surrounded the virus particles to generate emulsions that encapsulated the viral particles, whereas viral particles merely attached to the surfaces of the counterpart liposomes to form multiviral aggregates. Overall, these studies demonstrated that optimized DOTAP:DMPC mixed emulsions are potentially useful for adenoviral gene delivery due to less cytotoxicity and the unique ability to encapsulate the viral particle, highlighting the importance of nanoparticle formulation. PMID:29070949

  16. Therapeutic Efficacy of Adenoviral-Mediated p53 Gene Transfer Is Synergistically Enhanced by Combined Use of Antisense Oligodeoxynucleotide Targeting Clusterin Gene in a Human Bladder Cancer Model1

    PubMed Central

    Miyake, Hideaki; Yamanaka, Kazuki; Muramaki, Mototsugu; Hara, Isao; Gleave, Martin E

    2005-01-01

    Abstract To establish a more effective therapeutic strategy against advanced bladder cancer, we investigated the effects of combined treatment with antisense (AS) oligodeoxynucleotide (ODN) targeting the antiapoptotic gene clusterin and adenoviral-mediated p53 gene transfer (Ad5CMV-p53) using the human bladder cancer KoTCC-1 model. Clusterin expression in KoTCC-1 cells was highly upregulated by Ad5CMV-p53 treatment; however, AS clusterin ODN treatment further suppressed clusterin expression in KoTCC-1 cells after Ad5CMV-p53 treatment. AS clusterin ODN treatment synergistically enhanced the cytotoxic effect of Ad5CMV-p53, and DNA fragmentation characteristic of apoptosis was observed only after combined treatment with AS clusterin ODN and Ad5CMV-p53, but not after treatment with either agent alone. Administration of AS clusterin ODN and Ad5CMV-p53 into nude mice resulted in a significant inhibition of KoTCC-1 tumor growth as well as lymph node metastases compared to administration of either agent alone. Furthermore, combined treatment with AS clusterin ODN, Ad5CMV-p53, and cisplatin completely eradicated KoTCC-1 tumors and lymph node metastases in 60% and 100% of mice, respectively. These findings suggest that combined treatment with AS clusterin ODN and Ad5CMV-p53 could be a novel strategy to inhibit bladder cancer progression, and that further additional use of a chemotherapeutic agent may substantially enhance the efficacy of this combined regimen. PMID:15802022

  17. Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

    PubMed

    Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

    2002-10-01

    Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

  18. Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

    PubMed Central

    2011-01-01

    Background Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells. PMID

  19. Composite nanoparticles for gene delivery.

    PubMed

    Wang, Yuhua; Huang, Leaf

    2014-01-01

    Nanoparticle-mediated gene and siRNA delivery has been an appealing area to gene therapists when they attempt to treat the diseases by manipulating the genetic information in the target cells. However, the advances in materials science could not keep up with the demand for multifunctional nanomaterials to achieve desired delivery efficiency. Researchers have thus taken an alternative approach to incorporate various materials into single composite nanoparticle using different fabrication methods. This approach allows nanoparticles to possess defined nanostructures as well as multiple functionalities to overcome the critical extracellular and intracellular barriers to successful gene delivery. This chapter will highlight the advances of fabrication methods that have the most potential to translate nanoparticles from bench to bedside. Furthermore, a major class of composite nanoparticle-lipid-based composite nanoparticles will be classified based on the components and reviewed in details.

  20. Microbubble-assisted p53, RB, and p130 gene transfer in combination with radiation therapy in prostate cancer.

    PubMed

    Nande, Rounak; Greco, Adelaide; Gossman, Michael S; Lopez, Jeffrey P; Claudio, Luigi; Salvatore, Marco; Brunetti, Arturo; Denvir, James; Howard, Candace M; Claudio, Pier Paolo

    2013-06-01

    Combining radiation therapy and direct intratumoral (IT) injection of adenoviral vectors has been explored as a means to enhance the therapeutic potential of gene transfer. A major challenge for gene transfer is systemic delivery of nucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles) are viable candidates to enhance targeted delivery of systemically administered genes. Here we show that p53, pRB, and p130 gene transfer mediated by US cavitation of microbubbles at the tumor site resulted in targeted gene transduction and increased reduction in tumor growth compared to DU-145 prostate cancer cell xenografts treated intratumorally with adenovirus (Ad) or radiation alone. Microbubble-assisted/US-mediated Ad.p53 and Ad.RB treated tumors showed significant reduction in tumor volume compared to Ad.p130 treated tumors (p<0.05). Additionally, US mediated microbubble delivery of p53 and RB combined with external beam radiation resulted in the most profound tumor reduction in DU-145 xenografted nude mice (p<0.05) compared to radiation alone. These findings highlight the potential therapeutic applications of this novel image-guided gene transfer technology in combination with external beam radiation for prostate cancer patients with therapy resistant disease.

  1. Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

    PubMed Central

    Candolfi, Marianela; Xiong, Weidong; Yagiz, Kader; Liu, Chunyan; Muhammad, A. K. M. G.; Puntel, Mariana; Foulad, David; Zadmehr, Ali; Ahlzadeh, Gabrielle E.; Kroeger, Kurt M.; Tesarfreund, Matthew; Lee, Sharon; Debinski, Waldemar; Sareen, Dhruv; Svendsen, Clive N.; Rodriguez, Ron; Lowenstein, Pedro R.; Castro, Maria G.

    2010-01-01

    Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM. PMID:21030678

  2. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  3. Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.

    PubMed

    Robert, Marc-André; Zeng, Yue; Raymond, Benoît; Desfossé, Laurie; Mairey, Emilie; Tremblay, Jacques P; Massie, Bernard; Gilbert, Rénald

    2012-12-01

    Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.

  4. Polyethylenimine-mediated gene delivery: a mechanistic study.

    PubMed

    Kichler, A; Leborgne, C; Coeytaux, E; Danos, O

    2001-01-01

    Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7-74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes.

  5. Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides.

    PubMed

    Chae, Su Young; Kim, Hyun June; Lee, Min Sang; Jang, Yeon Lim; Lee, Yuhan; Lee, Soo Hyeon; Lee, Kyuri; Kim, Sun Hwa; Kim, Hong Tae; Chi, Sang-Cheol; Park, Tae Gwan; Jeong, Ji Hoon

    2011-09-09

    Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The evolution of adenoviral vectors through genetic and chemical surface modifications.

    PubMed

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-02-17

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

  7. Biological Gene Delivery Vehicles: Beyond Viral Vectors

    PubMed Central

    Seow, Yiqi; Wood, Matthew J

    2009-01-01

    Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications. PMID:19277019

  8. Biological gene delivery vehicles: beyond viral vectors.

    PubMed

    Seow, Yiqi; Wood, Matthew J

    2009-05-01

    Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications.

  9. Gene Delivery Strategies to Promote Spinal Cord Repair

    PubMed Central

    Walthers, Christopher M; Seidlits, Stephanie K

    2015-01-01

    Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed. PMID:25922572

  10. The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

    PubMed

    Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

    2012-12-15

    The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

  11. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

    PubMed Central

    Campos, Samuel K.; Barry, Michael A.

    2008-01-01

    Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting. PMID:17584037

  12. In vivo gene delivery and expression by bacteriophage lambda vectors.

    PubMed

    Lankes, H A; Zanghi, C N; Santos, K; Capella, C; Duke, C M P; Dewhurst, S

    2007-05-01

    Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

  13. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  14. New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

    PubMed

    Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

    2012-01-01

    The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

  15. New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization

    PubMed Central

    Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

    2012-01-01

    Background The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery. PMID:22802689

  16. Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

    PubMed

    Stachelek, S J; Song, C; Alferiev, I; Defelice, S; Cui, X; Connolly, J M; Bianco, R W; Levy, R J

    2004-01-01

    The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen

  17. The PPARγ2 A/B-Domain Plays a Gene-Specific Role in Transactivation and Cofactor Recruitment

    PubMed Central

    Bugge, Anne; Grøntved, Lars; Aagaard, Mads M.; Borup, Rehannah; Mandrup, Susanne

    2009-01-01

    We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5–8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARγ2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARγ lacking the A/B-domain (PPARγCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared with cells expressing full-length PPARγ2. Using chromatin immunoprecipitation, we demonstrate that PPARγ binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARγ-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARγ2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes. PMID:19282365

  18. Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

    PubMed

    Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

    2016-11-01

    Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. An effective method for adenoviral-mediated delivery of small interfering RNA into mesenchymal stem cells.

    PubMed

    Forte, Amalia; Napolitano, Marco A; Cipollaro, Marilena; Giordano, Antonio; Cascino, Antonino; Galderisi, Umberto

    2007-02-01

    Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies, due to their intrinsic ability to differentiate along different mesenchymal cell lineages, able to repair the diseased or injured tissue in which they are localized. The application of MSCs in therapies requires an in depth knowledge of their biology and of the molecular mechanisms leading to MSC multilineage differentiation. The knockdown of target genes through small interfering RNA (siRNA) carried by adenoviruses (Ad) represents a valid tool for the study of the role of specific molecules in cell biology. Unfortunately, MSCs are poorly transfected by conventional Ad serotype 5 (Ad5) vectors. We set up a method to obtain a very efficient transduction of rat MSCs with low doses of unmodified Ad5, carrying the siRNA targeted against the mRNA coding for Rb2/p130 (Ad-siRNA-Rb2), which plays a fundamental role in cell differentiation. This method allowed a 95% transduction rate of Ad-siRNA in MSC, along with a siRNA-mediated 85% decrease of Rb2/p130 mRNA and a 70% decrease of Rb2/p130 protein 48 h after transduction with 50 multiplicities of infection (MOIs) of Ad5. The effect on Rb2/p130 protein persisted 15 days after transduction. Finally, Ad-siRNA did not compromise the viability of transduced MSCs neither induced any cell cycle modification. The effective Ad-siRNA-Rb2 we constructed, together with the efficient method of delivery in MSCs we set up, will allow an in depth analysis of the role of Rb2/p130 in MSC biology and multilineage differentiation.

  20. Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

    PubMed

    Kolb, Andreas F; Knowles, Christopher; Pultinevicius, Patrikas; Harbottle, Jennifer A; Petrie, Linda; Robinson, Claire; Sorrell, David A

    2017-01-01

    Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

  1. Inhibition of Experimental Liver Cirrhosis in Mice by Telomerase Gene Delivery

    NASA Astrophysics Data System (ADS)

    Rudolph, Karl Lenhard; Chang, Sandy; Millard, Melissa; Schreiber-Agus, Nicole; DePinho, Ronald A.

    2000-02-01

    Accelerated telomere loss has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis. To test this hypothesis directly, telomerase-deficient mice, null for the essential telomerase RNA (mTR) gene, were subjected to genetic, surgical, and chemical ablation of the liver. Telomere dysfunction was associated with defects in liver regeneration and accelerated the development of liver cirrhosis in response to chronic liver injury. Adenoviral delivery of mTR into the livers of mTR-/- mice with short dysfunctional telomeres restored telomerase activity and telomere function, alleviated cirrhotic pathology, and improved liver function. These studies indicate that telomere dysfunction contributes to chronic diseases of continual cellular loss-replacement and encourage the evaluation of ``telomerase therapy'' for such diseases.

  2. Controlled superficial assembly of DNA-amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery.

    PubMed

    Oyane, Ayako; Araki, Hiroko; Nakamura, Maki; Shimizu, Yoshiki; Shubhra, Quazi T H; Ito, Atsuo; Tsurushima, Hideo

    2016-05-01

    Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

    PubMed

    Lopes, Cátia D F; Gonçalves, Nádia P; Gomes, Carla P; Saraiva, Maria J; Pêgo, Ana P

    2017-03-01

    Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Adenoviral Vector Immunity: Its Implications and circumvention strategies

    PubMed Central

    Ahi, Yadvinder S.; Bangari, Dinesh S.; Mittal, Suresh K.

    2014-01-01

    Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations. PMID:21453277

  5. Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes.

    PubMed

    Arangoa, M A; Düzgüneş, N; Tros de Ilarduya, C

    2003-01-01

    A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.

  6. N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

    PubMed

    Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

    2002-01-01

    It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

  7. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

    PubMed

    Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

    2002-02-05

    Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

  8. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

    PubMed Central

    Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

    2015-01-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  9. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications. PMID:28344260

  10. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  11. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naumov, Inna; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Kazanov, Dina

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1more » and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.« less

  12. INGN 201: Ad-p53, Ad5CMV-p53, adenoviral p53, p53 gene therapy--introgen, RPR/INGN 201.

    PubMed

    2007-01-01

    Introgen and its wholly owned European subsidiary Gendux AB are developing an adenoviral p53 gene therapy as a treatment for cancer in the US and Europe, respectively. Phase III trials in patients with head and neck cancer are ongoing, and a number of clinical trials in other cancer indications have been completed. INGN 201 is being reviewed by the EMEA for approval in Li-Fraumeni syndrome (LFS) under the provisions of exceptional circumstance; the therapy is available on a compassionate use basis to eligible LFS cancer patients under a protocol authorised by the US FDA. The p53 tumour suppressor gene is deleted or mutated in many tumour cells and is one of the most frequently mutated genes in human tumours. The p53 protein is one of the most intricate elements in the apoptotic signalling cascade, and a mutation in the gene encoding it is believed to result in a decreased ability of a cell to apoptose. Thus replacing this gene via adenovirally-mediated p53 gene therapy is hoped to result in increased apoptosis where it is administered.INGN 201 is available for licensing, although Introgen favours retaining partial or full rights to the therapy in the US. Introgen entered into a license agreement with The University of Texas System and MD Anderson Cancer Center in 1994. The technologies licenced include p53 and fus1 (INGN 401). The collaboration has yielded exclusive patent and licensing rights to numerous technologies. Introgen entered into a collaboration with Rhône-Poulenc Rorer Pharmaceuticals (now sanofi-aventis) to develop therapeutics based on p53 inhibition in October 1994. However, in June 2001 this relationship was restructured and Introgen assumed responsibility for the worldwide development of all p53 products including INGN 201, and acquired all marketing and commercialisation rights with respect to those products. Introgen initiated two phase III trials in head and neck cancer (in June 2000 and May 2001) at about 80 sites in the US, Canada and Europe

  13. Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

    PubMed Central

    Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

    2009-01-01

    Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

  14. Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

    PubMed Central

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

    2013-01-01

    The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  15. Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

    2004-11-01

    Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

  16. Convection-enhanced delivery of AAV2 in white matter--a novel method for gene delivery to cerebral cortex.

    PubMed

    Barua, N U; Woolley, M; Bienemann, A S; Johnson, D; Wyatt, M J; Irving, C; Lewis, O; Castrique, E; Gill, S S

    2013-10-30

    Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Synthesis of galactosyl compounds for targeted gene delivery.

    PubMed

    Ren, T; Zhang, G; Liu, D

    2001-11-01

    Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

  18. Construction of a star-shaped copolymer as a vector for FGF receptor-mediated gene delivery in vitro and in vivo.

    PubMed

    Li, Da; Ping, Yuan; Xu, Fujian; Yu, Hai; Pan, Hongming; Huang, Hongliang; Wang, Qingqing; Tang, Guping; Li, Jun

    2010-09-13

    The success of cancer gene therapy highly relies on the gene delivery vector with high transfection activity and low toxicity. In the present study, eight-armed polyethylene glycol (EAP) and low molecular weight (LMW) polyethylenimine (PEI) were used as basic units to construct the architecture of a new star-shaped EAP-PEI copolymer (EAPP). MC11, a peptide capable of selectively binding fibroblast growth factor receptor (FGFR) on tumor cell membranes, was further conjugated to EAPP to produce the vector EAPP-MC11 (EAPPM) to enhance tumor targetability. This tumor-targeting vector EAPPM was observed to retard the plasmids mobility at a nitrogen/phosphorus (N/P) ratio of 3. The vector could efficiently condense plasmids within 300 nm nanoparticles with a positive zeta potential at the N/P ratio of 20 or above. While the cytotoxicity of EAPPM polyplexes was similar to that of LMW PEI, it was significantly lower than that of PEI (25 kDa) in HepG2 and PC3 cell lines. In vitro gene transfection with pDNA mediated by EAPPM showed that the transfection efficiency increased 15 times in HepG2 cells but remained at a similar level in PC3 cells in comparison with that of EAPP. By systemic injection of EAPPM/pDNA complexes into a HepG2-bearing mice model, luciferase expression detected in lung, liver, and tumor tissues demonstrated EAPPM could deliver in a targeted manner a reporter gene into tumor tissues, where the luciferase expression of EAPPM was 4 times higher than that of EAPP and even 23 times higher than that of PEI (25 kDa). Furthermore, it was found that the systemic delivery of EAPPM/pCSK-α-interferon complexes in vivo were much more effective in inhibiting tumor growth than EAPP or PEI (25 kDa). These results clearly show that EAPPM is an efficient and safe vector for FGFR-mediated targeted gene delivery both in vitro and in vivo. With low cytotoxicity and high targetability, EAPPM may have great potential as a delivery vector for future cancer gene therapy

  19. Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

    PubMed Central

    Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

    2015-01-01

    Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

  20. Barriers to Liposomal Gene Delivery: from Application Site to the Target.

    PubMed

    Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

    2016-01-01

    Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

  1. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  2. Sonoporation, drug delivery, and gene therapy.

    PubMed

    Liang, H-D; Tang, J; Halliwell, M

    2010-01-01

    Ultrasound is a very effective modality for drug delivery and gene therapy because energy that is non-invasively transmitted through the skin can be focused deeply into the human body in a specific location and employed to release drugs at that site. Ultrasound cavitation, enhanced by injected microbubbles, perturbs cell membrane structures to cause sonoporation and increases the permeability to bioactive materials. Cavitation events also increase the rate of drug transport in general by augmenting the slow diffusion process with convective transport processes. Drugs and genes can be incorporated into microbubbles, which in turn can target a specific disease site using ligands such as the antibody. Drugs can be released ultrasonically from microbubbles that are sufficiently robust to circulate in the blood and retain their cargo of drugs until they enter an insonated volume of tissue. Local drug delivery ensures sufficient drug concentration at the diseased region while limiting toxicity for healthy tissues. Ultrasound-mediated gene delivery has been applied to heart, blood vessel, lung, kidney, muscle, brain, and tumour with enhanced gene transfection efficiency, which depends on the ultrasonic parameters such as acoustic pressure, pulse length, duty cycle, repetition rate, and exposure duration, as well as microbubble properties such as size, gas species, shell material, interfacial tension, and surface rigidity. Microbubble-augmented sonothrombolysis can be enhanced further by using targeting microbubbles.

  3. Adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter for colorectal cancer.

    PubMed

    Wang, Zhao-Xia; Bian, Hai-Bo; Yang, Jing-Song; De, Wei; Ji, Xiao-Hui

    2009-08-01

    Colorectal cancer is a most frequent type of gastrointestinal tract cancers. The prognosis of patients with colorectal cancer remains poor despite intensive interventions. Tumor specific promoter-directed gene therapy and adenoviral technology can be promising strategies for such advanced disease. This study was conducted to explore the possible therapeutic approach of Cox-2 promoter-directed suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with adenoviral technology for advanced colorectal cancer. Firstly, the activity of Cox-2 promoter was assessed by dual luciferase and enhanced green fluorescent protein reporter gene assays in colorectal cancer cell lines and normal human intestinal epithelial cell line. Then, the expression of coxsackievirus and adenovirus receptor (CAR) was detected in colorectal cancer cell lines. The Cox-2 promoter-directed HSV-tk/ganciclovir (GCV) system mediated by adenovirus (Ad-Cp-TK) was developed (Ad-CMVp-TK, Ad-null and no Ad as controls). In vitro cytoxicity, colony formation and apoptosis assays were performed using Ad-Cp-TK. An animal study was carried out in which BALB/C nude mice bearing tumors were treated with Ad-Cp-TK and GCV treatments. Results showed that Cox-2 promoter possessed high transcriptional activity in a tumor-specific manner. All colorectal cancer cells were detected CAR-positive. In vitro cytotoxic and colony formation assays showed that colorectal cancer cells infected with Ad-Cp-TK became more sensitive to GCV but the sensitivity of normal cells infected with Ad-Cp-TK to GCV were not altered. Moreover, the Ad-Cp-TK system combined with GCV treatment could significantly induce apoptosis of colorectal cancer cells but not normal intestinal epithelial cells. Furthermore, this system also significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice. Thus, adenovirus primary receptor was positive in colorectal cancer cells and adenovirus-mediated

  4. Antitumoral Cascade-Targeting Ligand for IL-6 Receptor-Mediated Gene Delivery to Glioma.

    PubMed

    Wang, Shanshan; Reinhard, Sören; Li, Chengyi; Qian, Min; Jiang, Huiling; Du, Yilin; Lächelt, Ulrich; Lu, Weiyue; Wagner, Ernst; Huang, Rongqin

    2017-07-05

    The effective treatment of glioma is largely hindered by the poor transfer of drug delivery systems across the blood-brain barrier (BBB) and the difficulty in distinguishing healthy and tumorous cells. In this work, for the first time, an interleukin-6 receptor binding I 6 P 7 peptide was exploited as a cascade-targeting ligand in combination with a succinoyl tetraethylene pentamine (Stp)-histidine oligomer-based nonviral gene delivery system (I 6 P 7 -Stp-His/DNA). The I 6 P 7 peptide provides multiple functions, including the cascade-targeting potential represented by a combined BBB-crossing and subsequent glioma-targeting ability, as well as a direct tumor-inhibiting effect. I 6 P 7 -Stp-His/DNA nanoparticles (NPs) mediated higher gene expression in human glioma U87 cells than in healthy human astrocytes and a deeper penetration into glioma spheroids than scrambled peptide-modified NPs. Transport of I 6 P 7 -modified, but not the control, NPs across the BBB was demonstrated in vitro in a transwell bEnd.3 cell model resulting in transfection of underlying U87 cells and also in vivo in glioma-bearing mice. Intravenous administration of I 6 P 7 -Stp-His/plasmid DNA (pDNA)-encoding inhibitor of growth 4 (pING4) significantly prolonged the survival time of orthotopic U87 glioma-bearing mice. The results denote that I 6 P 7 peptide is a roborant cascade-targeting ligand, and I 6 P 7 -modified NPs might be exploited for efficient glioma therapy. Copyright © 2017. Published by Elsevier Inc.

  5. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

    PubMed

    Keeney, Michael; Chung, Michael T; Zielins, Elizabeth R; Paik, Kevin J; McArdle, Adrian; Morrison, Shane D; Ransom, Ryan C; Barbhaiya, Namrata; Atashroo, David; Jacobson, Gunilla; Zare, Richard N; Longaker, Michael T; Wan, Derrick C; Yang, Fan

    2016-08-01

    Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016. © 2016 Wiley Periodicals, Inc.

  6. Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection.

    PubMed

    Kobulnik, Jeremy; Kuliszewski, Michael A; Stewart, Duncan J; Lindner, Jonathan R; Leong-Poi, Howard

    2009-10-27

    This study was designed to compare the efficacy of angiogenic gene delivery by ultrasound-mediated (UM) destruction of intravenous carrier microbubbles to direct intramuscular (IM) injections. Current trials of gene therapy for angiogenesis remain limited by suboptimal, invasive delivery techniques. Hind-limb ischemia was produced by iliac artery ligation in 99 rats. In 32 rats, UM delivery of green fluorescent protein (GFP)/vascular endothelial growth factor-165 (VEGF(165)) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF(165)/GFP plasmid. Remaining rats received no treatment. Before delivery (day 14 after ligation) and at days 17, 21, and 28 and week 8 after ligation, microvascular blood volume and microvascular blood flow to the proximal hind limbs were assessed by contrast-enhanced ultrasound (n = 8 per group). Total transfection was assessed by reverse transcriptase-polymerase chain reaction, and localization of transfection was determined by immunohistochemistry. By day 28, both IM and UM delivery of VEGF(165) produced significant increases in microvascular blood volume and microvascular blood flow. Whereas increases in microvascular blood volume were similar between treatment groups, microvascular blood flow was greater (p < 0.005) in UM-treated animals as compared with IM-treated animals, persisting to week 8. The VEGF(165)/GFP messenger ribonucleic acid expression was greater (p < 0.05) for IM-treated animals. A strong GFP signal was detected for both groups and was localized to focal perivascular regions and myocytes around injection sites for IM and to the vascular endothelium of arterioles/capillaries in a wider distribution for UM delivery. Despite lower transfection levels, UM delivery of VEGF(165) is as effective as IM injections. The UM delivery results in directed vascular transfection over a wider distribution, which may account for the more efficient angiogenesis.

  7. Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

    PubMed Central

    Fishbein, Ilia; Alferiev, Ivan; Bakay, Marina; Stachelek, Stanley J.; Sobolewski, Peter; Lai, Meizan; Choi, Hoon; Chen, I.-W.; Levy, Robert J.

    2012-01-01

    Background Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. Methods and Results We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37°C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial AdGFP expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Conclusions Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature. PMID:18413497

  8. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

    NASA Astrophysics Data System (ADS)

    Fields, Rachel Jennifer

    NPs to deliver difficult to gene therapy reagents, such as PNAs, to cells within the lung, I loaded NPs with PNA and DNA and administered them via the intranasal route. Triplex forming peptide nucleic acids (PNAs) are gene therapy reagents that can mediate site-specific homologous recombination with genomic DNA when successfully delivered to the nucleus of cells in combination with donor DNA oligos. Delivery of NPs resulted in EGFP expression in transgenic mice with an aberrant EGFP gene that could be corrected and effectively expressed with nuclear delivery of a PNA/DNA. This work represents the first successful use of PNA/DNA mediated homologous recombination to target cells of the lung.

  9. Cell-Mediated Drugs Delivery

    PubMed Central

    Batrakova, Elena V.; Gendelman, Howard E.; Kabanov, Alexander V.

    2011-01-01

    INTRODUCTION Drug targeting to sites of tissue injury, tumor or infection with limited toxicity is the goal for successful pharmaceutics. Immunocytes (including mononuclear phagocytes (dendritic cells, monocytes and macrophages), neutrophils, and lymphocytes) are highly mobile; they can migrate across impermeable barriers and release their drug cargo at sites of infection or tissue injury. Thus immune cells can be exploited as trojan horses for drug delivery. AREAS COVERED IN THIS REVIEW This paper reviews how immunocytes laden with drugs can cross the blood brain or blood tumor barriers, to facilitate treatments for infectious diseases, injury, cancer, or inflammatory diseases. The promises and perils of cell-mediated drug delivery are reviewed, with examples of how immunocytes can be harnessed to improve therapeutic end points. EXPERT OPINION Using cells as delivery vehicles enables targeted drug transport, and prolonged circulation times, along with reductions in cell and tissue toxicities. Such systems for drug carriage and targeted release represent a novel disease combating strategy being applied to a spectrum of human disorders. The design of nanocarriers for cell-mediated drug delivery may differ from those used for conventional drug delivery systems; nevertheless, engaging different defense mechanisms into drug delivery may open new perspectives for the active delivery of drugs. PMID:21348773

  10. Ultrasound-assisted non-viral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion

    PubMed Central

    Wang, Z; Zourelias, L; Wu, C; Edwards, PC; Trombetta, M; Passineau, MJ

    2015-01-01

    Rationale Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial utilizing Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system utilized in this pioneering trial preclude its advancement to a Phase II trial and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted non-viral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Findings Swine irradiated unilaterally with a 10Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 hours and one week post-treatment (1.62+/−0.48ml, 1.87+/−0.45ml) to pre-injury levels (1.34+/−0.14ml) in a manner comparable to Adenoviral delivery (2.32+/−0.6ml, 1.33+/−0.97ml). Conclusions UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model and is a candidate for advancement to a Phase I human clinical trial. PMID:25871828

  11. Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles

    PubMed Central

    2013-01-01

    Gene therapy provides an efficient approach for treatment of cardiovascular disease. To realize the therapeutic effect, both efficient delivery to the target cells and sustained expression of transgenes are required. Ultrasound targeted microbubble destruction (UTMD) technique has become a potential strategy for target-specific gene and drug delivery. When gene-loaded microbubble is injected, the ultrasound-mediated microbubble destruction may spew the transported gene to the targeted cells or organ. Meanwhile, high amplitude oscillations of microbubbles increase the permeability of capillary and cell membrane, facilitating uptake of the released gene into tissue and cell. Therefore, efficiency of gene therapy can be significantly improved. To date, UTMD has been successfully investigated in many diseases, and it has achieved outstanding progress in the last two decades. Herein, we discuss the current status of gene therapy of cardiovascular diseases, and reviewed the progress of the delivery of genes to cardiovascular system by UTMD. PMID:23594865

  12. Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2014-10-01

    The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body's natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality.

  13. Cancer gene therapy with targeted adenoviruses.

    PubMed

    Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

    2008-11-01

    Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

  14. Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes.

    PubMed

    Pohle, D; Kasch, R; Herlyn, P; Bader, R; Mittlmeier, T; Pützer, B M; Müller-Hilke, B

    2012-09-01

    The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins. Copyright © 2012 Wiley Periodicals, Inc.

  15. Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

    PubMed Central

    Cotten, M; Wagner, E; Zatloukal, K; Birnstiel, M L

    1993-01-01

    Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus. Images PMID:8099627

  16. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

    2015-09-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

  17. In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

    PubMed

    Moreno, Ana M; Fu, Xin; Zhu, Jie; Katrekar, Dhruva; Shih, Yu-Ru V; Marlett, John; Cabotaje, Jessica; Tat, Jasmine; Naughton, John; Lisowski, Leszek; Varghese, Shyni; Zhang, Kang; Mali, Prashant

    2018-04-25

    Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  18. Magnetic nanoparticles: Applications in gene delivery and gene therapy.

    PubMed

    Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

    2016-06-01

    Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

  19. New serine-derived gemini surfactants as gene delivery systems.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

    2015-01-01

    Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

    PubMed Central

    Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

    2016-01-01

    Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

  1. Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery.

    PubMed

    Martin, Timothy M; Wysocki, Beata J; Beyersdorf, Jared P; Wysocki, Tadeusz A; Pannier, Angela K

    2014-08-01

    Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

  2. Far-red light-mediated programmable anti-cancer gene delivery in cooperation with photodynamic therapy.

    PubMed

    Wang, Jinhui; He, Hua; Xu, Xin; Wang, Xiao; Chen, Yongbing; Yin, Lichen

    2018-07-01

    Effective anti-cancer therapy is hurdled by the complicated extracellular and intracellular barriers, and thus a smart gene vector that can enable programmable gene delivery is highly demanded. Photo-manipulation of gene delivery processes features spatial and temporal precision, while majority of current strategies utilizes short-wavelength UV/visible light with poor tissue penetration or high-power-density near-infrared (NIR) light that would cause undesired heat damage. Herein, an ROS-degradable polycation was designed and co-delivered with a photosensitizer (PS), thus realizing photo-programmable gene delivery using far-red light (661 nm) at low optical power density (down to 5 mW cm -2 ). Thioketal-crosslinked polyethylenimine (TK-PEI) was synthesized to condense p53 gene to form nanocomplexes (NCs), and hyaluronic acid (HA) modified with pheophytin a (Pha) was coated onto NCs to enhance their colloidal stability and enable cancer cell targeting. Short-time (8-min) light irradiation produced non-lethal amount of ROS to disrupt the endosomal membranes and facilitate p53 gene release via degradation of TK-PEI, which collectively enhanced p53 expression levels toward anti-cancer gene therapy. Long-time (30-min) light irradiation at the post-transfection state generated lethal amount of ROS, which cooperatively killed cancer cells to strengthen p53 gene therapy. To the best of our knowledge, this study represents the first example of an "one stone, three birds" approach to realize cooperative anti-cancer gene therapy using low-power-density, long-wavelength visible light as a single stimulus. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Ultrasound-mediated drug delivery for cardiovascular disease

    PubMed Central

    Sutton, Jonathan T; Haworth, Kevin J; Pyne-Geithman, Gail; Holland, Christy K

    2014-01-01

    Introduction Ultrasound (US) has been developed as both a valuable diagnostic tool and a potent promoter of beneficial tissue bioeffects for the treatment of cardiovascular disease. These effects can be mediated by mechanical oscillations of circulating microbubbles, or US contrast agents, which may also encapsulate and shield a therapeutic agent in the bloodstream. Oscillating microbubbles can create stresses directly on nearby tissue or induce fluid effects that effect drug penetration into vascular tissue, lyse thrombi or direct drugs to optimal locations for delivery. Areas covered The present review summarizes investigations that have provided evidence for US-mediated drug delivery as a potent method to deliver therapeutics to diseased tissue for cardiovascular treatment. In particular, the focus will be on investigations of specific aspects relating to US-mediated drug delivery, such as delivery vehicles, drug transport routes, biochemical mechanisms and molecular targeting strategies. Expert opinion These investigations have spurred continued research into alternative therapeutic applications, such as bioactive gas delivery and new US technologies. Successful implementation of US-mediated drug delivery has the potential to change the way many drugs are administered systemically, resulting in more effective and economical therapeutics, and less-invasive treatments. PMID:23448121

  4. Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

    PubMed

    Bak, Eun-Jung; Jho, Yeonsook; Woo, Gye-Hyeong

    2018-06-01

    Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

  5. Self-assembled pentablock copolymers for selective and sustained gene delivery

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Bingqi

    2011-05-15

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomesmore » of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.« less

  6. Evolving phage vectors for cell targeted gene delivery.

    PubMed

    Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

    2002-03-01

    We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

  7. Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2017-10-01

    mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

  8. Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling

    PubMed Central

    Cittadini, A; Monti, MG; Iaccarino, G; Di Rella, F; Tsichlis, PN; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

    2010-01-01

    The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the β-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca2+) handling, oxygen consumption, and β-adrenergic pathway. To this aim, Sprague–Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca2+ handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca2+–force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6±0.2 versus 2.7±0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca2+ available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca2+ responsiveness, documented by an increased maximal Ca2+-activated pressure (+19% versus controls) and a shift to the left of the Ca2+–force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca2+ ATPase protein levels were significantly increased in Ad.Akt rats. β-Adrenergic receptor density, affinity, kinase-1 levels, and

  9. Inhibition of myostatin gene expression in skeletal muscle of fish by in vivo electrically mediated dsRNA and shRNAi delivery.

    PubMed

    Terova, Genciana; Rimoldi, Simona; Bernardini, Giovanni; Saroglia, Marco

    2013-06-01

    Myostatin (MSTN), previously referred to as growth differentiation factor 8 (GDF8), is a negative regulator of skeletal muscle growth. In accordance with this role, natural mutations that inactivate the gene disrupting the function of the protein are associated with excessive muscle growth and double-muscling phenotype in several mammalian species. Recent studies using transgenic MSTN deficient zebrafish and medaka support the idea that this gene inhibits skeletal muscle growth even in fish. If the atrophic actions of mammalian MSTN are indeed conserved in fish, strategies capable of inhibiting the expression of this gene could be applied to enhance growth performance in livestock production. Gene silencing by RNA interference has emerged as a promising new method of inhibiting the expression of targeted genes and inducing knockdown of associated proteins both in vitro and in vivo. Accordingly, we investigated here whether double-stranded RNA (dsRNA) or different plasmids expressing short-hairpin interfering RNAs (shRNAs) against myostatin and transduced by in vivo electroporation would increase skeletal muscle mass in reared European sea bass. After 7 weeks of intramuscular injections on a weekly basis followed by in vivo electrically mediated dsRNA delivery, no increase in the condition factor (K) of fish was observed as compared to the controls. Analogously, mean body weight and K of sea bass injected with three shRNAs were not higher than those of the control fish. On the other hand, MSTN transcript quantification via real-time RT-PCR revealed a significant inhibition of gene expression in the muscle of the dsRNA-injected fish and in the muscle of fish injected with one of the three tested shRNA-expressing vector constructs. In conclusion, in vivo electric-mediated delivery of dsRNA- or shRNA-expressing vectors against MSTN inhibits MSTN gene expression in adult sea bass muscle, but this is associated with an inconsistent double-muscle phenotype.

  10. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors.

    PubMed

    Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

    2010-07-02

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  11. Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

    2015-03-02

    Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

  12. Gene delivery by a steroid-peptide nucleic acid conjugate.

    PubMed

    Rebuffat, Alexandre G; Nawrocki, Andrea R; Nielsen, Peter E; Bernasconi, Alessio G; Bernal-Mendez, Eloy; Frey, Brigitte M; Frey, Felix J

    2002-09-01

    We previously introduced a method called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the nuclear uptake of transfected DNA. Here, we describe a SMGD strategy with peptide nucleic acids (PNAs) that allowed linkage of a steroid molecule to a defined position in a plasmid without disturbing its gene expression. We synthesized and tested several bifunctional steroid derivatives [patent in process of nationalization] and finally selected the compound named DEX-bisPNA, a molecule consisting of a dexamethasone moiety linked to a PNA clamp (bisPNA) through a 30-atom chemical spacer. Dex-bisPNA binds to the glucocorticoid receptor (GR) as well as to reporter plasmids containing the corresponding PNA binding sites, translocates the GR from the cytoplasm into the nucleus, and increases the delivery of plasmid to the nucleus, resulting in enhanced GR-dependent expression of the reporter gene. The SMGD effect was more pronounced in growth-arrested cells than in proliferating cells. The specificity for the GR was shown by the reversion of the SMGD effect in the presence of dexamethasone as well as an enhanced expression in GR-positive cells but not in GR-negative cells. Thus, SMGD with PNA is a promising strategy for nonviral gene delivery into target tissues expressing specific steroid receptors.

  13. ROLE OF GENETIC SUSCEPTIBILITY TO LATENT ADENOVIRAL INFECTION AND DECREASED LUNG FUNCTION

    PubMed Central

    Kasuga, Ikuma; Hogg, James C.; Paré, Peter D.; Hayashi, Shizu; Sedgwick, Edward G.; Ruan, Jian; Wallace, Alison M.; He, Jian-Qing; Zhang, Xiaozhu; Sandford, Andrew J.

    2009-01-01

    Background Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin β5 (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function. Methods Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay. Results E1A positive individuals have a lower FEV1 compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV1 level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes. Conclusions Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD. PMID:19502044

  14. Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

    PubMed Central

    Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

    2013-01-01

    Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

  15. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

    PubMed

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  16. Vestibular regeneration--experimental models and clinical implications.

    PubMed

    Albu, Silviu; Muresanu, Dafin F

    2012-09-01

    Therapies aimed at the protection and/or regeneration of inner ear hair cells are of great interest, given the significant monetary and quality of life impact of balance disorders. Different viral vectors have been shown to transfect various cell types in the inner ear. The past decade has provided tremendous advances in the use of adenoviral vectors to achieve targeted treatment delivery. Several routes of delivery have been identified to introduce vectors into the inner ear while minimizing injury to surrounding structures. Recently, the transcription factor Atoh1 was determined to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo has demonstrated the ability to regenerate vestibular hair cells by causing transdifferentiation of neighbouring epithelial-supporting cells. Functional recovery of the vestibular system has also been documented following adenoviral-induced Atoh1 overexpression. Experiments demonstrating gene transfer in human vestibular epithelial cells reveal that the human inner ear is a suitable target for gene therapy. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  17. Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

    PubMed

    Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-08-01

    Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. © 2016 Federation of European Biochemical Societies.

  18. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  19. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    PubMed

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  20. Micro- and nanobubbles: a versatile non-viral platform for gene delivery.

    PubMed

    Cavalli, Roberta; Bisazza, Agnese; Lembo, David

    2013-11-18

    Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 μm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described. Copyright © 2013

  1. Right ventricular beneficial effects of beta adrenergic receptor kinase inhibitor (betaARKct) gene transfer in a rat model of severe pressure overload.

    PubMed

    Molina, Ezequiel J; Gupta, Dipin; Palma, Jon; Gaughan, John P; Macha, Mahender

    2009-06-01

    Heart failure is associated with abnormalities in betaAR cascade regulation, calcium cycling, expression of inflammatory mediators and apoptosis. Adenoviral mediated gene transfer of betaARKct has beneficial indirect effects on these pathologic processes upon the left ventricular myocardium. The concomitant biochemical changes that occur in the right ventricle have not been well characterized. Sprague-Dawley rats underwent aortic banding and were followed by echocardiography. After a decrease in fractional shortening of 25% from baseline, intracoronary injection of adenoviral-betaARKct (n=14) or adenoviral-beta-galactosidase (control, n=13) was performed. Rats were randomly euthanized on post-operative day 7, 14 or 21. Protein analysis including RV myocardial levels of betaARKct, betaARK1, SERCA(2a), inflammatory tissue mediators (IL-1, IL-6 and TNF-alpha), apoptotic markers (bax and bak), and MAP kinases (jnk, p38 and erk) was performed. ANOVA was employed for group comparison. Adenoviral-betaARKct treated animals showed increased expression of betaARKct and decreased levels of betaARK1 compared with controls. This treatment group also demonstrated normalization of SERCA(2a) expression and decreased levels of the inflammatory markers IL-1, IL-6 and TNF-alpha. The pro-apoptotic markers bax and bak were similarly improved. Ventricular levels of the MAP kinase jnk were increased. Differences were most significant 7 days after gene transfer, but the majority of these changes persisted at 21 days. These results suggest that attenuation of the pathologic mechanisms of beta adrenergic receptor desensitization, SERCA(2a) expression, inflammation and apoptosis, not only occur in the left ventricle but also in the right ventricular myocardium after intracoronary gene transfer of betaARKct during heart failure.

  2. Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

    PubMed Central

    Hooley, R P; Paterson, M; Brown, P; Kerr, K; Saunders, P T K

    2009-01-01

    Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. This paper describes a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems. PMID:18955374

  3. Ultrasound enhances retrovirus-mediated gene transfer.

    PubMed

    Naka, Toshio; Sakoda, Tsuyoshi; Doi, Takashi; Tsujino, Takeshi; Masuyama, Tohru; Kawashima, Seinosuke; Iwasaki, Tadaaki; Ohyanagi, Mitsumasa

    2007-01-01

    Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased

  4. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

    PubMed Central

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V. Bleu; Serrano, Elba E.

    2014-01-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology. PMID:21959846

  5. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

    PubMed

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.

  6. EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

    PubMed Central

    Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

    2013-01-01

    We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

  7. FUNCTIONAL NANOPARTICLES FOR MOLECULAR IMAGING GUIDED GENE DELIVERY

    PubMed Central

    Liu, Gang; Swierczewska, Magdalena; Lee, Seulki; Chen, Xiaoyuan

    2010-01-01

    Gene therapy has great potential to bring tremendous changes in treatment of various diseases and disorders. However, one of the impediments to successful gene therapy is the inefficient delivery of genes to target tissues and the inability to monitor delivery of genes and therapeutic responses at the targeted site. The emergence of molecular imaging strategies has been pivotal in optimizing gene therapy; since it can allow us to evaluate the effectiveness of gene delivery noninvasively and spatiotemporally. Due to the unique physiochemical properties of nanomaterials, numerous functional nanoparticles show promise in accomplishing gene delivery with the necessary feature of visualizing the delivery. In this review, recent developments of nanoparticles for molecular imaging guided gene delivery are summarized. PMID:22473061

  8. Microneedle-mediated vaccine delivery: Harnessing cutaneous immunobiology to improve efficacy

    PubMed Central

    Al-Zahrani, S; Zaric, M; McCrudden, C; Scott, C; Kissenpfennig, A; Donnelly, Ryan F.

    2014-01-01

    Introduction We describe the use of microneedle arrays for delivery to targets within the skin itself. Breaching the skin’s stratum corneum barrier raises the possibility of administration of vaccines, gene vectors, antibodies and even nanoparticles, all of which have at least their initial effect on populations of skin cells. Areas Covered Intradermal vaccine delivery, in particular, holds enormous potential for improved therapeutic outcomes for patients, particularly those in the developing world. Various vaccine-delivery strategies have been employed and here we discuss each one in turn. We also describe the importance of cutaneous immunobiology on the effect produced by microneedle-mediated intradermal vaccination. Expert Opinion Microneedle-mediated vaccine holds enormous potential for patient benefit. In order for microneedle vaccine strategies to fulfil their potential, however, the proportion of an immune response that is due to local action of delivered vaccines on skin antigen presenting cells and what is due to a systemic effect from vaccine reaching the systemic circulation must be determined. Moreover, industry will need to invest significantly in new equipment and instrumentation in order to mass produce microneedle vaccines consistently. Finally, microneedles will need to demonstrate consistent dose delivery across patient groups and match this to reliable immune responses before they will replace tried- and-tested needle-and-syringe based-approaches. PMID:22475249

  9. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma

    PubMed Central

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

    2016-01-01

    Background: Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. Study design: An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Materials and methods: Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. Results: In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Conclusion: Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. PMID:26884457

  10. Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis.

    PubMed

    Huang, Cuiyuan; Zhang, Hong; Bai, Ruidan

    2017-07-01

    Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble-mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.

  11. Non-viral gene delivery regulated by stiffness of cell adhesion substrates.

    PubMed

    Kong, Hyun Joon; Liu, Jodi; Riddle, Kathryn; Matsumoto, Takuya; Leach, Kent; Mooney, David J

    2005-06-01

    Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.

  12. Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain

    PubMed Central

    Mastorakos, Panagiotis; Song, Eric; Zhang, Clark; Berry, Sneha; Park, Hee Won; Kim, Young Eun; Park, Jong Sung; Lee, Seulki; Suk, Jung Soo; Hanes, Justin

    2016-01-01

    Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases. PMID:26680637

  13. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides.

    PubMed

    Chen, Yung-Jen; Liu, Betty Revon; Dai, Yun-Hao; Lee, Cheng-Yi; Chan, Ming-Huan; Chen, Hwei-Hsien; Chiang, Huey-Jenn; Lee, Han-Jung

    2012-02-10

    Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Microneedles As a Delivery System for Gene Therapy

    PubMed Central

    Chen, Wei; Li, Hui; Shi, De; Liu, Zhenguo; Yuan, Weien

    2016-01-01

    Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector) and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs), which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy. PMID:27303298

  15. Microneedle-mediated transdermal bacteriophage delivery

    PubMed Central

    Ryan, Elizabeth; Garland, Martin J.; Singh, Thakur Raghu Raj; Bambury, Eoin; O’Dea, John; Migalska, Katarzyna; Gorman, Sean P.; McCarthy, Helen O.; Gilmore, Brendan F.; Donnelly, Ryan F.

    2012-01-01

    Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific T4 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 × 106 PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 103 PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 103 PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. PMID:22750416

  16. Ionizing Radiation Enhances Adenoviral Vector Expressing mda-7/IL-24-mediated Apoptosis in Human Ovarian Cancer

    PubMed Central

    EMDAD, LUNI; SARKAR, DEVANAND; LEBEDEVA, IRINA V.; SU, ZAO-ZHONG; GUPTA, PANKAJ; MAHASRESHTI, PARAMESHWAR J.; DENT, PAUL; CURIEL, DAVID T.; FISHER, PAUL B.

    2007-01-01

    Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy. PMID:16646087

  17. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  18. External beam radiation therapy enhances mesenchymal stem cell-mediated sodium iodide symporter gene delivery.

    PubMed

    Schug, Christina; Sievert, Wolfgang; Urnauer, Sarah; Müller, Andrea Maria; Schmohl, Kathrin Alexandra; Wechselberger, Alexandra; Schwenk, Nathalie; Lauber, Kirsten; Schwaiger, Markus; Multhoff, Gabriele; Wagner, Ernst; Nelson, Peter J; Spitzweg, Christine

    2018-05-04

    The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium iodide symporter (NIS) to solid tumors. External beam radiation therapy (EBRT) may represent an ideal setting for the application of engineered MSC-based gene therapy as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7) (1-10 Gy) showed a strong dose-dependent increase in steady state mRNA levels of CXCL8, CXCL12/SDF-1, FGF2, PDGFβ, TGFβ1, TSP-1 and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration was tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index (yFMI), mean center of mass (yCoM) and mean directionality of MSCs towards supernatants was seen from irradiated as compared to nonirradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in qPCR and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2 or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using 123I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. Our results demonstrate that EBRT enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

  19. Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

    PubMed

    Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

    2009-03-09

    The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

  20. Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

    PubMed Central

    Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

    2015-01-01

    A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation. PMID:26225356

  1. Anti-adenoviral effect of anti-HIV agents in vitro in serotypes inducing keratoconjunctivitis.

    PubMed

    Uchio, Eiichi; Fuchigami, Aki; Kadonosono, Kazuaki; Hayashi, Akio; Ishiko, Hiroaki; Aoki, Koki; Ohno, Shigeaki

    2007-09-01

    Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

  2. Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery

    PubMed Central

    Kesharwani, Prashant; Iyer, Arun K.

    2015-01-01

    Advances in the application of nanotechnology in medicine have given rise to multifunctional smart nanocarriers that can be engineered with tunable physicochemical characteristics to deliver one or more therapeutic agent(s) safely and selectively to cancer cells, including intracellular organelle-specific targeting. Dendrimers having properties resembling biomolecules, with well-defined 3D nanopolymeric architectures, are emerging as a highly attractive class of drug and gene delivery vector. The presence of numerous peripheral functional groups on hyperbranched dendrimers affords efficient conjugation of targeting ligands and biomarkers that can recognize and bind to receptors overexpressed on cancer cells for tumor-cell-specific delivery. The present review compiles the recent advances in dendrimer-mediated drug and gene delivery to tumors by passive and active targeting principles with illustrative examples. PMID:25555748

  3. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

    PubMed Central

    Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

    2017-01-01

    Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

  4. Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier

    NASA Astrophysics Data System (ADS)

    Zamora, Genesis; Wang, Frederick; Sun, Chung-Ho; Trinidad, Anthony; Kwon, Young Jik; Cho, Soo Kyung; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2014-10-01

    The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either "naked" polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.

  5. Rapid Assembly of Customized TALENs into Multiple Delivery Systems

    PubMed Central

    Zhang, Zhengxing; Zhang, Siliang; Huang, Xin; Orwig, Kyle E.; Sheng, Yi

    2013-01-01

    Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated “user friendly” TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains. PMID:24244669

  6. Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

    PubMed

    Adam, Liana; Black, Peter C; Kassouf, Wassim; Eve, Beryl; McConkey, David; Munsell, Mark F; Benedict, William F; Dinney, Colin P N

    2007-05-01

    Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment

  7. Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

    PubMed Central

    Wulff, Holger; Krieger, Thorsten; Krüger, Karen; Stahmer, Ingrid; Thaiss, Friedrich; Schäfer, Hansjörg; Block, Andreas

    2007-01-01

    Background Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12. Results High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue. Conclusion VP16 transactivator-mediated and

  8. Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

    PubMed

    Ajithkumar, K C; Pramod, Kannissery

    2018-01-01

    Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

    PubMed

    Fitzgerald, Kathleen A; Guo, Jianfeng; Raftery, Rosanne M; Castaño, Irene Mencía; Curtin, Caroline M; Gooding, Matt; Darcy, Raphael; O' Brien, Fergal J; O' Driscoll, Caitriona M

    2016-09-25

    siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Gene therapy of uterine leiomyomas: adenovirus-mediated expression of dominant negative estrogen receptor inhibits tumor growth in nude mice.

    PubMed

    Al-Hendy, Ayman; Lee, Eun J; Wang, Hui Q; Copland, John A

    2004-11-01

    Leiomyomas (fibroids) are common estrogen-dependent uterine tumors with no effective medicinal treatment; hysterectomy is the mainstay of management. This study was undertaken to investigate a potential therapy for leiomyoma; we used a mutated dominant-negative estrogen receptor gene delivered via an adenoviral vector (Ad-ER-DN). Ad-ER-DN transduction, in both human and rat leiomyoma cell lines, induced an increase in both caspase-3 levels and BAX/Bcl-2 ratio with evident apoptosis in the TdT-mediated dUTP nick-end labeling assay. In nude mice, rat leiomyoma cells ex vivo transduced with Ad-ER-DN supported significantly smaller tumors compared with Ad-LacZ-treated cells 5 weeks after implantation. In mice treated by direct intratumor injection into preexisting lesions, Ad-ER-DN caused immediate overall arrest of tumor growth. The Ad-ER-DN-treated tumors demonstrated severely inhibited cell proliferation (BrdU index) and a marked increase in the number of apoptotic cells (TdT-mediated dUTP nick-end labeling index). Dominant-negative estrogen receptor gene therapy may provide a nonsurgical treatment option for women with symptomatic uterine fibroids who want to preserve their uteri.

  11. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluatedmore » an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.« less

  12. Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery

    PubMed Central

    Jones, Charles H.; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A.

    2015-01-01

    Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates. PMID:25941787

  13. Nonviral siRNA delivery for gene silencing in neurodegenerative diseases.

    PubMed

    Prakash, Satya; Malhotra, Meenakshi; Rengaswamy, Venkatesh

    2010-01-01

    Linking genes with the underlying mechanisms of diseases is one of the biggest challenges of genomics-driven drug discovery research. Designing an inhibitor for any neurodegenerative disease that effectively halts the pathogenicity of the disease is yet to be achieved. The challenge lies in crossing the blood-brain barrier (BBB)/blood-cerebrospinal fluid barrier (BCSFB) to reach the catalytic pockets of the enzyme/protein involved in the molecular mechanism of the disease process. Designing siRNA with exquisite specificity may result in selective suppression of the disease-linked gene. Although siRNA is the most promising method, it loses its potency in downregulating the gene due to its inherent instability, off-target effects, and lack of on-target effective delivery systems. Viral as well as nonviral delivery methods have been effectively tested in vivo for silencing of molecular targets and have resulted in significant efficacy in animal models of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anxiety, depression, encephalitis, glioblastoma, Huntington's disease, neuropathic pain, and spinocerebellar ataxia. To realize the full therapeutic potential of siRNA for neurodegenerative diseases, we need to overcome many hurdles and challenges such as selecting suitable tissue-specific delivery vectors, minimizing the off-target effects, and achieving distribution in sufficient concentrations at the target tissue without any side effects. Cationic nanoparticle-mediated targeted siRNA delivery for therapeutic purposes has gained considerable clinical importance as a result of its promising efficacy.

  14. Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

    PubMed Central

    White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

    2013-01-01

    We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

  15. Adenovector GAD65 gene delivery into the rat trigeminal ganglion produces orofacial analgesia

    PubMed Central

    Vit, Jean-Philippe; Ohara, Peter T; Sundberg, Christopher; Rubi, Blanca; Maechler, Pierre; Liu, Chunyan; Puntel, Mariana; Lowenstein, Pedro; Castro, Maria; Jasmin, Luc

    2009-01-01

    Background Our goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion. Results Injection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist. Conclusion Transfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya. PMID:19656360

  16. Adenovector GAD65 gene delivery into the rat trigeminal ganglion produces orofacial analgesia.

    PubMed

    Vit, Jean-Philippe; Ohara, Peter T; Sundberg, Christopher; Rubi, Blanca; Maechler, Pierre; Liu, Chunyan; Puntel, Mariana; Lowenstein, Pedro; Castro, Maria; Jasmin, Luc

    2009-08-05

    Our goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion. Injection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist. Transfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya.

  17. Gene delivery for cancer therapy.

    PubMed

    Zhang, Teng

    2014-01-01

    Gene therapy has potential in the treatment of human cancers. However, its clinical implication has only achieved little success due to the lack of an efficient gene delivery system. A major hurdle in the current available approaches is in the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. This review outlines the characteristics and utilities of several available gene delivery systems, including their advantages and drawbacks in the context of cancer treatment. A perspective of existing challenges and future directions is also included.

  18. Gene delivery of TIPE2 inhibits breast cancer development and metastasis via CD8+ T and NK cell-mediated antitumor responses.

    PubMed

    Zhang, Zhenhua; Liu, Li; Cao, Shousong; Zhu, Yizhun; Mei, Qibing

    2017-05-01

    Breast cancer is the second leading cause of cancer-related deaths in the female patients which was mainly caused by metastasis. Development of target gene therapy for breast cancer to suppress tumor progress and metastasis will improve the therapeutic options and be of great benefit to the patients. Tumor necrosis factor-alpha-induced protein 8-like 2 is a novel molecule for maintaining immune homeostasis and is involved in cancer development. In the present study, we overexpressed TIPE2 in breast cancer cells to investigate the role of TIPE2 in the development of breast cancer. Our results showed that overexpression of TIPE2 significantly inhibited the proliferation of 4T1 cells in vitro and in vivo. We constructed a non-viral targeted gene therapeutic system by using the minicircle plasmids expressing TIPE2. We found that the growth and metastasis of breast cancer was significantly inhibited by hydrodynamic gene delivery of TIPE2 plasmids in vivo. Mechanistically, TIPE2 increased T and NK cells, and decreased MDSCs. Gene delivery of TIPE2 up-regulated the production of IFN-γ and TNF-α by CD8 + T and NK cells in spleens and tumor microenvironment, and enhanced the cytotoxic activity of CD8 + T and NK cells. Taken together, TIPE2 inhibited breast cancer development and metastasis possibly via promoting CD8 + T and NK cell-mediated antitumor immune responses. Thus, the results indicate that TIPE2 may be a potential therapeutic target for breast cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors.

    PubMed

    Duan, Xiaoping; Guan, Hui; Cao, Ying; Kleinerman, Eugenie S

    2009-01-01

    This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery. MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein. Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal. MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors. Copyright (c) 2008 American Cancer Society.

  20. Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria.

    PubMed

    Strand, Mårten; Carlsson, Marcus; Uvell, Hanna; Islam, Koushikul; Edlund, Karin; Cullman, Inger; Altermark, Björn; Mei, Ya-Fang; Elofsson, Mikael; Willassen, Nils-Peder; Wadell, Göran; Almqvist, Fredrik

    2014-01-28

    Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure.

  1. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

    PubMed Central

    Novobrantseva, Tatiana I; Borodovsky, Anna; Wong, Jamie; Klebanov, Boris; Zafari, Mohammad; Yucius, Kristina; Querbes, William; Ge, Pei; Ruda, Vera M; Milstein, Stuart; Speciner, Lauren; Duncan, Rick; Barros, Scott; Basha, Genc; Cullis, Pieter; Akinc, Akin; Donahoe, Jessica S; Narayanannair Jayaprakash, K; Jayaraman, Muthusamy; Bogorad, Roman L; Love, Kevin; Whitehead, Katie; Levins, Chris; Manoharan, Muthiah; Swirski, Filip K; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G; de Fougerolles, Antonin; Nahrendorf, Matthias; Koteliansky, Victor

    2012-01-01

    Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells. PMID:23344621

  2. Modulation control over ultrasound-mediated gene delivery: evaluating the importance of standing waves.

    PubMed

    Hassan, Mariame A; Buldakov, Mikhail A; Ogawa, Ryohei; Zhao, Qing-Li; Furusawa, Yukihiro; Kudo, Nobuki; Kondo, Takashi; Riesz, Peter

    2010-01-04

    Low modulation frequencies from 0.5 to 100Hz were shown to alter the characteristics of the ultrasound field producing solution agitation (<5Hz; region of "ultrasound streaming" prevalence) or stagnancy (>5Hz; region of standing waves establishment) (Buldakov et al., Ultrason. Sonochem., 2009). In this study, the same conditions were used to depict the changes in exogenous DNA delivery in these regions. The luciferase expression data revealed that lower modulations were more capable of enhancing delivery at the expense of viability. On the contrary, the viability was conserved at higher modulations whereas delivery was found to be null. Cavitational activity and acoustic streaming were the effecters beyond the observed pattern and delivery enhancement was shown to be mediated mainly through sonopermeation. To promote transfection, the addition of calcium ions or an echo contrast agent (Levovist((R))) was proposed. Depending on the mechanism involved in each approach, differential enhancement was observed in both regions and at the interim zone (5Hz). In both cases, enhancement in standing waves field was significant reaching 16.0 and 3.3 folds increase, respectively. Therefore, it is concluded that although the establishment of standing waves is not the only prerequisite for high transfection rates, yet, it is a key element in optimization when other factors such as proximity and cavitation are considered.

  3. Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

    PubMed Central

    Zhang, Ling; Feng, Yan; Li, Zehong; Wu, GuangMou; Yue, Yuhuan; Li, Gensong; Cao, Yu; Zhu, Ping

    2015-01-01

    Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy. PMID:26556098

  4. Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery.

    PubMed

    Jones, Charles H; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A

    2015-07-01

    Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

    PubMed

    Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

    2016-03-20

    RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

  6. Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

    PubMed

    Chen, Zeming; Liu, Fuyao; Chen, Yanke; Liu, Jun; Wang, Xiaoying; Chen, Ann T; Deng, Gang; Zhang, Hongyi; Liu, Jie; Hong, Zhangyong; Zhou, Jiangbing

    2017-12-08

    Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

  7. Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

    PubMed

    Dasí, F; Benet, M; Crespo, J; Crespo, A; Aliño, S F

    2001-05-01

    The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

  8. Ultrasound mediated transdermal drug delivery.

    PubMed

    Azagury, Aharon; Khoury, Luai; Enden, Giora; Kost, Joseph

    2014-06-01

    Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injections. However, the stratum corneum serves as a barrier that limits the penetration of substances to the skin. Application of ultrasound (US) irradiation to the skin increases its permeability (sonophoresis) and enables the delivery of various substances into and through the skin. This review presents the main findings in the field of sonophoresis in transdermal drug delivery as well as transdermal monitoring and the mathematical models associated with this field. Particular attention is paid to the proposed enhancement mechanisms and future trends in the fields of cutaneous vaccination and gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Gene delivery in tissue engineering and regenerative medicine.

    PubMed

    Fang, Y L; Chen, X G; W T, Godbey

    2015-11-01

    As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle. © 2014 Wiley Periodicals, Inc.

  10. Tissue Bioeffects during Ultrasound-mediated Drug Delivery

    NASA Astrophysics Data System (ADS)

    Sutton, Jonathan

    Ultrasound has been developed as both a valuable diagnostic tool and a potent promoter of beneficial tissue bioeffects for the treatment of cardiovascular disease. Vascular effects can be mediated by mechanical oscillations of circulating microbubbles, or ultrasound contrast agents, which may also encapsulate and shield a therapeutic agent in the bloodstream. Oscillating microbubbles can create stresses directly on nearby tissue or induce fluid effects that effect drug penetration into vascular tissue, lyse thrombi, or direct drugs to optimal locations for delivery. These investigations have spurred continued research into alternative therapeutic applications, such as bioactive gas delivery. This dissertation addresses a fundamental hypothesis in biomedical ultrasound: ultrasound-mediated drug delivery is capable of increasing the penetration of drugs across different physiologic barriers within the cardiovascular system, such as the vascular endothelium, blood clots, and smooth muscle cells.

  11. Development of an adenoviral vector with robust expression driven by p53

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bajgelman, Marcio C.; Biotechnology Program, Biomedical Sciences Institute, University of Sao Paulo; Millennium Institute-Gene Therapy Network, Ministry of Science and Technology

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG servedmore » as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.« less

  12. Glutathione-responsive nano-transporter-mediated siRNA delivery: silencing the mRNA expression of Ras.

    PubMed

    Doss, C George Priya; Debottam, S; Debajyoti, C

    2013-06-01

    Gene therapy through antisense technology via intracellular delivery of a gene-silencing element is a promising approach to treat critical diseases like cancers. Ras acts as molecular switch, considered as one of the proto-oncogenes whose modification or mutation may promote tumor formation. The recent trends of nano-carrier-based drug delivery have gained superiority and proved to be 100 times more potent in drug delivery compared to standard therapies. The nano-based drug delivery has provided the basis of achieving successful target-specific drug delivery. Glutathione (GSH) is considered as one of the best and ubiquitous internal stimulus for swift destabilization of nano-transporters inside cells to accomplish proficient intracellular drug release. This concept has given a new hope to oncologists of modifying the existing drugs to be delivered to their desired destination. RNA interference is a primary tool in functional genomics to selectively silence messenger RNA (mRNA) expression, which can be exploited quickly to develop novel drugs against lethal disease target. Silencing of mRNA molecules using siRNA has also come of age to become one of the latest weapons developed in the concept of gene therapy. However, this strategy has severely failed to achieve target specificity especially to a tumor cell. In this context, we have proposed the incorporation of an antisense siRNA packed inside a GSH-responsive nano-transporter to be delivered specifically to a tumor cell against the sense mRNA of the Ras protein. It will limit the Ras-mediated activation of other proteins and transcription factors. Thus, it will knock down several differential gene expressions being regulated by Ras-activated pathways like enzyme-linked receptor kinase pathway. Henceforth, gene silencing technology through nano-drug delivery can be combined as a single weapon to terminate malignancy.

  13. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    PubMed

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Aptamer modification improves the adenoviral transduction of malignant glioma cells.

    PubMed

    Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

    2013-12-01

    Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  15. Gene delivery with viral vectors for cerebrovascular diseases

    PubMed Central

    Gan, Yu; Jing, Zheng; Stetler, R. Anne; Cao, Guodong

    2017-01-01

    Recent achievements in the understanding of molecular events involved in the pathogenesis of central nervous system (CNS) injury have made gene transfer a promising approach for various neurological disorders, including cerebrovascular diseases. However, special obstacles, including the post-mitotic nature of neurons and the blood-brain barrier (BBB), constitute key challenges for gene delivery to the CNS. Despite the various limitations in current gene delivery systems, a spectrum of viral vectors has been successfully used to deliver genes to the CNS. Furthermore, recent advancements in vector engineering have improved the safety and delivery of viral vectors. Numerous viral vector-based clinical trials for neurological disorders have been initiated. This review will summarize the current implementation of viral gene delivery in the context of cerebrovascular diseases including ischemic stroke, hemorrhagic stroke and subarachnoid hemorrhage (SAH). In particular, we will discuss the potentially feasible ways in which viral vectors can be manipulated and exploited for use in neural delivery and therapy. PMID:23276981

  16. Intracellular trafficking of hybrid gene delivery vectors.

    PubMed

    Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

    2015-06-10

    Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing.

    PubMed

    Gómez-Lechón, M José; Tolosa, Laia; Donato, M Teresa

    2017-02-01

    Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an 'artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

  18. Modification of nanostructured calcium carbonate for efficient gene delivery.

    PubMed

    Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

    2014-06-01

    In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.

    PubMed

    Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum

    2017-01-01

    The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cell-penetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines.

  20. How controlled release technology can aid gene delivery.

    PubMed

    Jo, Jun-Ichiro; Tabata, Yasuhiko

    2015-01-01

    Many types of gene delivery systems have been developed to enhance the level of gene expression. Controlled release technology is a feasible gene delivery system which enables genes to extend the expression duration by maintaining and releasing them at the injection site in a controlled manner. This technology can reduce the adverse effects by the bolus dose administration and avoid the repeated administration. Biodegradable biomaterials are useful as materials for the controlled release-based gene delivery technology and various biodegradable biomaterials have been developed. Controlled release-based gene delivery plays a critical role in a conventional gene therapy and genetic engineering. In the gene therapy, the therapeutic gene is released from biodegradable biomaterial matrices around the tissue to be treated. On the other hand, the intracellular controlled release of gene from the sub-micro-sized matrices is required for genetic engineering. Genetic engineering is feasible for cell transplantation as well as research of stem cells biology and medicine. DNA hydrogel containing a sequence of therapeutic gene and the exosome including the individual specific nucleic acids may become candidates for controlled release carriers. Technologies to deliver genes to cell aggregates will play an important role in the promotion of regenerative research and therapy.

  1. Electrostatic Surface Modifications to Improve Gene Delivery

    PubMed Central

    Shmueli, Ron B.; Anderson, Daniel G.

    2010-01-01

    Importance of the field Gene therapy has the potential to treat a wide variety of diseases including genetic diseases and cancer. Areas covered in this review This review introduces biomaterials used for gene delivery and then focuses on the use of electrostatic surface modifications to improve gene delivery materials. These modifications have been used to stabilize therapeutics in vivo, add cell-specific targeting ligands, and promote controlled release. Coatings of nanoparticles and microparticles as well as non-particulate surface coatings are covered in this review. Electrostatic principles are crucial for the development of multilayer delivery structures fabricated by the layer-by-layer method. What the reader will gain The reader will gain knowledge about the composition of biomaterials used for surface modifications and how these coatings and multilayers can be utilized to improve spatial control and efficiency of delivery. Examples are shown for the delivery of nucleic acids, including DNA and siRNA, to in vitro and in vivo systems. Take home message The versatile and powerful approach of electrostatic coatings and multilayers will lead to the development of enhanced gene therapies. PMID:20201712

  2. The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

    NASA Astrophysics Data System (ADS)

    Hasan, Mahadi; Tarashima, Noriko; Fujikawa, Koki; Ohgita, Takashi; Hama, Susumu; Tanaka, Tamotsu; Saito, Hiroyuki; Minakawa, Noriaki; Kogure, Kentaro

    2016-01-01

    An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

  3. Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

    PubMed

    Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

    2018-03-16

    Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

  4. Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

    PubMed

    Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

    2009-11-01

    Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

  5. Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

    PubMed

    Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

    2018-05-14

    Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

  6. Carbon nanotubes part II: a remarkable carrier for drug and gene delivery

    PubMed Central

    Karimi, Mahdi; Solati, Navid; Ghasemi, Amir; Estiar, Mehrdad Asghari; Hashemkhani, Mahshid; Kiani, Parnian; Mohamed, Elmira; Saeidi, Ahad; Taheri, Mahdiar; Avci, Pinar; Aref, Amir R; Amiri, Mohammad; Baniasadi, Fazel; Hamblin, Michael R

    2015-01-01

    Introduction Carbon nanotubes (CNT) have recently been studied as novel and versatile drug and gene delivery vehicles. When CNT are suitably functionalized, they can interact with various cell types and are taken up by endocytosis. Areas covered Anti-cancer drugs cisplatin and doxorubicin have been delivered by CNT, as well as methotrexate, taxol and gemcitabine. The delivery of the antifungal compound amphotericin B and the oral administration of erythropoietin have both been assisted using CNT. Frequently, targeting moieties such as folic acid, epidermal growth factor or various antibodies are attached to the CNT-drug nanovehicle. Different kinds of functionalization (e.g., polycations) have been used to allow CNT to act as gene delivery vectors. Plasmid DNA, small interfering RNA and micro-RNA have all been delivered by CNT vehicles. Significant concerns are raised about the nanotoxicology of the CNT and their potentially damaging effects on the environment. Expert opinion CNT-mediated drug delivery has been studied for over a decade, and both in vitro and in vivo studies have been reported. The future success of CNTs as vectors in vivo and in clinical application will depend on achievement of efficacious therapy with minimal adverse effects and avoidance of possible toxic and environmentally damaging effects. PMID:25613837

  7. Investigation of a thiolated polymer in gene delivery

    NASA Astrophysics Data System (ADS)

    Bacalocostantis, Irene

    Thiol-containing bioreducible polymers show significant potential as delivery vectors in gene therapy, a rapidly growing field which seeks to treat genetic-based disorders by delivering functional synthetic genes to diseased cells. Studies have shown that thiolated polymers exhibit improved biodegradability and prolonged in vivo circulation times over non-thiolated polymers. However, the extent to which thiol concentrations impact the carrier's delivery potential has not been well explored. The aim of this dissertation is to investigate how relative concentrations of free thiols and disulfide crosslinks impact a polymeric carriers delivery performance with respect to DNA packaging, complex stability, cargo protection, gene release, internalization efficiency and cytotoxicity. To accomplish this goal, several fluorescent polymers containing varying concentrations of thiol groups were synthesized by conjugating thiol-pendant chains onto the primary amines of cationic poly(allylamine). In vitro delivery assays and characterization techniques were employed to assess the effect of thiols in gene delivery.

  8. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  9. Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis.

    PubMed

    Park, Shin-Young; Kim, Kyoung-Hwa; Gwak, Eun-Hye; Rhee, Sang-Hoon; Lee, Jeong-Cheol; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2015-01-01

    Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects. © 2014 Wiley Periodicals, Inc.

  10. Bioreducible Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery.

    PubMed

    Cai, Xiaojun; Jin, Rongrong; Wang, Jiali; Yue, Dong; Jiang, Qian; Wu, Yao; Gu, Zhongwei

    2016-03-09

    Polymeric vectors have shown great promise in the development of safe and efficient gene delivery systems; however, only a few have been developed in clinical settings due to poor transport across multiple physiological barriers. To address this issue and promote clinical translocation of polymeric vectors, a new type of polymeric vector, bioreducible fluorinated peptide dendrimers (BFPDs), was designed and synthesized by reversible cross-linking of fluorinated low generation peptide dendrimers. Through masterly integration all of the features of reversible cross-linking, fluorination, and polyhedral oligomeric silsesquioxane (POSS) core-based peptide dendrimers, this novel vector exhibited lots of unique features, including (i) inactive surface to resist protein interactions; (ii) virus-mimicking surface topography to augment cellular uptake; (iii) fluorination-mediated efficient cellular uptake, endosome escape, cytoplasm trafficking, and nuclear entry, and (iv) disulfide-cleavage-mediated polyplex disassembly and DNA release that allows efficient DNA transcription. Noteworthy, all of these features are functionally important and can synergistically facilitate DNA transport from solution to the nucleus. As a consequences, BFPDs showed excellent gene transfection efficiency in several cell lines (∼95% in HEK293 cells) and superior biocompatibility compared with polyethylenimine (PEI). Meanwhile BFPDs provided excellent serum resistance in gene delivery. More importantly, BFPDs offer considerable in vivo gene transfection efficiency (in muscular tissues and in HepG2 tumor xenografts), which was approximately 77-fold higher than that of PEI in luciferase activity. These results suggest bioreducible fluorinated peptide dendrimers are a new class of highly efficient and safe gene delivery vectors and should be used in clinical settings.

  11. Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

    PubMed

    Zheng, Hao; Tang, Cui; Yin, Chunhua

    2015-06-01

    Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

  12. Functionalized nanoparticles for AMF-induced gene and drug delivery

    NASA Astrophysics Data System (ADS)

    Biswas, Souvik

    The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

  13. Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector.

    PubMed

    Campbell, Samuel; Suwan, Keittisak; Waramit, Sajee; Aboagye, Eric Ofori; Hajitou, Amin

    2018-04-21

    The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the α ν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

  14. Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery.

    PubMed

    Beekers, Ines; van Rooij, Tom; Verweij, Martin D; Versluis, Michel; de Jong, Nico; Trietsch, Sebastiaan J; Kooiman, Klazina

    2018-04-01

    Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3-D cell culture, perfusion flow, and membrane-free soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh-speed camera (17 million frames/s) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated drug delivery. When insonified at frequencies between 1 and 2 MHz, microbubbles in the OrganoPlate experienced larger oscillation amplitudes resulting from higher local pressures. Microbubbles responded similarly in both systems when insonified at frequencies between 2 and 4 MHz. Numerical simulations performed with a 3-D finite-element model of ultrasound propagation into the OrganoPlate and the OptiCell showed the same frequency-dependent behavior. The predictable and homogeneous pressure field in the OrganoPlate demonstrates its potential to develop an in vitro 3-D cell culture model, well suited to study ultrasound-mediated drug delivery.

  15. Prolonging pulse duration in ultrasound-mediated gene delivery lowers acoustic pressure threshold for efficient gene transfer to cells and small animals.

    PubMed

    Tran, Dominic M; Harrang, James; Song, Shuxian; Chen, Jeremy; Smith, Bryn M; Miao, Carol H

    2018-06-10

    While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 μs-36 ms and PNPs spanning 0.5-2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 μs-22 ms and PNPs spanning 0.5-2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  17. [Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

    PubMed

    Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

    2008-06-01

    This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

  18. Ultrasound-guided drug delivery in cancer

    PubMed Central

    2017-01-01

    Recent advancements in ultrasound and microbubble (USMB) mediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs and genes to target tissues while reducing systemic dose and toxicity. The mechanism behind enhanced delivery of therapeutics is sonoporation, the formation of openings in the vasculature, induced by ultrasound-triggered oscillations and destruction of microbubbles. In this review, progress and challenges of USMB mediated drug delivery are summarized, with special focus on cancer therapy. PMID:28607323

  19. Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

    PubMed Central

    Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K.; Champaneri, Shivam A.; Taylor, Sarah; Davidson, Brian P.; Zhao, Yan; Klibanov, Alexander L.; Kuliszewski, Michael A.; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R.

    2013-01-01

    OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with

  20. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. © 2012 The Author. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

  1. Dual peptide-mediated targeted delivery of bioactive siRNAs to oral cancer cells in vivo.

    PubMed

    Alexander-Bryant, Angela A; Zhang, Haiwen; Attaway, Christopher C; Pugh, William; Eggart, Laurence; Sansevere, Robert M; Andino, Lourdes M; Dinh, Lu; Cantini, Liliana P; Jakymiw, Andrew

    2017-09-01

    Despite significant advances in cancer treatment, the prognosis for oral cancer remains poor in comparison to other cancer types, including breast, skin, and prostate. As a result, more effective therapeutic modalities are needed for the treatment of oral cancer. Consequently, in the present study, we examined the feasibility of using a dual peptide carrier approach, combining an epidermal growth factor receptor (EGFR)-targeting peptide with an endosome-disruptive peptide, to mediate targeted delivery of small interfering RNAs (siRNAs) into EGFR-overexpressing oral cancer cells and induce silencing of the targeted oncogene, cancerous inhibitor of protein phosphatase 2A (CIP2A). Fluorescence microscopy, real-time PCR, Western blot analysis, and in vivo bioimaging of mice containing orthotopic xenograft tumors were used to examine the ability of the dual peptide carrier to mediate specific delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells/tissues. Co-complexation of the EGFR-targeting peptide, GE11R9, with the endosome-disruptive 599 peptide facilitated the specific uptake of siRNAs into oral cancer cells overexpressing EGFR in vitro with optimal gene silencing observed at a 60:30:1 (GE11R9:599:siRNA) molar ratio. Furthermore, when administered systemically to mice bearing xenograft oral tumors, this dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone and significantly enhanced CIP2A silencing. Herein we provide the first report demonstrating the clinical potential of a dual peptide strategy for siRNA-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing oral cancer cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Fe₃O₄ Nanoparticles in Targeted Drug/Gene Delivery Systems.

    PubMed

    Shen, Lazhen; Li, Bei; Qiao, Yongsheng

    2018-02-23

    Fe₃O₄ nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe₃O₄ NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe₃O₄ NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe₃O₄ NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe₃O₄ NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe₃O₄ NPs targeting drug/gene delivery systems.

  3. Therapeutic gene editing: delivery and regulatory perspectives.

    PubMed

    Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

    2017-06-01

    Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues.

  4. Therapeutic gene editing: delivery and regulatory perspectives

    PubMed Central

    Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

    2017-01-01

    Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues. PMID:28392568

  5. The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

    PubMed

    Pastor, Marie; Johnen, Sandra; Harmening, Nina; Quiviger, Mickäel; Pailloux, Julie; Kropp, Martina; Walter, Peter; Ivics, Zoltán; Izsvák, Zsuzsanna; Thumann, Gabriele; Scherman, Daniel; Marie, Corinne

    2018-06-01

    The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Exploring the role of peptides in polymer-based gene delivery.

    PubMed

    Sun, Yanping; Yang, Zhen; Wang, Chunxi; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

    2017-09-15

    Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. Ultrasound-mediated ocular delivery of therapeutic agents: a review.

    PubMed

    Lafond, Maxime; Aptel, Florent; Mestas, Jean-Louis; Lafon, Cyril

    2017-04-01

    Due to numerous anatomical and physiological barriers, ocular drug delivery remains a major limitation in the treatment of diseases such as glaucoma, macular degeneration or inflammatory diseases. To date, only invasive approaches provide clinically effective results. Ultrasound can be defined as the propagation of a high-frequency sound wave exposing the propagation media to mechanical and thermal effects. Ultrasound has been proposed as a non-invasive physical agent for increasing therapeutic agent delivery in various fields of medicine. Areas covered: An update on recent advances in transscleral and transcorneal ultrasound-mediated drug delivery is presented. Efficient drug delivery is achieved in vitro, ex vivo and in vivo for various types of materials. Numerous studies indicate that efficacy is related to cavitation. Although slight reversible effects can be observed on the corneal epithelium, efficient drug delivery can be performed without causing damage to the cornea. Expert opinion: Recent developments prove the potential of ultrasound-mediated ocular drug delivery. Cavitation appears to be a preponderant mechanism, opening a way to treatment monitoring by cavitation measurement. Even if no clinical studies have yet been performed, the promising results summarized here are promoting developments toward clinical applications, particularly in assessing the safety of the technique.

  8. Adenoviral bcl-2 transfer improves survival and early graft function after ischemia and reperfusion in rat liver transplantation.

    PubMed

    Rentsch, Markus; Kienle, Klaus; Mueller, Thomas; Vogel, Mandy; Jauch, Karl Walter; Püllmann, Kerstin; Obed, Aiman; Schlitt, Hans J; Beham, Alexander

    2005-11-27

    Primary graft dysfunction due to ischemia and reperfusion injury represents a major problem in liver transplantation. The related cell stress may induce apoptosis, which can be suppressed by bcl-2. The purpose of the study was to investigate the effect of adenoviral bcl-2 gene transfer on early graft function and survival in rat liver transplantation. An adenoviral construct that transfers bcl-2 under the control of a tetracycline inducible promoter was generated (advTetOn bcl-2) and used with a second adenovirus that transfers the repressor protein (advCMV Rep). Forty-eight hours before explantation, donor rats were treated with advTetOn bcl-2/ advCMV Rep (n=7) and doxycyclin, with the control adenoviral construct advCMV GFP (n=8) or with doxycyclin alone (n=8). Liver transplantation was performed following 16 hours of cold storage (UW). Bcl-2 expression and intrahepatic apoptosis was assessed. Bile flow was monitored 90 min posttransplantation. The endpoint for survival was 7 days. Bcl-2 was expressed in hepatocytes and sinusoidal lining cells. This was associated with a significant reduction of apoptotic sinusoidal lining cells and hepatocytes after 24 hours and 7 days. Bile production was significantly higher following bcl-2 pretreatment. Furthermore, bcl-2 transfer resulted in significantly improved survival (100% vs. 50% both control groups). Adenoviral bcl-2 transfer results in protein expression in hepatocytes and sinusoidal lining cells resulting in early graft function and survival enhancement after prolonged ischemia and reperfusion injury. The inhibition of apoptosis in the context of liver transplantation might be a reasonable approach in the treatment of graft dysfunction.

  9. Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

    PubMed

    Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

    2010-02-01

    We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

  10. Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

    PubMed

    Kuboki, T; Nakanishi, T; Kanyama, M; Sonoyama, W; Fujisawa, T; Kobayashi, K; Ikeda, T; Kubo, T; Yamashita, A; Takigawa, M

    1999-09-01

    Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint

  11. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material.

    PubMed

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  12. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material

    NASA Astrophysics Data System (ADS)

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  13. Design strategies and applications of circulating cell-mediated drug delivery systems.

    PubMed

    Su, Yixue; Xie, Zhiwei; Kim, Gloria B; Dong, Cheng; Yang, Jian

    2015-01-01

    Drug delivery systems, particularly nanomaterial-based drug delivery systems, possess a tremendous amount of potential to improve diagnostic and therapeutic effects of drugs. Controlled drug delivery targeted to a specific disease is designed to significantly improve the pharmaceutical effects of drugs and reduce their side effects. Unfortunately, only a few targeted drug delivery systems can achieve high targeting efficiency after intravenous injection, even with the development of numerous surface markers and targeting modalities. Thus, alternative drug and nanomedicine targeting approaches are desired. Circulating cells, such as erythrocytes, leukocytes, and stem cells, present innate disease sensing and homing properties. Hence, using living cells as drug delivery carriers has gained increasing interest in recent years. This review highlights the recent advances in the design of cell-mediated drug delivery systems and targeting mechanisms. The approaches of drug encapsulation/conjugation to cell-carriers, cell-mediated targeting mechanisms, and the methods of controlled drug release are elaborated here. Cell-based "live" targeting and delivery could be used to facilitate a more specific, robust, and smart payload distribution for the next-generation drug delivery systems.

  14. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

    PubMed Central

    Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

    2009-01-01

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

  15. [HSV-1 based vector mediated IL-1Rα gene for knee osteoarthritis in rabbits].

    PubMed

    Wu, Yi; Li, Jianming; Kong, Ying; Chen, Ding; Liu, Bo; Wang, Wanchun

    2013-06-01

    To investigate the effect and mechanism of herpes simplex virus type 1 (HSV-1) based vector mediated interlukin-1 receptor antagonist (IL-1Rα) gene for knee osteoarthritis in rabbits. HSV-1 vectors containing IL-1Rα genes were constructed and injected into the joint space of the osteoarthritis knee in rabbits for 4 weeks. The rabbits were sacrificed, and the knees were lavaged, dissected and the effect of transgene expression was analyzed. Levels of IL-1Rα and IL-1 expression in the recovered lavage fluids were measured with a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were observed with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The blank control group was injected pHSV-LacZ vector into rabbit knees. Intra-articular delivery of pHSV-IL-1Rα-LacZ resulted in a significant inhibition of IL-1 level and cartilage degradation compared with those in the blank control group (P<0.05). pHSV-LacZ is an ideal vector to mediate intra-articular gene delivery in the rabbit model of osteoarthritis. Continuous intra-articular expression of IL-1Rα can treat knee osteoarthritis by inhibiting IL-1.

  16. Non-viral gene delivery strategies for gene therapy: a "ménage à trois" among nucleic acids, materials, and the biological environment. Stimuli-responsive gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Pezzoli, Daniele; Candiani, Gabriele

    2013-03-01

    Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription-translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

  17. In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK.

    PubMed

    Marini, F C; Cannon, J P; Belmont, J W; Shillitoe, E J; Lapeyre, J N

    1995-09-01

    We evaluated the ability of a replication-deficient, recombinant adenoviral vector to transfer the bifunctional gene GAL-TEK, which expresses a marking/therapeutic gene product, to naturally occurring cat fibrosarcomas in situ. GAL-TEK contains an in-frame fusion of the bacterial LacZ gene for histochemical marking of tumors with beta-galactosidase (beta-Gal) and the HSV tk gene for enzyme-prodrug activation of the prodrug ganciclovir (GCV) to induce selective tumor cell killing. GAL-TEK bifunctional marking and cell killing activities were tested in vitro after adenoviral vector infection of HT1080 human fibrosarcoma cells. The tk activity of GAL-TEK is shown to be almost as potent as HSV tk to catalyze conversion of GCV to GCV nucleotides and promote selective cell killing. Using 8 cats with recurring 2.5-cm2 fibrosarcomas that either arose spontaneously or were induced by vaccine, we determined experimentally the administration routes and times required for optimum GAL-TEK gene transfer by beta-Gal histological staining and reverse transcriptase polymerase chain reaction to the multiple compartments of the growing fibrosarcomas consonant with minimizing collateral infection of neighboring tissues and other unwanted side effects.

  18. Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery.

    PubMed

    Dobson, J

    2006-02-01

    The recent emphasis on the development of non-viral transfection agents for gene delivery has led to new physics and chemistry-based techniques, which take advantage of charge interactions and energetic processes. One of these techniques which shows much promise for both in vitro and in vivo transfection involves the use of biocompatible magnetic nanoparticles for gene delivery. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles, which are then focused to the target site/cells via high-field/high-gradient magnets. The technique promotes rapid transfection and, as more recent work indicates, excellent overall transfection levels as well. The advantages and difficulties associated with magnetic nanoparticle-based transfection will be discussed as will the underlying physical principles, recent studies and potential future applications.

  19. Over-expression of angiotensin II type 2 receptor gene induces cell death in lung adenocarcinoma cells.

    PubMed

    Pickel, Lara; Matsuzuka, Takaya; Doi, Chiyo; Ayuzawa, Rie; Maurya, Dharmendra Kumar; Xie, Sheng-Xue; Berkland, Cory; Tamura, Masaaki

    2010-02-01

    The endogenous angiotensin II (Ang II) type 2 receptor (AT 2) has been shown to mediate apoptosis in cardiovascular tissues. Thus, the aim of this study was to explore the anti-cancer effect of AT 2 over-expression on lung adenocarcinoma cells in vitro using adenoviral (Ad), FuGENE, and nanoparticle vectors. All three gene transfection methods efficiently transfected AT 2 cDNA into lung cancer cells but caused minimal gene transfection in normal lung epithelial cells. Ad-AT 2 significantly attenuated multiple human lung cancer cell growth (A549 and H358) as compared to the control viral vector, Ad-LacZ, when cell viability was examined by direct cell count. Examination of annexin V by flow cytometry revealed the activation of the apoptotic pathway via AT 2 over-expression. Western Blot analysis confirmed the activation of caspase-3. Similarly, poly (lactide-co-glycolic acid) (PLGA) biodegradable nanoparticles encapsulated AT 2 plasmid DNA were shown to be effectively taken up into the lung cancer cell. Nanoparticle-based AT 2 gene transfection markedly increased AT 2 expression and resultant cell death in A549 cells. These results indicate that AT 2 over-expression effectively attenuates growth of lung adenocarcinoma cells through intrinsic apoptosis. Our results also suggest that PLGA nanoparticles can be used as an efficient gene delivery vector for lung adenocarcinoma targeted therapy.

  20. EMX2 gene expression predicts liver metastasis and survival in colorectal cancer.

    PubMed

    Aykut, Berk; Ochs, Markus; Radhakrishnan, Praveen; Brill, Adrian; Höcker, Hermine; Schwarz, Sandra; Weissinger, Daniel; Kehm, Roland; Kulu, Yakup; Ulrich, Alexis; Schneider, Martin

    2017-08-22

    The Empty Spiracles Homeobox (EMX-) 2 gene has been associated with regulation of growth and differentiation in neuronal development. While recent studies provide evidence that EMX2 regulates tumorigenesis of various solid tumors, its role in colorectal cancer remains unknown. We aimed to assess the prognostic significance of EMX2 expression in stage III colorectal adenocarcinoma. Expression levels of EMX2 in human colorectal cancer and adjacent mucosa were assessed by qRT-PCR technology, and results were correlated with clinical and survival data. siRNA-mediated knockdown and adenoviral delivery-mediated overexpression of EMX2 were performed in order to investigate its effects on the migration of colorectal cancer cells in vitro. Compared to corresponding healthy mucosa, colorectal tumor samples had decreased EMX2 expression levels. Furthermore, EMX2 down-regulation in colorectal cancer tissue was associated with distant metastasis (M1) and impaired overall patient survival. In vitro knockdown of EMX2 resulted in increased tumor cell migration. Conversely, overexpression of EMX2 led to an inhibition of tumor cell migration. EMX2 is frequently down-regulated in human colorectal cancer, and down-regulation of EMX2 is a prognostic marker for disease-free and overall survival. EMX2 might thus represent a promising therapeutic target in colorectal cancer.

  1. Neonatal adenoviral infection: a seventeen year experience and review of the literature.

    PubMed

    Ronchi, Andrea; Doern, Christopher; Brock, Evangeline; Pugni, Lorenza; Sánchez, Pablo J

    2014-03-01

    To describe the clinical manifestations and short-term outcomes of adenoviral infections in neonates and review all published cases to better determine impact and treatment outcomes. Retrospective cohort study of all neonates hospitalized at Children's Medical Center (CMC) and Parkland Memorial Hospital (PMH), Dallas, TX with laboratory-confirmed adenoviral infection from January 1,1995-December 31, 2012. Neonates were identified by review of the CMC Virology Laboratory's prospective database of all positive adenovirus tests performed in the inpatient and ambulatory settings, and at PMH, of a prospective neonatal database that included all neonatal intensive care unit admissions. Patients also were identified by discharge International Classification of Disease, 9th edition codes for adenoviral infection. The medical records were reviewed, and a review of the English literature was performed. During 17 years, 26 neonates had adenoviral infection (25, CMC; 1, PMH). The principle reasons for hospitalization were respiratory signs (88%) and temperature instability (65%). Five (19%) had disseminated disease and 4 (80%) of these infants died. Ribavirin or cidofovir treatment, as well as immune globulin intravenous, did not improve outcomes except in 1 neonate. Literature review (n = 72) combined with our data found that disseminated infection was associated with death (68% vs 21% with localized infection, P < .001). In addition, neonates <14 days of age were more likely to have disseminated disease (44% vs 12%, P = .004) and death (48% vs 8%; P < .001). Adenoviral infection in hospitalized neonates was associated with severe morbidity and mortality, especially when infection was disseminated and involved the respiratory tract. Development of new therapeutic strategies is needed. Copyright © 2014 Mosby, Inc. All rights reserved.

  2. Polymeric Carriers for Gene Delivery: Chitosan and Poly(amidoamine) Dendrimers

    PubMed Central

    Xu, Qingxing; Wang, Chi-Hwa; Pack, Daniel Wayne

    2012-01-01

    Gene therapy is a potential medical solution that promises new treatments and may hold the cure for many different types of diseases and disorders of the human race. However, gene therapy is still a growing medical field and the technology is still in its infancy. The main challenge for gene therapy is to find safe and effective vectors that are able to deliver genes to the specific cells and get them to express inside the cells. Due to safety concerns, synthetic delivery systems, rather than viral vectors, are preferred for gene delivery and significant efforts have been focused on the development of this field. However, we are faced with problems like low gene transfer efficiency, cytotoxicity and lack of cell-targeting capability for these synthetic delivery systems. Over the years, we have seen a variety of new and effective polymers which have been designed and synthesized specifically for gene delivery. Moreover, various strategies that aimed at enhancing their physicochemical properties, improving transfection efficiency, reducing cytotoxicity as well as incorporating functional groups that offer better targetability and higher cellular uptake are established. Here, we look at two potential polymeric carriers, chitosan and poly(amidoamine) dendrimers, which have been widely reported for gene delivery. For chitosan, the interest arises from their availability, excellent non-cytotoxicity profile, biodegradability and ease of modification. For poly(amidoamine) dendrimers, the interest arises from their ease of synthesis with controlled structure and size, minimal cytotoxicity, biodegradability and high transfection efficiencies. The latest developments on these polymers for gene delivery will be the main focus of this article. PMID:20618156

  3. Peptides for Specific Intracellular Delivery and Targeting of Nanoparticles: Implications for Developing Nanoparticle-Mediated Drug Delivery

    DTIC Science & Technology

    2010-01-01

    for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist. Update 8(6), 381–402 (2005). 89 Smith BR, Cheng Z...component can be realized. Select examples from the literature have already demonstrated the feasibility of generating hybrid NP–peptide constructs in...peptide-mediated delivery of NP-based imaging agents (fluorescence and magnetic resonance), drug-delivery vehicles, therapeutic proteins and nucleic

  4. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    NASA Astrophysics Data System (ADS)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  5. Gene Therapy for Posttraumatic Osteoarthritis

    DTIC Science & Technology

    2017-10-01

    are currently no useful treatments. To provide a clear assessment of the clinical potential of this technology we are testing the following hypothesis...efficacy of scAAV-mediated gene delivery of IL-1Ra for treatment of OA. We will test the hypothesis that scAAV-mediated gene delivery of IL-1Ra to...1Ra) Post -traumatic OA (PTOA) Self-complimentary AAV (scAAV) Cartilage Synovium Gene Transfer Large animal model 6 2. ACCOMPLISHMENTS

  6. Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

    PubMed

    Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K; Champaneri, Shivam A; Taylor, Sarah; Davidson, Brian P; Zhao, Yan; Klibanov, Alexander L; Kuliszewski, Michael A; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R

    2012-12-01

    Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa

  7. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Gene Delivery to the Airway

    PubMed Central

    Keiser, Nicholas W.; Engelhardt, John F.

    2013-01-01

    This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. PMID:23853081

  9. Current and future technological advances in transdermal gene delivery.

    PubMed

    Chen, Xianfeng

    2017-12-19

    Transdermal gene delivery holds significant advantages as it is able to minimize the problems of systemic administration such as enzymatic degradation, systemic toxicity, and poor delivery to target tissues. This technology has the potential to transform the treatment and prevention of a range of diseases. However, the skin poses a great barrier for gene delivery because of the "bricks-and-mortar" structure of the stratum corneum and the tight junctions between keratinocytes in the epidermis. This review systematically summarizes the typical physical and chemical approaches to overcome these barriers and facilitate gene delivery via skin for applications in vaccination, wound healing, skin cancers and skin diseases. Next, the advantages and disadvantages of different approaches are discussed and the insights for future development are provided. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Reducible, Dibromomaleimide-linked Polymers for Gene Delivery

    PubMed Central

    Tan, James-Kevin Y.; Choi, Jennifer L.; Wei, Hua; Schellinger, Joan G.; Pun, Suzie H.

    2014-01-01

    Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azidealkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino) ethyl methacrylate (DMAEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azidefluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles. PMID:26214195

  11. Adenoviral Gene Transfer of Hepatic Stimulator Substance Confers Resistance Against Hepatic Ischemia–Reperfusion Injury by Improving Mitochondrial Function

    PubMed Central

    Jiang, Shu-Jun; Li, Wen

    2013-01-01

    Abstract Hepatic stimulator substance (HSS) has been suggested to protect liver cells from various toxins. However, the precise role of HSS in hepatic ischemia–reperfusion (I/R) injury remains unknown. This study aims to elucidate whether overexpression of HSS could attenuate hepatic ischemia–reperfusion injury and its possible mechanisms. Both in vivo hepatic I/R injury in mice and in vitro hypoxia–reoxygenation (H/R) in a cell model were used to evaluate the effect of HSS protection after adenoviral gene transfer. Moreover, a possible mitochondrial mechanism of HSS protection was investigated. Efficient transfer of the HSS gene into liver inhibited hepatic I/R injury in mice, as evidenced by improvement in liver function tests, the preservation of hepatic morphology, and a reduction in hepatocyte apoptosis. HSS overexpression also inhibited H/R-induced cell death, as detected by cell viability and cell apoptosis assays. The underlying mechanism of this hepatic protection might involve the attenuation of mitochondrial dysfunction and mitochondrial-dependent cell apoptosis, as shown by the good preservation of mitochondrial ultrastructure, mitochondrial membrane potential, and the inhibition of cytochrome c leakage and caspase activity. Moreover, the suppression of H/R-induced mitochondrial ROS production and the maintenance of mitochondrial respiratory chain complex activities may participate in this mechanism. This new function of HSS expands the possibility of its application for the prevention of I/R injury, such as hepatic resection and liver transplantation in clinical practice. PMID:23461564

  12. Chitosan for gene delivery and orthopedic tissue engineering applications.

    PubMed

    Raftery, Rosanne; O'Brien, Fergal J; Cryan, Sally-Ann

    2013-05-15

    Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering.

  13. Selective intraarterial gene delivery into a canine meningioma.

    PubMed

    Chauvet, A E; Kesava, P P; Goh, C S; Badie, B

    1998-05-01

    The goal of this study was to evaluate gene delivery to a benign brain tumor. A recombinant adenovirus vector bearing the Escherichia coli beta-galactosidase reporter gene was selectively injected into the vascular supply of a spontaneously occurring canine olfactory groove meningioma. The tumor and a small amount of peritumoral brain tissue were removed 5 days after viral injection and stained with X-Gal to assess gene delivery. The authors noted significant beta-galactosidase gene expression by the tumor, but not by surrounding brain tissue. No obvious viral-related cytotoxicity was noted. The authors found that meningiomas can be successfully transduced by adenovirus vectors by using endovascular techniques.

  14. A chemistry/physics pathway with nanofibrous scaffolds for gene delivery.

    PubMed

    Wan, Fen; Tang, Zhaohui; He, Weidong; Chu, Benjamin

    2010-10-21

    This perspective is to introduce a new pathway for non-viral gene delivery by taking advantage of nanofibrous scaffolds as gene storage devices, gene carriers and homing devices. During gene delivery to the target, the DNA has to be protected in order to pass through a set of barriers before reaching the nucleus. The DNA can form a complex with polycations, and numerous publications exist on how to stabilize the DNA fragments by natural and synthetic materials. Electrospun nanofibrous scaffolds can be used to store the DNA, especially in the form of a more stabilized polyplex, and then to deliver the DNA (polyplex) to cells that are attached to the scaffold. While each essential step has been tested experimentally, the overall yet untested process, especially for in vivo experiments, may lead to a promising specific approach for gene/drug storage and delivery. The pathway described herein is based mainly on our understanding of the physics and chemistry of gene storage and delivery processes, in contrast to using pure biological concepts. Novel biodegradable, biocompatible nanofibrous materials with imbedded DNA (e.g., in the polyplex form) can then be designed to fabricate an intelligent scaffold for gene delivery. To achieve the above goal, the first step is to stabilize the DNA so that it can be incorporated into nanofibrous scaffolds. In this respect, we shall discuss the different methods of DNA/gene condensation and complex formation, and then explain the strategy used to incorporate DNA into electrospun nanofibers. Solvent-induced DNA condensation and then encapsulation were achieved. However, the released naked DNA was not sufficiently protected for gene transfection in cells. The objective of the current perspective is to suggest that, instead of the solvent-induced DNA condensation, one can combine the recently developed polyplex formation by using branched polyethyleneimine (bPEI). More importantly, free bPEI can be incorporated into the nanofibers

  15. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector.

    PubMed

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-10-31

    Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation.

  16. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector

    PubMed Central

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-01-01

    Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation. PMID:16259632

  17. Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

    PubMed

    Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

    2006-08-01

    Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

  18. Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay.

    PubMed

    Senoo, M; Matsubara, Y; Fujii, K; Nagasaki, Y; Hiratsuka, M; Kure, S; Uehara, S; Okamura, K; Yajima, A; Narisawa, K

    2000-04-01

    Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period. We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan-polymerase chain reaction (TaqMan-PCR) assay. We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus. Histochemical analysis showed limited transgene expression in fetal tissues. We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein. The highest beta-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland. The transgene expression was also present in kidney, intestine, and placenta to a lesser degree. No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs. Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver. The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues. The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer. Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.

  19. AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

    PubMed

    Kiyota, T; Ingraham, K L; Swan, R J; Jacobsen, M T; Andrews, S J; Ikezu, T

    2012-07-01

    Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

  20. Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

    PubMed Central

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny

    2012-01-01

    Abstract Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

  1. Physical non-viral gene delivery methods for tissue engineering.

    PubMed

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  2. Physical non-viral gene delivery methods for tissue engineering

    PubMed Central

    Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

    2016-01-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

  3. Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

    PubMed

    Molas, M; Gómez-Valadés, A G; Vidal-Alabró, A; Miguel-Turu, M; Bermudez, J; Bartrons, R; Perales, J C

    2003-10-01

    Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

  4. The copolymer of Poly(2-dimethylaminoethyl methacrylate) and methacrylated chondroitin sulfate with low cytotoxicity for gene delivery.

    PubMed

    Lo, Yu-Lun; Wang, Yao-Sheng; Wang, Li-Fang

    2013-11-01

    Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) is one of the most potent synthetic nonviral gene-delivery vectors because of its high transfection efficiency. However, the cytotoxicity of PDMAEMA is a major concern for its clinical applications. An anionic crosslinker is synthesized based on a natural polysaccharide, chondroitin sulfate (CS), by introducing methacrylate groups (CSMA). By systematically adjusting the substitution degree of methacrylation on CS and the weight percent of CSMA and PDMAEMA, sol-type copolymers are obtained as a gene-delivery vector. The combination of CS and PDMAEMA is expected not only to reduce the cytotoxicity of PDMAEMA, but also to facilitate better transfection efficiency than PDMAEMA because of the recognition of CS by CD44 receptors on cell surfaces. Two CSMA-modified PDMAEMA copolymers with different CSMA constituents are selected and their polyplexes prepared with plasmid DNA. The cytotoxicity and gene transfection efficiency of the polyplexes are tested and compared with those of PDMAEMA/pDNA. The copolymers of CSMA and PDMAEMA show significantly improved cell viability as compared with PDMAEMA. Their formed polyplexes with pDNA also show lower cytotoxicity than does PDMAEMA/pDNA. The transfection efficiency remarkably increases as the CSMA-modified PDMAEMA/pDNA polyplex is prepared at a weight ratio of 2.4. The internalization mechanism of CSMA-modified PDMAEMA/pDNA in HEK 293T cells is mainly based on caveolae-mediated endocytosis. However, both caveolae-mediated and CD44-mediated endocytosis mechanisms are involved in U87 cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

    PubMed

    Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

    2018-06-05

    The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Gene delivery for periodontal tissue engineering: current knowledge - future possibilities.

    PubMed

    Chen, Fa-Ming; Ma, Zhi-Wei; Wang, Qin-Tao; Wu, Zhi-Fen

    2009-08-01

    The cellular and molecular events of periodontal healing are coordinated and regulated by an elaborate system of signaling molecules, pointing to a primary strategy for functional periodontal compartment regeneration to replicate components of the natural cellular microenvironment by providing an artificial extracellular matrix (ECM) and by delivering growth factors. However, even with optimal carriers, the localized delivery of growth factors often requires a large amount of protein to stimulate significant effects in vivo, which increases the risk and unwanted side effects. A simple and relatively new approach to bypassing this dilemma involves converting cells into protein producing factories. This is done by a so-called gene delivery method, where therapeutic agents to be delivered are DNA plasmids that include the gene encoding desired growth factors instead of recombinant proteins. As localized depots of genes, novel gene delivery systems have the potential to release their cargo in a sustained and controlled manner and finally provide time- and space- dependent levels of encoded proteins during all stages of tissue regrowth, offering great versatility in their application and prompting new tissue engineering strategy in periodontal regenerative medicine. However, gene therapy in Periodontology is clearly in its infancy. Significant efforts still need to be made in developing safe and effective delivery platforms and clarifying how gene delivery, in combination with tissue engineering, may mimic the critical aspects of natural biological processes occurring in periodontal development and repair. The aim of this review is to trace an outline of the state-of-the-art in the application of gene delivery and tissue engineering strategies for periodontal healing and regeneration.

  7. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    NASA Astrophysics Data System (ADS)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  8. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  9. Treatment of Adenoviral Acute Respiratory Distress Syndrome Using Cidofovir With Extracorporeal Membrane Oxygenation.

    PubMed

    Lee, Minhyeok; Kim, Seulgi; Kwon, Oh Jung; Kim, Ji Hye; Jeong, Inbeom; Son, Ji Woong; Na, Moon Jun; Yoon, Yoo Sang; Park, Hyun Woong; Kwon, Sun Jung

    2017-03-01

    Adenovirus infections are associated with respiratory (especially upper respiratory) infection and gastrointestinal disease and occur primarily in infants and children. Although rare in adults, severe lower respiratory adenovirus infections including pneumonia are reported in specific populations, such as military recruits and immunocompromised patients. Antiviral treatment is challenging due to limited clinical experience and lack of well-controlled randomized trials. Several previously reported cases of adenoviral pneumonia showed promising efficacy of cidofovir. However, few reports discussed the efficacy of cidofovir in acute respiratory distress syndrome (ARDS). We experienced 3 cases of adenoviral pneumonia associated with ARDS and treated with cidofovir and respiratory support, including extracorporeal membrane oxygenation (ECMO). All 3 patients showed a positive clinical response to cidofovir and survival at 28 days. Cidofovir with early ECMO therapy may be a therapeutic option in adenoviral ARDS. A literature review identified 15 cases of adenovirus pneumonia associated with ARDS.

  10. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

    PubMed

    Gaj, Thomas; Staahl, Brett T; Rodrigues, Gonçalo M C; Limsirichai, Prajit; Ekman, Freja K; Doudna, Jennifer A; Schaffer, David V

    2017-06-20

    Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Delivery of gene silencing agents for breast cancer therapy

    PubMed Central

    2013-01-01

    The discovery of RNA interference has opened the door for the development of a new class of cancer therapeutics. Small inhibitory RNA oligos are being designed to specifically suppress expression of proteins that are traditionally considered nondruggable, and microRNAs are being evaluated to exert broad control of gene expression for inhibition of tumor growth. Since most naked molecules are not optimized for in vivo applications, the gene silencing agents need to be packaged into delivery vehicles in order to reach the target tissues as their destinations. Thus, the selection of the right delivery vehicles serves as a crucial step in the development of cancer therapeutics. The current review summarizes the status of gene silencing agents in breast cancer and recent development of candidate cancer drugs in clinical trials. Nanotechnology-based delivery vectors for the formulation and packaging of gene silencing agents are also described. PMID:23659575

  12. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery

    PubMed Central

    Zhou, Jiangbing; Liu, Jie; Cheng, Christopher J.; Patel, Toral R.; Weller, Caroline E.; Piepmeier, Joseph M.; Jiang, Zhaozhong; Saltzman, W. Mark

    2014-01-01

    Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density. PMID:22138789

  13. Micelles and Nanoparticles for Ultrasonic Drug and Gene Delivery

    PubMed Central

    Husseini, Ghaleb A.; Pitt, William G.

    2008-01-01

    Drug delivery research employing micelles and nanoparticles has expanded in recent years. Of particular interest is the use of these nanovehicles that deliver high concentrations of cytotoxic drugs to diseased tissues selectively, thus reducing the agent’s side effects on the rest of the body. Ultrasound, traditionally used in diagnostic medicine, is finding a place in drug delivery in connection with these nanoparticles. In addition to their non-invasive nature and the fact that they can be focused on targeted tissues, acoustic waves have been credited with releasing pharmacological agents from nanocarriers, as well as rendering cell membranes more permeable. In this article, we summarize new technologies that combine the use of nanoparticles with acoustic power both in drug and gene delivery. Ultrasonic drug delivery from micelles usually employs polyether block copolymers, and has been found effective in vivo for treating tumors. Ultrasound releases drug from micelles, most probably via shear stress and shock waves from collapse of cavitation bubbles. Liquid emulsions and solid nanoparticles are used with ultrasound to deliver genes in vitro and in vivo. The small packaging allows nanoparticles to extravasate into tumor tissues. Ultrasonic drug and gene delivery from nano-carriers has tremendous potential because of the wide variety of drugs and genes that could be delivered to targeted tissues by fairly non-invasive means. PMID:18486269

  14. Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

    PubMed

    Wang, Fei; Wang, Yitong; Wang, Hui; Shao, Naimin; Chen, Yuanyuan; Cheng, Yiyun

    2014-11-01

    Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Tailoring the dendrimer core for efficient gene delivery.

    PubMed

    Hu, Jingjing; Hu, Ke; Cheng, Yiyun

    2016-04-15

    Dendrimers have been widely used as non-viral gene vectors due to well-defined chemical structures, high density of cationic charges and ease of surface modification. Although a large number of studies have reported the important roles of dendrimer architecture, component, generation and surface functionality in gene delivery, the effect of dendrimer core on this issue still remains unclear. Recent literatures suggest that a slight alternation in dendrimer core has a profound effect in the transfection efficacy and biocompatibility. In this review, we will discuss the transfection mechanism of dendrimers with different types of cores in respect of flexibility, hydrophobicity and functionality. We hope to open a possibility of designing efficient dendrimers for gene delivery by choosing a proper dendrimer core. As a branch of researches on dendrimers and dendritic polymers, the design of biocompatible and high efficient polymeric gene carriers has attracted increasing attentions during these years. Although the effect of dendrimer generation, species, architecture and surface functionality on gene delivery have been widely reported, the effect of dendrimer core on this issue still remains unclear. Recent literatures suggest that a minor variation on the dendrimer core has a profound effect in the transfection efficacy and biocompatibility. This critical review summarized the dendrimers with different types of cores and discussed the transfection mechanism with particular focus on the flexibility, hydrophobicity, and functionality. It is hoped to provide a new insight to design efficient and safe dendrimer-based gene vectors by choosing a proper core. To the best of our knowledge, this is the first review on the effect of dendrimer core on gene delivery. The findings obtained in this filed are of central importance in the design of efficient polymeric gene vectors. This article will appeal a wide readership such as physical chemist, dendrimer chemist, biological

  16. LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

    PubMed

    Shepelev, Mikhail V; Korobko, Elena V; Vinogradova, Tatiana V; Kopantsev, Eugene P; Korobko, Igor V

    2013-03-04

    Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

  17. Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

    PubMed

    Duisit, G; Salvetti, A; Moullier, P; Cosset, F L

    1999-01-20

    We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

  18. Recent Advances in Nanoparticle-Mediated Delivery of Anti-Inflammatory Phytocompounds

    PubMed Central

    Conte, Raffaele; Marturano, Valentina; Peluso, Gianfranco; Calarco, Anna; Cerruti, Pierfrancesco

    2017-01-01

    Phytocompounds have been used in medicine for decades owing to their potential in anti-inflammatory applications. However, major difficulties in achieving sustained delivery of phyto-based drugs are related to their low solubility and cell penetration, and high instability. To overcome these disadvantages, nanosized delivery technologies are currently in use for sustained and enhanced delivery of phyto-derived bioactive compounds in the pharmaceutical sector. This review focuses on the recent advances in nanocarrier-mediated drug delivery of bioactive molecules of plant origin in the field of anti-inflammatory research. In particular, special attention is paid to the relationship between structure and properties of the nanocarrier and phytodrug release behavior. PMID:28350317

  19. Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

    PubMed

    Zhang, Yuefeng; Yang, Bin; Liu, Yajie; Qin, Wenjie; Li, Chao; Wang, Lantian; Zheng, Wen; Wu, Yulian

    2016-05-01

    RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

  20. Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

    PubMed

    Kneidinger, Doris; Ibrišimović, Mirza; Lion, Thomas; Klein, Reinhard

    2012-06-01

    Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Potential of Gene Therapy for the Treatment of Pituitary Tumors

    PubMed Central

    Goya, R G.; Sarkar, D.K.; Brown, O.A.; Hereñú, C.B.

    2010-01-01

    Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy—the transfer of genetic material for therapeutic purposes—has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary

  2. PLGA/polymeric liposome for targeted drug and gene co-delivery.

    PubMed

    Wang, Hanjie; Zhao, Peiqi; Su, Wenya; Wang, Sheng; Liao, Zhenyu; Niu, Ruifang; Chang, Jin

    2010-11-01

    Chemotherapy is one of the most effective approaches to treat cancers in the clinic, but the problems, such as multidrug resistance (MDR), low bioavailability and toxicity, severely constrain the further application of chemotherapy. Our group recently reported that cationic PLGA/folate coated PEGlated polymeric liposome core-shell nanoparticles (PLGA/FPL NPs). It was self-assembled from a hydrophobic PLGA core and a hydrophilic folate coated PEGlated lipid shell for targeting co-delivery of drug and gene. Hydrophobic drugs can be incorporated into the core and the cationic shell of the drug-loaded nanoparticles can be used to bind DNA. The drug-loaded PLGA/FPL NPs/DNA complexes offer advantages to overcome these problems mentioned above, such as co-delivery of drugs and DNA to improving the chemosensitivity of cancer cells at a gene level, and targeting delivery of drug to the cancer tissue that enhance the bioavailability and reduce the toxicity. The experiment showed that nanoparticles have core-shell structure with nanosize, sustained drug release profile and good DNA-binding ability. Importantly, the core-shell nanoparticles achieve the possibility of co-delivering drugs and genes to the same cells with high gene transfection and drug delivery efficiency. Our data suggest that the PLGA/FPL NPs may be a useful drug and gene co-delivery system. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Fe3O4 Nanoparticles in Targeted Drug/Gene Delivery Systems

    PubMed Central

    Shen, Lazhen; Li, Bei; Qiao, Yongsheng

    2018-01-01

    Fe3O4 nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe3O4 NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe3O4 NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe3O4 NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe3O4 NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe3O4 NPs targeting drug/gene delivery systems. PMID:29473914

  4. A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer's disease.

    PubMed

    Rafii, Michael S; Baumann, Tiffany L; Bakay, Roy A E; Ostrove, Jeffrey M; Siffert, Joao; Fleisher, Adam S; Herzog, Christopher D; Barba, David; Pay, Mary; Salmon, David P; Chu, Yaping; Kordower, Jeffrey H; Bishop, Kathie; Keator, David; Potkin, Steven; Bartus, Raymond T

    2014-09-01

    Nerve growth factor (NGF) is an endogenous neurotrophic-factor protein with the potential to restore function and to protect degenerating cholinergic neurons in Alzheimer's disease (AD), but safe and effective delivery has proved unsuccessful. Gene transfer, combined with stereotactic surgery, offers a potential means to solve the long-standing delivery obstacles. An open-label clinical trial evaluated the safety and tolerability, and initial efficacy of three ascending doses of the genetically engineered gene-therapy vector adeno-associated virus serotype 2 delivering NGF (AAV2-NGF [CERE-110]). Ten subjects with AD received bilateral AAV2-NGF stereotactically into the nucleus basalis of Meynert. AAV2-NGF was safe and well-tolerated for 2 years. Positron emission tomographic imaging and neuropsychological testing showed no evidence of accelerated decline. Brain autopsy tissue confirmed long-term, targeted, gene-mediated NGF expression and bioactivity. This trial provides important evidence that bilateral stereotactic administration of AAV2-NGF to the nucleus basalis of Meynert is feasible, well-tolerated, and able to produce long-term, biologically active NGF expression, supporting the initiation of an ongoing multicenter, double-blind, sham-surgery-controlled trial. Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

  5. Fusogenic-Oligoarginine Peptide-Mediated Delivery of siRNAs Targeting the CIP2A Oncogene into Oral Cancer Cells

    PubMed Central

    Cantini, Liliana; Attaway, Christopher C.; Butler, Betsy; Andino, Lourdes M.; Sokolosky, Melissa L.; Jakymiw, Andrew

    2013-01-01

    Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer. PMID:24019920

  6. Selective gene silencing by viral delivery of short hairpin RNA

    PubMed Central

    2010-01-01

    RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells. PMID:20858246

  7. Epidermal growth factor targeting of bacteriophage to the choroid plexus for gene delivery to the central nervous system via cerebrospinal fluid.

    PubMed

    Gonzalez, Ana Maria; Leadbeater, Wendy; Podvin, Sonia; Borboa, Alexandra; Burg, Michael; Sawada, Ritsuko; Rayner, James; Sims, Karen; Terasaki, Tetsuya; Johanson, Conrad; Stopa, Edward; Eliceiri, Brian; Baird, Andrew

    2010-11-04

    Because the choroid plexus normally controls the production and composition of cerebrospinal fluid and, as such, its many functions of the central nervous system, we investigated whether ligand-mediated targeting could deliver genes to its secretory epithelium. We show here that when bacteriophages are targeted with epidermal growth factor, they acquire the ability to enter choroid epithelial cells grown in vitro as cell cultures, ex vivo as tissue explants or in vivo by intracerebroventricular injection. The binding and internalization of these particles activate EGF receptors on targeted cells, and the dose- and time-dependent internalization of particles is inhibited by the presence of excess ligand. When the phage genome is further reengineered to contain like green fluorescent protein or firefly luciferase under control of the cytomegalovirus promoter, gene expression is detectable in the choroid plexus and ependymal epithelium by immunohistochemistry or by noninvasive imaging, respectively. Taken together, these data support the hypothesis that reengineered ligand-mediated gene delivery should be considered a viable strategy to increase the specificity of gene delivery to the central nervous system and bypass the blood-brain barrier so as to exploit the biological effectiveness of the choroid plexus as a portal of entry into the brain. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Solid Lipid Nanoparticles as Efficient Drug and Gene Delivery Systems: Recent Breakthroughs

    PubMed Central

    Ezzati Nazhad Dolatabadi, Jafar; Valizadeh, Hadi; Hamishehkar, Hamed

    2015-01-01

    In recent years, nanomaterials have been widely applied as advanced drug and gene delivery nanosystems. Among them, solid lipid nanoparticles (SLNs) have attracted great attention as colloidal drug delivery systems for incorporating hydrophilic or lipophilic drugs and various macromolecules as well as proteins and nucleic acids. Therefore, SLNs offer great promise for controlled and site specific drug and gene delivery. This article includes general information about SLN structures and properties, production procedures, characterization. In addition, recent progress on development of drug and gene delivery systems using SLNs was reviewed. PMID:26236652

  9. Gene therapy in the inner ear using adenovirus vectors.

    PubMed

    Husseman, Jacob; Raphael, Yehoash

    2009-01-01

    Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression. Copyright (c) 2009 S. Karger AG, Basel.

  10. Gene delivery to skin using biolistics.

    PubMed

    Heiser, William C

    2006-06-01

    INTRODUCTION Biolistics ("biological ballistics") or particle bombardment provides a rapid and simple physical procedure for delivering genes into cells (Klein et al. 1987; Yang et al. 1990). The technique has many advantages-plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells.

  11. A Self-Assembled Coumarin-Anchored Dendrimer for Efficient Gene Delivery and Light-Responsive Drug Delivery.

    PubMed

    Wang, Hui; Miao, Wujun; Wang, Fei; Cheng, Yiyun

    2018-06-11

    The assembly of low molecular weight polymers into highly efficient and nontoxic nanostructures has broad applicability in gene delivery. In this study, we reported the assembly of coumarin-anchored low generation dendrimers in aqueous solution via hydrophobic interactions. The synthesized material showed significantly improved DNA binding and gene delivery, and minimal toxicity on the transfected cells. Moreover, the coumarin moieties in the assembled nanostructures endow the materials with light-responsive drug delivery behaviors. The coumarin substitutes in the assembled nanostructures were cross-linked with each other upon irradiation at 365 nm, and the cross-linked assemblies were degraded upon further irradiation at 254 nm. As a result, the drug-loaded nanoparticle showed a light-responsive drug release behavior and light-enhanced anticancer activity. The assembled nanoparticle also exhibited a complementary anticancer activity through the codelivery of 5-fluorouracil and a therapeutic gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study provided a facile strategy to develop light-responsive polymers for the codelivery of therapeutic genes and anticancer drugs.

  12. Gene Delivery in Neuro-Oncology.

    PubMed

    Dixit, Karan; Kumthekar, Priya

    2017-09-02

    Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis despite aggressive multimodal management thus novel treatments are urgently needed. Gene therapy is a versatile treatment strategy being investigated in multiple cancers including GBM. In gene therapy, a variety of vectors or "carriers" are used to deliver genes designed for different anti-tumoral effects. Gene delivery vehicles and approaches to treatment will be addressed in this review. The most commonly studied vectors are viral based, however, driven by advances in biomedical engineering, mesenchymal and neural stem cells, as well as multiple different types of nanoparticles have been developed to improve tumor tropism and also increase gene transfer into tumor cells. Different genes have been studied including suicide genes, which convert non-toxic prodrug into cytotoxic drug; immunomodulatory genes, which stimulate the immune system; and tumor suppressor genes which repair the defect that allow cells to divide unchecked. Gene therapy may be a promising treatment strategy in neuro-oncology as it is versatile and flexible due to the ability to tailor vectors and genes for specific therapeutic activity. Pre-clinical studies and clinical trials have demonstrated feasibility and safety of gene therapy; however, further studies are required to determine efficacy.

  13. Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

    PubMed

    Rowland, Christopher R; Glass, Katherine A; Ettyreddy, Adarsh R; Gloss, Catherine C; Matthews, Jared R L; Huynh, Nguyen P T; Guilak, Farshid

    2018-05-30

    Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore

  14. Systemic Gene Delivery Transduces the Enteric Nervous System of Guinea Pigs and Cynomolgus Macaques

    PubMed Central

    Gombash, Sara E; Cowley, Christopher J; Fitzgerald, Julie A; Lepak, Christina A; Neides, Mitchell G; Hook, Kathryn; Todd, Levi J; Wang, Guo-Du; Mueller, Christian; Kaspar, Brian K; Bielefeld, Eric C; Fischer, Andrew J; Wood, Jackie D; Foust, Kevin D

    2017-01-01

    Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or PBS. Piglets were euthanized three weeks post-injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9 treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders. PMID:28771235

  15. Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

    PubMed

    Gombash, S E; Cowley, C J; Fitzgerald, J A; Lepak, C A; Neides, M G; Hook, K; Todd, L J; Wang, G-D; Mueller, C; Kaspar, B K; Bielefeld, E C; Fischer, A J; Wood, J D; Foust, K D

    2017-10-01

    Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

  16. HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

    PubMed

    Kanagavelu, Saravana; Termini, James M; Gupta, Sachin; Raffa, Francesca N; Fuller, Katherine A; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S; Stone, Geoffrey W

    2014-01-01

    Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia

  17. Smart Micro/Nano-robotic Systems for Gene Delivery.

    PubMed

    Pedram, Alireza; Pishkenari, Hossein Nejat

    2017-01-01

    Small scale robotics have attracted growing attention for the prospect of targeting and accessing cell-sized sites, necessary for high precision biomedical applications and drug/gene delivery. The loss of controlled gene therapy, inducing systemic side effects and reduced therapeutic efficiency, can be settled utilizing these intelligent carriers. Newly proposed solutions for the main challenges of control, power supplying, gene release and final carrier extraction/degradation have shifted these smart miniature robots to the point of being employed for practical applications of transferring oligonucleotides (pDNA, siRNA, mRNA, etc.) in near future. In this paper, different scenarios and their endeavors to address the vital working demands and steps, in particular, carrier attachment and release, cell internalization, manipulation concerns as well as actuation systems are discussed.This review highlights some promising experimental results showing controlled gene release of robotic systems in comparison with current non-specific gene delivery methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Dendrimers as Carriers for siRNA Delivery and Gene Silencing: A Review

    PubMed Central

    Huang, Weizhe; He, Ziying

    2013-01-01

    RNA interference (RNAi) was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA) are used to specifically downregulate the expression of the targeted gene, known as the term “gene silencing.” One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc.) of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system. PMID:24288498

  19. Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

    PubMed

    Kia, Azadeh; Yata, Teerapong; Hajji, Nabil; Hajitou, Amin

    2013-10-22

    Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  20. Sustained delivery of siRNA/mesoporous silica nanoparticle (siRNA/MSN) complexes from nanofiber scaffolds for long-term gene silencing.

    PubMed

    Pinese, Coline; Lin, Junquan; Milbreta, Ulla; Li, Mingqiang; Wang, Yucai; Leong, Kam W; Chew, Sing Yian

    2018-06-08

    A low toxicity and efficient delivery system is needed to deliver small interfering RNAs (siRNA) in vitro and in vivo. The use of mesoporous silica nanoparticles (MSN) is becoming increasingly common due to its biocompatibility, tunable pore size and customizable properties. However, bolus delivery of siRNA/MSN complexes remains suboptimal, especially when a sustained and long-term administration is required. Here, we utilized electrospun scaffolds for sustained delivery of siRNA/MSN-PEI through surface adsorption and nanofiber encapsulation. As a proof-of-concept, we targeted collagen type I expression to modulate fibrous capsule formation. Surface adsorption of siRNA/MSN-PEI provided sustained availability of siRNA for at least 30 days in vitro. As compared to conventional bolus delivery, such scaffold-mediated transfection provided more effective gene silencing (p < 0.05). On the contrary, a longer sustained release was attained (at least 5 months) when siRNA/MSN-PEI complexes were encapsulated within the electrospun fibers. In vivo subcutaneous implantation and biodistribution analysis of these scaffolds revealed that siRNA remained localized up to ∼290 μm from the implants. Finally, a fibrous capsule reduction of ∼45.8 % was observed after 4 weeks in vivo as compared to negative scrambled siRNA treatment. Taken together, these results demonstrate the efficacy of scaffold-mediated sustained delivery of siRNA/MSN-PEI for long-term non-viral gene silencing applications. The bolus delivery of siRNA/ Mesoporous Silica Nanoparticles (MSN) complexes shows high efficiency to silence protein agonists of tumoral processes as cancer treatments. However, in tissue engineering area, scaffold mediated delivery is desired to achieve a local and sustained release of therapeutics. We showed the feasibility and the efficacy of siRNA/MSN delivered from electrospun scaffolds through surface adsorption and nanofiber encapsulation. We showed that this method enhances si

  1. Nano-scale gene delivery systems; current technology, obstacles, and future directions.

    PubMed

    Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

    2018-01-07

    Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

  2. Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

    PubMed Central

    Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

    2018-01-01

    ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the

  3. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    PubMed Central

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-01-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

  4. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    NASA Astrophysics Data System (ADS)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  5. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

    PubMed Central

    Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

    2016-01-01

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release. PMID:26883682

  6. Characterisation of gene delivery using liposomal bubbles and ultrasound

    NASA Astrophysics Data System (ADS)

    Koshima, Risa; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Nomura, Tetsuya; Negishi, Yoichi; Utoguchi, Naoki; Kudo, Nobuki; Maruyama, Kazuo

    2011-09-01

    The combination of nano/microbubbles and ultrasound is a novel technique for a non-viral gene deliver. We have previously developed novel ultrasound sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumors in vivo. The delivery of genes by bubble liposomes depended on the intensity of the applied ultrasound. The transfection efficiency plateaued at 0.7 W/cm2 ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, bubble liposomes were able to introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells. We conclude that the combination of Bubble liposomes and ultrasound is a good method for gene transfer in vivo.

  7. Therapeutic neovascularization by nanotechnology-mediated cell-selective delivery of pitavastatin into the vascular endothelium.

    PubMed

    Kubo, Mitsuki; Egashira, Kensuke; Inoue, Takahiro; Koga, Jun-ichiro; Oda, Shinichiro; Chen, Ling; Nakano, Kaku; Matoba, Tetsuya; Kawashima, Yoshiaki; Hara, Kaori; Tsujimoto, Hiroyuki; Sueishi, Katsuo; Tominaga, Ryuji; Sunagawa, Kenji

    2009-06-01

    Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.

  8. Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.

    PubMed

    Gonzalez-Fernandez, Tomas; Tierney, Erica G; Cunniffe, Grainne M; O'Brien, Fergal J; Kelly, Daniel J

    2016-05-01

    Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-β3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-β3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-β3-pBMP2 codelivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these

  9. Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

    PubMed

    Manunta, Maria D I; McAnulty, Robin J; Tagalakis, Aristides D; Bottoms, Stephen E; Campbell, Frederick; Hailes, Helen C; Tabor, Alethea B; Laurent, Geoffrey J; O'Callaghan, Christopher; Hart, Stephen L

    2011-01-01

    Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

  10. Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources: a comprehensive study.

    PubMed

    Boura, Joana S; Santos, Francisco Dos; Gimble, Jeffrey M; Cardoso, Carla M P; Madeira, Catarina; Cabral, Joaquim M S; Silva, Cláudia Lobato da

    2013-02-01

    Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

  11. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.

    PubMed

    Kessler, P D; Podsakoff, G M; Chen, X; McQuiston, S A; Colosi, P C; Matelis, L A; Kurtzman, G J; Byrne, B J

    1996-11-26

    Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

  12. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

    PubMed Central

    Kessler, Paul D.; Podsakoff, Gregory M.; Chen, Xiaojuan; McQuiston, Susan A.; Colosi, Peter C.; Matelis, Laura A.; Kurtzman, Gary J.; Byrne, Barry J.

    1996-01-01

    Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the β-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies. PMID:8943064

  13. Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

    PubMed Central

    Jakubczak, John L.; Rollence, Michele L.; Stewart, David A.; Jafari, Jonathon D.; Von Seggern, Dan J.; Nemerow, Glen R.; Stevenson, Susan C.; Hallenbeck, Paul L.

    2001-01-01

    A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector. PMID:11222722

  14. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

    2016-01-01

    Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

  15. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    PubMed

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-01-06

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

    PubMed

    Martin, Irene; Dohmen, Christian; Mas-Moruno, Carlos; Troiber, Christina; Kos, Petra; Schaffert, David; Lächelt, Ulrich; Teixidó, Meritxell; Günther, Michael; Kessler, Horst; Giralt, Ernest; Wagner, Ernst

    2012-04-28

    In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer. This journal is © The Royal Society of Chemistry 2012

  17. Ex vivo adenoviral gene transfer of constitutively activated STAT3 reduces post-transplant liver injury and promotes regeneration in a 20% rat partial liver transplant model.

    PubMed

    Huda, Kamrul A S M; Guo, Lei; Haga, Sanae; Murata, Hiroshi; Ogino, Tetsuya; Fukai, Moto; Yagi, Takahito; Iwagaki, Hiromi; Tanaka, Noriaki; Ozaki, Michitaka

    2006-05-01

    Signal transducer and activator of transcription-3 (STAT3) is one of the most important transcription factors for liver regeneration. This study was designed to examine the effects of constitutively activated STAT3 (STAT3-C) on post-transplant liver injury and regeneration in a rat 20% partial liver transplant (PLTx) model by ex vivo adenoviral gene transfer. Adenovirus encoding the STAT3-C gene was introduced intraportally into liver grafts and clamped for 30 min during cold preservation. After orthotopic PLTx, liver graft/body weights and serum biochemistry were monitored, and both a histological study and DNA binding assay were performed. STAT3-C protein expression and its binding to DNA in the liver graft were confirmed by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. This treatment modality promoted post-Tx liver regeneration effectively and rapidly. The serum levels of alanine aminotransferase/aspartate aminotransferase (AST/ALT) and bilirubin decreased in rats with STAT3-C. However, albumin (a marker of liver function) did not. Ex vivo gene transfer of STAT3-C to liver grafts reduced post-Tx injury and promoted liver regeneration. Thus, the activation of STAT3 in the liver graft may be a potentially effective clinical strategy for improving the outcome of small-for-size liver transplantation.

  18. Lipid-polymer hybrid nanoparticle-mediated therapeutics delivery: advances and challenges.

    PubMed

    Bose, Rajendran J C; Ravikumar, Rramaswamy; Karuppagounder, Vengadeshprabu; Bennet, Devasier; Rangasamy, Sabarinathan; Thandavarayan, Rajarajan A

    2017-08-01

    With rapid advances in nanomedicine, lipid-polymer hybrid nanoparticles (LPHNPs) have emerged as promising nanocarriers for several biomedical applications, including therapeutics delivery and biomedical imaging. Significant research has been dedicated to biomimetic or targeting functionalization, as well as controlled and image-guided drug-release capabilities. Despite this research, the clinical translation of LPHNP-mediated therapeutics delivery has progressed incrementally. In this review, we discuss the recent advances in and challenges to the development and application of LPHNPs, present examples to demonstrate the advantages of LPHNPs in therapeutics delivery and imaging applications, and discuss the translational obstacles to LPHNP technology. Copyright © 2017. Published by Elsevier Ltd.

  19. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

    PubMed

    Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

    2018-05-04

    Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Malaria Prevention by New Technology: Vectored Delivery of Antibody Genes

    DTIC Science & Technology

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0401 TITLE: Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes PRINCIPAL INVESTIGATOR: Gary...CONTRACT NUMBER Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes 5b. GRANT NUMBER W81XWH-15-1-0401 5c. PROGRAM ELEMENT...whole animals. Using a specific technology originally applied to expression of HIV antibodies, we demonstrated that mice can be protected from

  1. PDGF-B Gene Therapy Accelerates Bone Engineering and Oral Implant Osseointegration

    PubMed Central

    Chang, Po-Chun; Seol, Yang-Jo; Cirelli, Joni A; Pellegrini, Gaia R.; Jin, Qiming; Franco, Lea M.; Goldstein, Steven A.; Chandler, Lois A.; Sosnowski, Barbara; Giannobile, William V.

    2009-01-01

    Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due in part to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5×108 or 5.5×109 pfu/ml), Ad encoding luciferase (Ad-Luc; 5.5×109 pfu/ml; control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg/ml). Bone repair and osseointegration were measured via backscattered SEM, histomorphometry, microcomputed tomography, and biomechanical assessments. Further, a panel of local and systemic safety assessments was performed. Results demonstrated bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared to Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B demonstrates regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable to rhPDGF-BB protein delivery in vivo. PMID:19741730

  2. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches. © 2013.

  3. Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence

    NASA Astrophysics Data System (ADS)

    Xue, Jingwei; Zhao, Zekai; Zhang, Lei; Xue, Lingjing; Shen, Shiyang; Wen, Yajing; Wei, Zhuoyuan; Wang, Lu; Kong, Lingyi; Sun, Hongbin; Ping, Qineng; Mo, Ran; Zhang, Can

    2017-07-01

    Cell-mediated drug-delivery systems have received considerable attention for their enhanced therapeutic specificity and efficacy in cancer treatment. Neutrophils (NEs), the most abundant type of immune cells, are known to penetrate inflamed brain tumours. Here we show that NEs carrying liposomes that contain paclitaxel (PTX) can penetrate the brain and suppress the recurrence of glioma in mice whose tumour has been resected surgically. Inflammatory factors released after tumour resection guide the movement of the NEs into the inflamed brain. The highly concentrated inflammatory signals in the brain trigger the release of liposomal PTX from the NEs, which allows delivery of PTX into the remaining invading tumour cells. We show that this NE-mediated delivery of drugs efficiently slows the recurrent growth of tumours, with significantly improved survival rates, but does not completely inhibit the regrowth of tumours.

  4. Male-Mediated Gene Flow in Patrilocal Primates

    PubMed Central

    Schubert, Grit; Stoneking, Colin J.; Arandjelovic, Mimi; Boesch, Christophe; Eckhardt, Nadin; Hohmann, Gottfried; Langergraber, Kevin; Lukas, Dieter; Vigilant, Linda

    2011-01-01

    Background Many group–living species display strong sex biases in dispersal tendencies. However, gene flow mediated by apparently philopatric sex may still occur and potentially alters population structure. In our closest living evolutionary relatives, dispersal of adult males seems to be precluded by high levels of territoriality between males of different groups in chimpanzees, and has only been observed once in bonobos. Still, male–mediated gene flow might occur through rare events such as extra–group matings leading to extra–group paternity (EGP) and female secondary dispersal with offspring, but the extent of this gene flow has not yet been assessed. Methodology/Principal Findings Using autosomal microsatellite genotyping of samples from multiple groups of wild western chimpanzees (Pan troglodytes verus) and bonobos (Pan paniscus), we found low genetic differentiation among groups for both males and females. Characterization of Y–chromosome microsatellites revealed levels of genetic differentiation between groups in bonobos almost as high as those reported previously in eastern chimpanzees, but lower levels of differentiation in western chimpanzees. By using simulations to evaluate the patterns of Y–chromosomal variation expected under realistic assumptions of group size, mutation rate and reproductive skew, we demonstrate that the observed presence of multiple and highly divergent Y–haplotypes within western chimpanzee and bonobo groups is best explained by successful male–mediated gene flow. Conclusions/Significance The similarity of inferred rates of male–mediated gene flow and published rates of EGP in western chimpanzees suggests this is the most likely mechanism of male–mediated gene flow in this subspecies. In bonobos more data are needed to refine the estimated rate of gene flow. Our findings suggest that dispersal patterns in these closely related species, and particularly for the chimpanzee subspecies, are more variable than

  5. Effects of pressure characteristics on transfection efficiency in laser-induced stress wave-mediated gene delivery

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Ashida, Hiroshi; Obara, Minoru

    2013-07-01

    Laser-induced stress waves (LISWs) generated by irradiating a light-absorbing medium with a pulsed laser can transiently increase the permeability of cell membranes for gene delivery. In this study, we investigated the effects of pressure characteristics of LISWs upon gene transfection efficiency using lasers with different pulse durations: a 6-ns pulsed Nd:YAG laser and 20-ns and 200-µs pulsed ruby lasers. LISWs were generated by irradiating a black rubber disk, on which a transparent plastic sheet was adhered for confinement of the laser-produced plasma. Rat dorsal skin was injected with plasmid DNA coding for luciferase, to which LISWs were applied. With nanosecond laser pulses, transfection efficiency increased linearly with increasing positive peak pressure in the range of 35 to 145 MPa, the corresponding impulse ranging from 10 to 40 Paṡs. With 200-µs laser pulses, on the other hand, efficient gene expression was observed by the application of LISWs even with a 10-fold-lower peak pressure (˜5 MPa), the corresponding impulse being as large as 430 Paṡs. These results indicate that even at low peak pressures, efficient transfection can be achieved by extending the pressure duration and hence by increasing the impulse of LISWs, while the averaged expression efficiencies were relatively low.

  6. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  7. DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

    PubMed

    Cole, Grace; Ali, Ahlam A; McCrudden, Cian M; McBride, John W; McCaffrey, Joanne; Robson, Tracy; Kett, Vicky L; Dunne, Nicholas J; Donnelly, Ryan F; McCarthy, Helen O

    2018-06-01

    Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Adenoviral infection in a collection of juvenile inland bearded dragons (Pogona vitticeps).

    PubMed

    Doneley, R J T; Buckle, K N; Hulse, L

    2014-01-01

    Juvenile inland bearded dragons (Pogona vitticeps) from a breeding collection in south-east Queensland were presented at age 6-10 weeks with neurological signs, poor growth and occasional deaths. Histopathological examination revealed that six of eight lizards had multifocal non-suppurative hepatitis associated with 5-10 μm diameter, smudgy, basophilic, hyaline intranuclear inclusion bodies that marginated the nuclear chromatin. These histological lesions were considered consistent with adenoviral hepatitis. Infection with adenovirus was confirmed positive in one of the eight dragons by PCR for adenoviral DNA. DNA was extracted from formalin-fixed paraffin-embedded pooled tissues of the juvenile inland bearded dragons and tested using a nested-PCR protocol with primers specific for identification of adenovirus. Sequencing of the one PCR-positive dragon showed 95% nucleotide sequence alignment with agamid atadenovirus 1. Further investigation involved testing the breeding population, including the parents of the affected juveniles. Blood and cloacal samples were collected from the adult population, DNA was extracted and tested by PCR for adenovirus. There was a high percentage of positive results from the samples collected from the breeding population. This is the first reported group outbreak of adenoviral disease in bearded dragons in Australia. © 2014 Australian Veterinary Association.

  9. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-11-01

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

  10. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors.

    PubMed

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-11-17

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

  11. The magnetofection method: using magnetic force to enhance gene delivery.

    PubMed

    Plank, Christian; Schillinger, Ulrike; Scherer, Franz; Bergemann, Christian; Rémy, Jean-Serge; Krötz, Florian; Anton, Martina; Lausier, Jim; Rosenecker, Joseph

    2003-05-01

    In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.

  12. Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

    PubMed

    Yokoo, T; Kamimura, K; Suda, T; Kanefuji, T; Oda, M; Zhang, G; Liu, D; Aoyagi, Y

    2013-08-01

    The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time-pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time-pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time-pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml(-1)) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

  13. Lumbar spine intervertebral disc gene delivery: a pilot study in lewis rats.

    PubMed

    Damle, Sheela R; Rawlins, Bernard A; Boachie-Adjei, Oheneba; Crystal, Ronald G; Hidaka, Chisa; Cunningham, Matthew E

    2013-02-01

    Basic research toward understanding and treating disc pathology in the spine has utilized numerous animal models, with delivery of small molecules, purified factors, and genes of interest. To date, gene delivery to the rat lumbar spine has only been described utilizing genetically programmed cells in a matrix which has required partial disc excision, and expected limitation of treatment diffusion into the disc. This study was designed to develop and describe a surgical technique for lumbar spine exposure and disc space preparation, and use of a matrix-free method for gene delivery. Naïve or genetically programmed isogeneic bone marrow stromal cells were surgically delivered to adolescent male Lewis rat lumbar discs, and utilizing quantitative biochemical and qualitative immunohistological assessments, the implanted cells were detected 3 days post-procedure. Statistically significant differences were noted for recovery of the β-galactosidase marker gene comparing delivery of naïve or labeled cells (10(5) cells per disc) from the site of implantation, and between delivery of 10(5) or 10(6) labeled cells per disc at the site of implantation and the adjacent vertebral body. Immunohistology confirmed that the β-galactosidase marker was detected in the adjacent vertebra bone in the zone of surgical implantation. The model requires further testing in larger cohorts and with biologically active genes of interest, but the observations from the pilot experiments are very encouraging that this will be a useful comparative model for basic spine research involving gene or cell delivery, or other locally delivered therapies to the intervertebral disc or adjacent vertebral bodies in rats.

  14. Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

    2007-04-01

    We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

  15. Ultrasound-mediated drug delivery by gas bubbles generated from a chemical reaction.

    PubMed

    Lee, Sungmun; Al-Kaabi, Leena; Mawart, Aurélie; Khandoker, Ahsan; Alsafar, Habiba; Jelinek, Herbert F; Khalaf, Kinda; Park, Ji-Ho; Kim, Yeu-Chun

    2018-02-01

    Highly echogenic and ultrasound-responsive microbubbles such as nitrogen and perfluorocarbons have been exploited as ultrasound-mediated drug carriers. Here, we propose an innovative method for drug delivery using microbubbles generated from a chemical reaction. In a novel drug delivery system, luminol encapsulated in folate-conjugated bovine serum albumin nanoparticles (Fol-BSAN) can generate nitrogen gas (N 2 ) by chemical reaction when it reacts with hydrogen peroxide (H 2 O 2 ), one of reactive oxygen species (ROS). ROS plays an important role in the initiation and progression of cancer and elevated ROS have been observed in cancer cells both in vitro and in vivo. High-intensity focussed ultrasound (HIFU) is used to burst the N 2 microbubbles, causing site-specific delivery of anticancer drugs such as methotrexate. In this research, the drug delivery system was optimised by using water-soluble luminol and Mobil Composition of Matter-41 (MCM-41), a mesoporous material, so that the delivery system was sensitive to micromolar concentrations of H 2 O 2 . HIFU increased the drug release from Fol-BSAN by 52.9 ± 2.9% in 10 minutes. The cytotoxicity of methotrexate was enhanced when methotrexate is delivered to MDA-MB-231, a metastatic human breast cancer cell line, using Fol-BSAN with HIFU. We anticipate numerous applications of chemically generated microbubbles for ultrasound-mediated drug delivery.

  16. Mutated-leptin gene transfer induces increases in body weight by electroporation and hydrodynamics-based gene delivery in mice.

    PubMed

    Xiang, Lan; Murai, Atsushi; Muramatsu, Tatsuo

    2005-12-01

    To investigate whether in vivo gene transfer causes leptin-antagonistic effects on food intake, animal body weight and fat tissue weight, the R128Q mutated-leptin gene, an R to Q substitution at position 128 of mouse leptin, was transferred into mouse liver and leg muscle by electroporation and hydrodynamics-based gene delivery. Mutated-leptin gene transfer by electroporation caused significant increases in body weight at 5 days and after (5.4% increase relative to control; p<0.05). Hydrodynamics-based gene delivery of the mutated-leptin gene also caused an increase in body weight (3.0% increase relative to control; p<0.05). Mutated-leptin gene transfer by electroporation significantly increased the tissue weight of epididymal white fat and neuropeptide Y mRNA expression in the hypothalamus compared with those of the control group 3 weeks after gene transfer (p<0.05). These results suggest that mutated-leptin gene transfer successfully produced leptin-antagonistic effects by modulating the central regulator of energy homeostasis. Also, the extent of leptin-antagonistic effects by electroporation was much higher than hydrodynamics-based gene delivery, with at least single gene transfer.

  17. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  18. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    PubMed

    Tolmachov, Oleg E

    2015-01-01

    are then attracted to and internalized into the intended target cells via the expressed cognate strongly binding extra-cellular receptor, causing escalation of gene transfer into these cells and increasing the copy number of the therapeutic gene expression modules. Such self-focusing swarms of gene vectors can be either homogeneous, with 'scout' and 'therapeutic' members of the swarm being structurally identical, or, alternatively, heterogeneous (split), with 'scout' and 'therapeutic' members of the swarm being structurally specialized. It is hoped that the proposed self-focusing cell-targeted gene vector swarms with receptor-mediated intra-swarm signalling could be particularly effective in 'top-up' gene delivery scenarios, achieving high-level and sustained expression of therapeutic transgenes that are prone to shut-down through degradation and silencing. Crucially, in contrast to low-precision 'general location' vector guidance by diffusible chemo-attractants, ear-marking non-diffusible receptors can provide high-accuracy targeting of therapeutic vector particles to the specific cell, which has undergone a 'successful cell-specific hit' by a 'scout' vector particle. Opportunities for cell targeting could be expanded, since in the proposed model of self-focusing it could be possible to probe a broad selection of intra-cellular determinants of cell-specificity and not just to rely exclusively on extra-cellular markers of cell-specificity. By employing such self-focusing gene vectors for the improvement of cell-targeted delivery of therapeutic genes, e.g., in cancer therapy or gene addition therapy of recessive genetic diseases, it could be possible to broaden a leeway for the reduction of the vector load and, consequently, to minimize undesired vector cytotoxicity, immune reactions, and the risk of inadvertent genetic modification of germline cells in genetic treatment in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

    PubMed

    Kang, Mijeong; Kim, Bongsoo

    2015-01-01

    Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

  20. Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

    PubMed

    Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

    2018-05-01

    Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  1. A sight on protein-based nanoparticles as drug/gene delivery systems.

    PubMed

    Salatin, Sara; Jelvehgari, Mitra; Maleki-Dizaj, Solmaz; Adibkia, Khosro

    2015-01-01

    Polymeric nanomaterials have extensively been applied for the preparation of targeted and controlled release drug/gene delivery systems. However, problems involved in the formulation of synthetic polymers such as using of the toxic solvents and surfactants have limited their desirable applications. In this regard, natural biomolecules including proteins and polysaccharide are suitable alternatives due to their safety. According to literature, protein-based nanoparticles possess many advantages for drug and gene delivery such as biocompatibility, biodegradability and ability to functionalize with targeting ligands. This review provides a general sight on the application of biodegradable protein-based nanoparticles in drug/gene delivery based on their origins. Their unique physicochemical properties that help them to be formulated as pharmaceutical carriers are also discussed.

  2. Innovations in gene and growth factor delivery systems for diabetic wound healing

    PubMed Central

    Laiva, Ashang Luwang; O'Brien, Fergal J.

    2017-01-01

    Abstract The rise in lower extremity amputations due to nonhealing of foot ulcers in diabetic patients calls for rapid improvement in effective treatment regimens. Administration of growth factors (GFs) are thought to offer an off‐the‐shelf treatment; however, the dose‐ and time‐dependent efficacy of the GFs together with the hostile environment of diabetic wound beds impose a major hindrance in the selection of an ideal route for GF delivery. As an alternative, the delivery of therapeutic genes using viral and nonviral vectors, capable of transiently expressing the genes until the recovery of the wounded tissue offers promise. The development of implantable biomaterial dressings capable of modulating the release of either single or combinatorial GFs/genes may offer solutions to this overgrowing problem. This article reviews the state of the art on gene and protein delivery and the strategic optimization of clinically adopted delivery strategies for the healing of diabetic wounds. PMID:28482114

  3. Recent Advances in Skin Penetration Enhancers for Transdermal Gene and Drug Delivery.

    PubMed

    Amjadi, Morteza; Mostaghaci, Babak; Sitti, Metin

    2017-01-01

    There is a growing interest in transdermal delivery systems because of their noninvasive, targeted, and on-demand delivery of gene and drugs. However, efficient penetration of therapeutic compounds into the skin is still challenging largely due to the impermeability of the outermost layer of the skin, known as stratum corneum. Recently, there have been major research activities to enhance the skin penetration depth of pharmacological agents. This article reviews recent advances in the development of various strategies for skin penetration enhancement. We show that approaches such as ultrasound waves, laser, and microneedle patches have successfully been employed to physically disrupt the stratum corneum structure for enhanced transdermal delivery. Rather than physical approaches, several non-physical route have also been utilized for efficient transdermal delivery across the skin barrier. Finally, we discuss some clinical applications of transdermal delivery systems for gene and drug delivery. This paper shows that transdermal delivery devices can potentially function for diverse healthcare and medical applications while further investigations are still necessary for more efficient skin penetration of gene and drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

    PubMed Central

    Kagey, Michael H.; Newman, Jamie J.; Bilodeau, Steve; Zhan, Ye; Orlando, David A.; van Berkum, Nynke L.; Ebmeier, Christopher C.; Goossens, Jesse; Rahl, Peter B.; Levine, Stuart S.; Taatjes, Dylan J.; Dekker, Job; Young, Richard A.

    2010-01-01

    Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell. PMID:20720539

  5. Selenium nanoparticles: potential in cancer gene and drug delivery.

    PubMed

    Maiyo, Fiona; Singh, Moganavelli

    2017-05-01

    In recent decades, colloidal selenium nanoparticles have emerged as exceptional selenium species with reported chemopreventative and therapeutic properties. This has sparked widespread interest in their use as a carrier of therapeutic agents with results displaying synergistic effects of selenium with its therapeutic cargo and improved anticancer activity. Functionalization remains a critical step in selenium nanoparticles' development for application in gene or drug delivery. In this review, we highlight recent developments in the synthesis and functionalization strategies of selenium nanoparticles used in cancer drug and gene delivery systems. We also provide an update of recent preclinical studies utilizing selenium nanoparticles in cancer therapeutics.

  6. Placental Abruption and Perinatal Mortality With Preterm Delivery as a Mediator: Disentangling Direct and Indirect Effects

    PubMed Central

    Ananth, Cande V.; VanderWeele, Tyler J.

    2011-01-01

    The authors use recent methodology in causal inference to disentangle the direct and indirect effects that operate through a mediator in an exposure-response association paradigm. They demonstrate how total effects can be partitioned into direct and indirect effects even when the exposure and mediator interact. The impact of bias due to unmeasured confounding on the exposure-response association is assessed through a series of sensitivity analyses. These methods are applied to a problem in perinatal epidemiology to examine the extent to which the effect of abruption on perinatal mortality is mediated through preterm delivery. Data on over 26 million US singleton births (1995–2002) were utilized. Risks of mortality among abruption and nonabruption births were 102.7 and 6.2 per 1,000 births, respectively. Risk ratios of the natural direct and indirect (preterm delivery-mediated) effects of abruption on mortality were 10.18 (95% confidence interval: 9.80, 10.58) and 1.35 (95% confidence interval: 1.33, 1.38), respectively. The proportion of increased mortality risk mediated through preterm delivery was 28.1%, with even higher proportions associated with deliveries at earlier gestational ages. Sensitivity analyses underscore that the qualitative conclusions of some mediated effects and substantial direct effects are reasonably robust to unmeasured confounding of a fairly considerable magnitude. PMID:21430195

  7. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

    PubMed

    Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

    2005-06-01

    Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

  8. Randomized, Controlled, Phase 2 Trial of Povidone-Iodine/Dexamethasone Ophthalmic Suspension for Treatment of Adenoviral Conjunctivitis.

    PubMed

    Pepose, Jay S; Ahuja, Arjun; Liu, Wenlei; Narvekar, Abhijit; Haque, Reza

    2018-05-19

    To evaluate the efficacy/safety of an ophthalmic suspension of povidone-iodine (PVP-I) 0.6% and dexamethasone 0.1% in patients with acute adenoviral conjunctivitis. Multicenter, randomized, vehicle-controlled, double-masked trial. Adults with a positive Rapid Pathogen Screening Adeno-Detector Plus TM test were randomized 1:1:1 to PVP-I 0.6%/dexamethasone 0.1%, PVP-I 0.6%, or vehicle, bilaterally 4 times daily for 5 days (days 1-5). Patients were evaluated on days 3, 6, and 12 (+1-day window). Efficacy measures included clinical resolution and adenoviral eradication. Overall, 144 patients were included in the efficacy analysis (PVP-I/dexamethasone, n = 48; PVP-I, n = 50; vehicle, n = 46). The proportion of patients with clinical resolution (primary study eye with last observation carried forward [LOCF]) at the day 6 visit was higher with PVP-I/dexamethasone (31.3%) than vehicle (10.9%; P = .0158) and PVP-I (18.0%; P = nonsignificant). The proportion with adenoviral eradication (primary study eye with LOCF) was higher with PVP-I/dexamethasone than vehicle at the day 3 (35.4% vs 8.7%; P = .0019) and day 6 (79.2% vs 56.5%; P = .0186) visits and versus PVP-I (day 3 visit, 32.0%; day 6 visit, 62.0%; each P = nonsignificant). Treatment-emergent adverse events (AEs) occurred in 69.0% (vehicle), 62.7% (PVP-I), and 53.4% (PVP-I/dexamethasone) of patients in the safety dataset. Discontinuation owing to AEs occurred in 37 patients (vehicle, n = 16; PVP-I, n = 12; PVP-I/dexamethasone, n = 9). PVP-I/dexamethasone appeared safe and well tolerated, and significantly improved clinical resolution and adenoviral eradication in patients with acute adenoviral conjunctivitis. Copyright © 2018. Published by Elsevier Inc.

  9. Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats.

    PubMed

    Huang, Shun-Ping; Lin, Po-Kang; Liu, Jorn-Hon; Khor, Chin-Ni; Lee, Yih-Jing

    2004-01-01

    Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats. Copyright 2004 National Science Council, ROC and S. Karger AG, Basel

  10. Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

    PubMed

    Rey-Rico, Ana; Cucchiarini, Magali

    2016-04-01

    Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

  11. Splenic gene delivery system using self-assembling nano-complex with phosphatidylserine analog.

    PubMed

    Kurosaki, Tomoaki; Nakasone, Chihiro; Kodama, Yukinobu; Egashira, Kanoko; Harasawa, Hitomi; Muro, Takahiro; Nakagawa, Hiroo; Kitahara, Takashi; Higuchi, Norihide; Nakamura, Tadahiro; Sasaki, Hitoshi

    2015-01-01

    The recognition of phosphatidylserine on the erythrocyte membrane mediates erythrophagocytosis by resident spleen macrophages. The application of phosphatidylserine to a gene vector may be a novel approach for splenic drug delivery. Therefore, we chose 1,2-dioleoyl-sn-glycero-3-phospho-L-serin (DOPS) as an analogue of phosphatidylserine for splenic gene delivery of plasmid DNA (pDNA). In the present study, we successfully prepared a stable pDNA ternary complex using DOPS and polyethyleneimine (PEI) and evaluated its efficacy and safety. The pDNA/PEI complex had a positive charge and showed high transgene efficacy, although it caused cytotoxicity and agglutination. The addition of DOPS changed the ζ-potential of the pDNA/PEI complex to negative. It is known that anionic complexes are not taken up well by cells. Surprisingly, however, the pDNA/PEI/DOPS complex showed relatively high transgene efficacy in vitro. Fluorescence microscope observation revealed that the pDNA/PEI/DOPS complex internalized the cells while maintaining the complex formation. The injection of the pDNA/PEI complex killed most mice within 24 h at high doses, although all mice in the pDNA/PEI/DOPS complex group survived. The ternary complex with DOPS showed markedly better safety compared with the pDNA/PEI complex. The pDNA/PEI/DOPS complex showed high gene expression selectively in the spleen after intravenous injection into mice. Thus the ternary complex with DOPS can be used to deliver pDNA to the spleen, in which immune cells are abundant. It appears to have an excellent safety level, although further study to determine the mechanism of action is necessary.

  12. Nanomedicines for Back of the Eye Drug Delivery, Gene Delivery, and Imaging

    PubMed Central

    Kompella, Uday B.; Amrite, Aniruddha C.; Ravi, Rashmi Pacha; Durazo, Shelley A.

    2013-01-01

    Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology. Nanomedicines, products of nanotechnology, having at least one dimension in the nanoscale include nanoparticles, micelles, nanotubes, and dendrimers, with and without targeting ligands, are making a significant impact in the fields of ocular drug delivery, gene delivery, and imaging, the focus of this review. Key applications of nanotechnology discussed in this review include a) bioadhesive nanomedicines; b) functionalized nanomedicines that enhance target recognition and/or cell entry; c) nanomedicines capable of controlled release of the payload; d) nanomedicines capable of enhancing gene transfection and duration of transfection; f) nanomedicines responsive to stimuli including light, heat, ultrasound, electrical signals, pH, and oxidative stress; g) diversely sized and colored nanoparticles for imaging, and h) nanowires for retinal prostheses. Additionally, nanofabricated delivery systems including implants, films, microparticles, and nanoparticles are described. Although the above nanomedicines may be administered by various routes including topical, intravitreal, intravenous, transscleral, suprachoroidal, and subretinal routes, each nanomedicine should be tailored for the disease, drug, and site of administration. In addition to the nature of materials used in nanomedicine design, depending on the site of nanomedicine administration, clearance and toxicity are expected to differ. PMID:23603534

  13. Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells.

    PubMed

    Liang, Gaofeng; Kan, Shu; Zhu, Yanliang; Feng, Shuying; Feng, Wenpo; Gao, Shegan

    2018-01-01

    Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells. In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation. The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1-Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle. Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of miRNA-loaded exosomes that recognize specific biomarkers on the tumor cell.

  14. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    PubMed Central

    Yata, Teerapong; Lee, Koon-Yang; Dharakul, Tararaj; Songsivilai, Sirirurg; Bismarck, Alexander; Mintz, Paul J; Hajitou, Amin

    2014-01-01

    Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage. PMID:25118171

  15. Synthetic Virology: Engineering Viruses for Gene Delivery

    PubMed Central

    Guenther, Caitlin M.; Kuypers, Brianna E.; Lam, Michael T.; Robinson, Tawana M.; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or “bionic” viruses, feature engineered components, or “parts”, that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies – rational, combinatorial, and pseudo-rational – have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavours will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. PMID:25195922

  16. Synthetic virology: engineering viruses for gene delivery.

    PubMed

    Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

  17. In vivo evaluation of a cancer therapy strategy combining HSV1716-mediated oncolysis with gene transfer and targeted radiotherapy.

    PubMed

    Sorensen, Annette; Mairs, Robert J; Braidwood, Lynne; Joyce, Craig; Conner, Joe; Pimlott, Sally; Brown, Moira; Boyd, Marie

    2012-04-01

    Oncolytic herpes viruses show promise for cancer treatment. However, it is unlikely that they will fulfill their therapeutic potential when used as monotherapies. An alternative strategy is to use these viruses not only as oncolytic agents but also as a delivery mechanism of therapeutic transgenes to enhance tumor cell killing. The herpes simplex virus 1 deletion mutant HSV1716 is a conditionally replicating oncolytic virus that selectively replicates in and lyses dividing tumor cells. It has a proven safety profile in clinical trials and has demonstrated efficacy as a gene-delivery vehicle. To enhance its therapeutic potential, we have engineered HSV1716 to convey the noradrenaline transporter (NAT) gene (HSV1716/NAT), whose expression endows infected cells with the capacity to accumulate the noradrenaline analog metaiodobenzylguanidine (MIBG). Thus, the NAT gene-infected cells are susceptible to targeted radiotherapy using radiolabeled (131)I-MIBG, a strategy that has already shown promise for combined targeted radiotherapy-gene therapy in cancer cells after plasmid-mediated transfection. We used HSV1716/NAT as a dual cell lysis-gene delivery vehicle for targeting the NAT transgene to human tumor xenografts in vivo. In tumor xenografts that did not express NAT, intratumoral or intravenous injection of HSV1716/NAT induced the capacity for active uptake of (131)I-MIBG. Administration of HSV1716/NAT and (131)I-MIBG resulted in decreased tumor growth and enhanced survival relative to injection of either agent alone. Efficacy was dependent on the scheduling of delivery of the 2 agents. These findings support a role for combination radiotherapy-gene therapy for cancer using HSV1716 expressing the NAT transgene and targeted radionuclide therapy.

  18. The evolution of heart gene delivery vectors

    PubMed Central

    Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

    2012-01-01

    Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

  19. Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

    PubMed

    Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

    2017-02-13

    Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

  20. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

    PubMed Central

    Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

    1993-01-01

    To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

  1. Theory and in vivo application of electroporative gene delivery.

    PubMed

    Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

    2000-09-01

    Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

  2. Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo.

    PubMed

    Sapet, Cédric; Pellegrino, Christophe; Laurent, Nicolas; Sicard, Flavie; Zelphati, Olivier

    2012-05-01

    Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

  3. Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

    PubMed

    Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

    2016-03-01

    Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

  4. Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-β type II receptor (sTGFβRII) gene transfer.

    PubMed

    Sharma, Ajay; Rodier, Jason T; Tandon, Ashish; Klibanov, Alexander M; Mohan, Rajiv R

    2012-01-01

    /µg DNA and 1,640±100 pg/ml sTGFβRII protein during these assays. The PEI-mediated sTGFβRII delivery remarkably attenuated TGFβ1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFβRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFβRII overexpression by either method was significantly higher than the naked vector transfection. Furthermore, PEI- or Lipofectamine-mediated sTGFβRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2-6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death. This study demonstrated that PEI-DNA nanoparticles are an attractive vector for the development of nonviral corneal gene therapy approaches and that the sTGFβRII gene delivery into keratocytes could be used to control corneal fibrosis in vivo.

  5. Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis.

    PubMed

    von Degenfeld, Georges; Banfi, Andrea; Springer, Matthew L; Blau, Helen M

    2003-10-01

    Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of 'growth' factors with 'maturation' factors like PDGF-BB, angiopoietin-1 or TGF-beta. Myoblast-mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long-lasting expression, and can be considered as a relatively rapid form of 'adult transgenesis' in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different 'growth' and 'maturation' factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline-inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell-based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

  6. In vivo correction of anaemia in β-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery

    PubMed Central

    Bahal, Raman; Ali McNeer, Nicole; Quijano, Elias; Liu, Yanfeng; Sulkowski, Parker; Turchick, Audrey; Lu, Yi-Chien; Bhunia, Dinesh C.; Manna, Arunava; Greiner, Dale L.; Brehm, Michael A.; Cheng, Christopher J.; López-Giráldez, Francesc; Ricciardi, Adele; Beloor, Jagadish; Krause, Diane S.; Kumar, Priti; Gallagher, Patrick G.; Braddock, Demetrios T.; Mark Saltzman, W.; Ly, Danith H.; Glazer, Peter M.

    2016-01-01

    The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration. PMID:27782131

  7. Erythrocyte membrane based cationic polymer-mcDNA complexes as an efficient gene delivery system.

    PubMed

    Huang, Ping; Zhao, Jing; Wei, Chiju; Hou, Xiaohu; Chen, Pingzhang; Tan, Yan; He, Cheng-Yi; Wang, Zhiyong; Chen, Zhi-Ying

    2016-12-20

    Gene therapy has great promise for the treatment of obtained and inherited serious diseases. However, the lack of safe and efficient gene delivery systems remains a barrier for their clinical application. Here, we reported a potential gene delivery vehicle composed of the erythrocyte membrane and cationic polymers, for example the XtremeGENE from Roche and the ε-caprolactone modified polyethylenimine. In addition to high efficiency, this system showed negligible cytotoxicity compared to the two cationic polymers alone in various cell lines, including human embryonic kidney cells (293T), human liver cancer cells (Huh7 and HepG2), murine dendritic cells (DC2.4) and human umbilical cord mesenchymal stem cells (Hu-MSCs). Moreover, the results of confocal laser scanning microscopy and flow cytometry suggested that the cell uptake of this gene vector was improved and might be introduced by the fusion interaction between the erythrocyte membrane and targeted cells.Thus, all the results revealed that the erythrocyte membrane based gene delivery system might be able to serve as an excellent gene delivery system.

  8. Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

    NASA Astrophysics Data System (ADS)

    Bhise, Nupura S.

    Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

  9. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    PubMed

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. The evolution of heart gene delivery vectors.

    PubMed

    Wasala, Nalinda B; Shin, Jin-Hong; Duan, Dongsheng

    2011-10-01

    Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. Copyright © 2011 John Wiley & Sons, Ltd.

  11. Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

    PubMed Central

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-01-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  12. Current Strategies in the Modification of PLGA-based Gene Delivery System.

    PubMed

    Ramezani, Mohammad; Ebrahimian, Mahboubeh; Hashemi, Maryam

    2017-01-01

    Successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and genes efficiently. This formulation has several advantages in comparison with other formulations including improvement in solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as a gene carrier, there exist many challenges. PLGA NPs could protect the encapsulated DNA from in vivo degradation but the DNA release is slow and the negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce cytotoxicity, to enhance delivery efficiency and to target specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for the modification of PLGA particles applied in gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Specific gene delivery to liver sinusoidal and artery endothelial cells.

    PubMed

    Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

    2013-09-19

    Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

  14. A clinical algorithm identifies high risk pediatric oncology and bone marrow transplant patients likely to benefit from treatment of adenoviral infection.

    PubMed

    Williams, Kirsten Marie; Agwu, Allison L; Dabb, Alix A; Higman, Meghan A; Loeb, David M; Valsamakis, Alexandra; Chen, Allen R

    2009-11-01

    Adenoviral infections cause morbidity and mortality in blood and marrow transplantation and pediatric oncology patients. Cidofovir is active against adenovirus, but must be used judiciously because of its nephrotoxicity and unclear indications. Therefore, before introducing cidofovir use during an adenoviral outbreak, we developed a clinical algorithm to distinguish low risk patients from those who merited cidofovir therapy because of significant adenoviral disease and high risk for death. This study was conducted to determine whether the algorithm accurately predicted severe adenovirus disease and whether selective cidofovir treatment was beneficial. A retrospective analysis of a pediatric oncology/blood and marrow transplantation cohort prealgorithm and postalgorithm implementation was performed. Twenty patients with adenovirus infection were identified (14 high risk and 6 low risk). All low-risk patients cleared their infections without treatment. Before algorithm implementation, all untreated high-risk patients died, 4 out of 5 (80%), from adenoviral infection. In contrast, cidofovir reduced adenovirus-related mortality in the high-risk group postalgorithm implementation (9 patients treated, 1 patient died; RR 0.14, P<0.05) and all treated high-risk patients cleared their virus. The clinical algorithm accurately identified patients at high risk for severe fatal adenoviral disease who would benefit from selective use of cidofovir.

  15. Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirus-mediated transgene expression.

    PubMed

    Taura, Kojiro; Yamamoto, Yuzo; Nakajima, Akio; Hata, Koichiro; Uchinami, Hiroshi; Yonezawa, Kei; Hatano, Etsuro; Nishino, Norikazu; Yamaoka, Yoshio

    2004-05-01

    Histone deacetylase inhibitors (HDIs) are known to enhance adenovirus (Ad)-mediated transgene expression. Recently, novel HDIs, including cyclic hydroxamic-acid-containing peptide 31 (CHAP31) and FR901228 (FK228), have been developed. The effects of these two novel HDIs on Ad-transduced or endogenous gene expression were investigated. Acetylation of core histones and the expression of the coxsackie and adenovirus receptor (CAR) in HDI-treated cells were examined using Western blot and a quantitative reverse transcription polymerase chain reaction (TaqMan RT-PCR), respectively. Their in vivo effect on adenoviral gene expression was investigated in BALB/c mice. Both compounds enhanced and prolonged Ad-mediated beta-galactosidase expression more effectively than did trichostatin A, a classic HDI. The same effect was observed in Ad-transduced heat shock protein 72 (HSP72), but not in hyperthermia-induced endogenous expression of HSP72, suggesting that the effect is specific for transduced gene expression. Hyperacetylation of core histones induced by HDIs was considered responsible for the augmentative effects of gene expression. Intravenous administration of either CHAP31 or FR901228 enhanced beta-galactosidase expression in mice infected with AdLacZ. CHAP31 and FR901228 amplified Ad-mediated transgene expression. The enhancement of transgene expression by HDIs may result in fewer vector doses for necessary gene expression, helping to alleviate disadvantages caused by Ad vectors. This could be a useful tool in overcoming current limitations of gene therapy using adenovirus vectors. Copyright 2004 John Wiley & Sons, Ltd.

  16. Human gene therapy: novel approaches to improve the current gene delivery systems.

    PubMed

    Cucchiarini, Magali

    2016-06-01

    Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.

  17. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

  18. Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo.

    PubMed

    Chumakova, Olga V; Liopo, Anton V; Andreev, Valery G; Cicenaite, Inga; Evers, B Mark; Chakrabarty, Shilla; Pappas, Todd C; Esenaliev, Rinat O

    2008-03-18

    The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

  19. Polyethyleneimine grafted short halloysite nanotubes for gene delivery.

    PubMed

    Long, Zheru; Zhang, Jun; Shen, Yan; Zhou, Changren; Liu, Mingxian

    2017-12-01

    Inorganic nanoparticles have attracted much attentions in gene delivery because of their desirable characteristics including low toxicity, well-controlled characteristics, high gene delivery efficiency, and multi-functionalities. Here, natural occurred halloysite nanotubes (HNTs) were developed as a novel non-viral gene vector. To increase the efficiency of endocytosis, HNTs were firstly shortened into an appropriate size (~200nm). Then polyethyleneimine (PEI) was grafted onto HNTs to bind green fluorescence protein (GFP) labeled pDNA. The structure and physical-chemical properties of PEI grafted HNTs (PEI-g-HNTs) were characterized by various methods. PEI-g-HNTs show lower cytotoxicity than PEI. PEI-g-HNTs are positively charged and can bind DNA tightly at designed N/P ratio from 5:1 to 40:1. PEI-g-HNTs/pDNA complexes show much higher transfection efficiency towards both 293T and HeLa cells compared with PEI/pDNA complexes at the equivalent N/P ratio. The transfection efficiencies of PEI-g-HNTs/pDNA complex towards HeLa cell can reach to 44.4% at N/P ratio of 20. PEI-g-HNTs/pDNA complexes possess a higher GFP protein expression than PEI/pDNA from simple western immunoblots. So, PEI-g-HNTs are potential gene vectors with good biocompatibility and high transfection efficiency, which have promising applications in cancer gene therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Novel Approaches for Retinal Drug and Gene Delivery

    PubMed Central

    Kim, Stephen J.

    2014-01-01

    The ARVO 2014 minisymposium on “Novel Approaches for Retinal Drug and Gene Delivery” was held on May 6, 2014 in Orlando, FL. The main intent of the symposium was to review recent advances in retinal drug and gene delivery with specific emphasis on novel approaches that address current limitations and have the potential to translate into clinical practice. The symposium was sponsored by Translational Vision Science and Technology. PMID:25346872

  1. Noninvasive, targeted gene therapy for acute spinal cord injury using LIFU-mediated BDNF-loaded cationic nanobubble destruction.

    PubMed

    Song, Zhaojun; Ye, Yongjie; Zhang, Zhi; Shen, Jieliang; Hu, Zhenming; Wang, Zhigang; Zheng, Jiazhuang

    2018-02-12

    Various gene delivery systems have been widely studied for the acute spinal cord injury (SCI) treatment. In the present study, a novel type of brain-derived neurotrophic factor (BDNF)-loaded cationic nanobubbles (CNBs) conjugated with MAP-2 antibody (mAb MAP-2 /BDNF/CNBs) was prepared to provide low-intensity focused ultrasound (LIFU)-targeted gene therapy. In vitro experiments, the ultrasound-targeted tranfection to BDNF overexpressioin in neurons and efficiently inhibition neuronal apoptosis have been demonstrated, and the elaborately designed mAb MAP-2 /BDNF/CNBs can specifically target to the neurons. Furthermore, in a acute SCI rat model, LIFU-mediated mAb MAP-2 /BDNF/CNBs transfection significantly increased BDNF expression, attenuated histological injury, decreased neurons loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in SCI rats. LIFU-mediated mAb MAP-2 /BDNF/CNBs destruction significantly increase transfection efficiency of BDNF gene both in vitro and in vivo, and has a significant neuroprotective effect on the injured spinal cord. Therefore, the combination of LIFU irradiation and gene therapy through mAb MAP-2 /BDNF/CNBs can be considered as a novel non-invasive and targeted treatment for gene therapy of SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Dual delivery of rhPDGF-BB and bone marrow mesenchymal stromal cells expressing the BMP2 gene enhance bone formation in a critical-sized defect model.

    PubMed

    Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2013-11-01

    Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

  3. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

    PubMed

    Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

    2015-10-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

  4. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  5. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    NASA Astrophysics Data System (ADS)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  6. Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras-associating Domains of Phospholipase Cϵ*

    PubMed Central

    Dusaban, Stephanie S.; Kunkel, Maya T.; Smrcka, Alan V.; Brown, Joan Heller

    2015-01-01

    Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses. PMID:26350460

  7. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα.

    PubMed

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-08-31

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer.

  8. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα

    PubMed Central

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-01-01

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer. PMID:27576898

  9. Hyaluronic Acid Enhances Gene Delivery into the Cochlea

    PubMed Central

    Shibata, Seiji B.; Cortez, Sarah R.; Wiler, James A.; Swiderski, Donald L.

    2012-01-01

    Abstract Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application. PMID:22074321

  10. Hyaluronic acid enhances gene delivery into the cochlea.

    PubMed

    Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

    2012-03-01

    Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

  11. Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

    PubMed

    Liu, Shuo; Niger, Corinne; Koh, Eugene Y; Stains, Joseph P

    2015-01-01

    Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like

  12. Quantitative measurement of delivery and gene silencing activities of siRNA polyplexes containing pyridylthiourea-grafted polyethylenimines.

    PubMed

    Pinel, Sophie; Aman, Emmanuel; Erblang, Felix; Dietrich, Jonathan; Frisch, Benoit; Sirman, Julien; Kichler, Antoine; Sibler, Annie-Paule; Dontenwill, Monique; Schaffner, Florence; Zuber, Guy

    2014-05-28

    The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H(+)/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of "simple"/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom-up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Novel gemini cationic lipids with carbamate groups for gene delivery

    PubMed Central

    Zhao, Yi-Nan; Qureshi, Farooq; Zhang, Shu-Biao; Cui, Shao-Hui; Wang, Bing; Chen, Hui-Ying; Lv, Hong-Tao; Zhang, Shu-Fen; Huang, Leaf

    2014-01-01

    To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study. PMID:25045521

  14. Smart Carbon Nanotubes with Laser-Controlled Behavior in Gene Delivery and Therapy through a Non-Digestive Trafficking Pathway.

    PubMed

    Kong, Fenfen; Liu, Fei; Li, Wei; Guo, Xiaomeng; Wang, Zuhua; Zhang, Hanbo; Li, Qingpo; Luo, Lihua; Du, Yongzhong; Jin, Yi; You, Jian

    2016-12-01

    Near-infrared (NIR) laser-controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a "photothermal transfection" agent is obtained by wrapping poly(ethylenimine)-cholesterol derivatives (PEI-Chol) around single-walled carbon nanotubes (SWNTs). The PEI-Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae-mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser-mediated photothermal transfection of PCS 10K /plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor-growth inhibition in vivo than naked pTP53, PEI 25K /pTP53, and PCS 10K /pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae-mediated cellular uptake of the complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Combinatorial therapy with adenoviral-mediated PTEN and a PI3K inhibitor suppresses malignant glioma cell growth in vitro and in vivo by regulating the PI3K/AKT signaling pathway.

    PubMed

    Nan, Yang; Guo, Liyun; Song, Yunpeng; Wang, Le; Yu, Kai; Huang, Qiang; Zhong, Yue

    2017-08-01

    Glioblastoma is a highly invasive and challenging tumor of the central nervous system. The mutation/deletion of the tumor suppressor phosphatase and tensin homolog (PTEN) gene is the main genetic change identified in glioblastomas. PTEN plays a critical role in tumorigenesis and has been shown to be an important therapeutic target. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is commonly used to inhibit glioma cell growth via regulation of the PI3K/AKT signaling pathway. In this study, we examined the growth inhibitory effects of a combinatorial therapy of adenoviral-mediated PTEN (Ad-PTEN) and LY294002 on LN229 and U251 glioma cells in vitro and on tumor xenografts in vivo. In vitro, LN229 and U251 glioma cells were treated by combinatorial therapy with Ad-PTEN and LY294002. The growth ability was determined by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Cell invasive ability was analyzed by transwell invasion assay and cell apoptosis analysis via FITC-Annexin V analysis. In vivo, U251 subcutaneous glioblastoma xenograft was used to assay anti-tumor effect of combinatorial therapy with Ad-PTEN and LY294002 by mean volume of tumors, immunohistochemistry and TUNEL method. The combinatorial treatment clearly suppressed cell proliferation, arrested the cell cycle, reduced cell invasion and promoted cell apoptosis compared with the Ad-PTEN or LY294002 treatment alone. The treatment worked by inhibiting the PI3K/AKT pathway. In addition, the growth of U251 glioma xenografts treated with the combination of Ad-PTEN and LY294002 was significantly inhibited compared with those treated with Ad-PTEN or LY294002 alone. Our data indicated that the combination of Ad-PTEN and LY294002 effectively suppressed the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach for glioma gene therapy that warrants further investigation.

  16. Benefits of gene transduction of granulocyte macrophage colony-stimulating factor in cancer vaccine using genetically modified dendritic cells.

    PubMed

    Ojima, Toshiyasu; Iwahashi, Makoto; Nakamura, Masaki; Matsuda, Kenji; Nakamori, Mikihito; Ueda, Kentaro; Naka, Teiji; Katsuda, Masahiro; Miyazawa, Motoki; Yamaue, Hiroki

    2007-10-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) is a key cytokine for the generation and stimulation of dendritic cells (DCs), and it may also play a pivotal role in promoting the survival of DCs. In this study, the feasibility of creating a cancer vaccine using DCs adenovirally transduced with the carcinoembryonic antigen (CEA) gene and the GM-CSF gene was examined. In addition, the effect of the co-transduction of GM-CSF gene on the lifespan of these genetically modified DCs was determined. A cytotoxic assay using peripheral blood mononuclear cell (PBMC)-derived cytotoxic T lymphocytes (CTLs) was performed in a 4-h 51Cr release assay. The apoptosis of DCs was examined by TdT-mediated dUTP-FITC nick end labeling (TUNEL) assay. CEA-specific CTLs were generated from PBMCs stimulated with genetically modified DCs expressing CEA. The cytotoxicity of these CTLs was augmented by co-transduction of DCs with the GM-CSF gene. Co-transduction of the GM-CSF gene into DCs inhibited apoptosis of these DCs themselves via up-regulation of Bcl-x(L) expression, leading to the extension of the lifespan of these DCs. Furthermore, the transduction of the GM-CSF gene into DCs also suppressed the incidence of apoptosis of DCs induced by transforming growth factor-beta1 (TGFbeta-1). Immunotherapy using these genetically modified DCs may therefore be useful with several advantages as follows: i) adenoviral toxicity to DCs can be reduced; ii) the lifespan of vaccinated DCs can be prolonged; and iii) GM-CSF may protect DCs from apoptosis induced by tumor-derived TGFbeta-1 in the regional lymph nodes.

  17. AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

    PubMed

    Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

    2014-10-30

    For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

    PubMed

    Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

    2011-06-01

    Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

  19. Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

    PubMed Central

    Kilpatrick, Lauren A.; Li, Qian; Yang, John; Goddard, John C; Fekete, Donna M.; Lang, Hainan

    2010-01-01

    Murine models are ideal for studying cochlear gene transfer as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, due to its small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAV) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear and allows for near-complete preservation of low and middle frequency hearing. In the present study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6, and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus (CMV) promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells (IHCs) were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness. PMID:21209625

  20. Carbon Nanotubes in Drug and Gene Delivery

    NASA Astrophysics Data System (ADS)

    Karimi, Mahdi; Ghasemi, Amir; Mirkiani, Soroush; Moosavi Basri, Seyed Masoud; Hamblin, Michael R.

    2017-10-01

    Recent important discoveries and developments in nanotechnology have had a remarkable and ever-increasing impact on many industries, especially materials science, pharmaceuticals, and biotechnology. Within this book, the authors describe different features of carbon nanotubes, survey the properties of both the multi-walled and single-walled varieties, and cover their applications in drug and gene delivery.

  1. Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-gamma-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector.

    PubMed

    Huang, Hongliang; Yu, Hai; Tang, Guping; Wang, Qingqing; Li, Jun

    2010-03-01

    Gene delivery is one of the critical steps for gene therapy. Non-viral vectors have many advantages but suffered from low gene transfection efficiency. Here, in order to develop new polymeric gene vectors with low cytotoxicity and high gene transfection efficiency, we synthesized a cationic polymer composed of low molecular weight polyethylenimine (PEI) of molecular weight of 600 Da cross-linked by 2-hydroxypropyl-gamma-cyclodextrin (HP gamma-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. The oligopeptide can target to HER2, the human epidermal growth factor receptor 2, which is often over expressed in many breast and ovary cancers. The new gene vector was expected to be able to target delivery of genes to HER2 positive cancer cells for gene therapy. The new gene vector was composed of chemically bonded HP gamma-CD, PEI (600 Da), and MC-10 peptide at a molar ratio of 1:3.3:1.2. The gene vector could condense plasmid DNA at an N/P ratio of 6 or above. The particle size of HP gamma-CD-PEI-P/DNA complexes at N/P ratios 40 was around 170-200 nm, with zeta potential of about 20 mV. The gene vector showed very low cytotoxicity, strong targeting specificity to HER2 receptor, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. The delivery of therapeutic IFN-alpha gene mediated by the new gene vector and the therapeutic efficiency were also studied in mice animal model. The animal study results showed that the new gene vector HP gamma-CD-PEI-P significantly enhanced the anti-tumor effect on tumor-bearing nude mice as compared to PEI (25 kDa), HP gamma-CD-PEI, and other controls, indicating that this new polymeric gene vector is a potential candidate for cancer gene therapy. (c) 2009 Elsevier Ltd. All rights reserved.

  2. Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene

    PubMed Central

    George, Marina A.; El-Shorbagy, Haidan M.; Bassiony, Heba; Farroh, Khaled Y.; Youssef, Tareq; Salaheldin, Taher A.

    2017-01-01

    Background Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. Aim of work The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Methods and results Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Conclusion Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV. PMID:28746382

  3. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

    PubMed Central

    Nitta, Sachiko Kaihara; Numata, Keiji

    2013-01-01

    There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin), protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin). The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed. PMID:23344060

  4. Cyclodextrin-cross-linked diaminotriazine-based hydrogen bonding strengthened hydrogels for drug and reverse gene delivery.

    PubMed

    Hu, Xiufeng; Wang, Ning; Liu, Lu; Liu, Wenguang

    2013-01-01

    A hydrogen bonding strengthened hydrogel was prepared by radical copolymerization of poly(ethylene glycol) methacrylated β-cyclodextrin (PEG-β-CD) and 2-vinyl-4,6-diamino-1,3,5-triazine (VDT) monomer. PEG-β-CD served not only as a cross-linker, but also as a built-in solubilizing agent of the hydrophobic drug in the gel. Increasing VDT content resulted in a notable enhancement in the mechanical strengths of hydrogels whose equilibrium water contents could be modulated from 75% to 85% by varying the ratio of PEG-β-CD cross-linker. It was shown that copolymerizing more PEG-β-CDs could load higher amount of ibuprofen (IBU) in the gels and contribute to a slower release rate of IBU. Plasmid DNA could be anchored onto the surface of hydrogels due to the hydrogen bonding between the base pairs and diaminotriazine, thereby mediating efficient reverse gene transfection of luciferase gene in COS-7 cells cultured on the gel surface. The cytocompatible PEG-β-CD-cross-linked PVDT hydrogels with multifunction of drug and gene delivery hold a potential as tissue engineering scaffold.

  5. Focused ultrasound-mediated drug delivery through the blood-brain barrier

    PubMed Central

    Burgess, Alison; Shah, Kairavi; Hough, Olivia; Hynynen, Kullervo

    2015-01-01

    Despite recent advances in blood-brain barrier (BBB) research, it remains a significant hurdle for the pharmaceutical treatment of brain diseases. Focused ultrasound (FUS) is one method to transiently increase permeability of the BBB to promote drug delivery to specific brain regions. An introduction to the BBB and a brief overview of the methods which can be used to circumvent the BBB to promote drug delivery is provided. In particular, we discuss the advantages and limitations of FUS technology and the efficacy of FUS-mediated drug delivery in models of disease. MRI for targeting and evaluating FUS treatments, combined with administration of microbubbles, allows for transient, reproducible BBB opening. The integration of a real-time acoustic feedback controller has improved treatment safety. Successful clinical translation of FUS has the potential to transform the treatment of brain disease worldwide without requiring the development of new pharmaceutical agents. PMID:25936845

  6. Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

    2015-10-01

    Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

  7. Advances in using chitosan-based nanoparticles for in vitro and in vivo drug and gene delivery.

    PubMed

    Duceppe, Nicolas; Tabrizian, Maryam

    2010-10-01

    This review aims to provide an overview of state-of-the-art chitosan-based nanosized carriers for the delivery of therapeutic agents. Chitosan nanocarriers are smart delivery systems owing to the possibility of their property alterations with various approaches, which would confer them with the possibility of spatiotemporal delivery features. The focus of this review is principally on those aspects that have not often been addressed in other reviews. These include the influence of physicochemical properties of chitosan on delivery mechanisms and chitosan modification with a variety of ligand moieties specific for cell surface receptors to increase recognition and uptake of nanocarriers into cells through receptor-mediated endocytosis. Multiple examples that demonstrate the advantages of chitosan-based nanocarriers over other delivery systems of therapeutic agents are highlighted. Particular emphasis is given to the alteration of material properties by functionalization or combination with other polymers for their specific applications. Finally, structural and experimental parameters influencing transfection efficiency of chitosan-based nanocarriers are presented for both in vitro and in vivo gene delivery. The readers will acquire knowledge of parameters influencing the properties of the chitosan-based nanocarriers for delivery of therapeutic agents (genetic material or drugs) in vitro and in vivo. They will get a better idea of the strategies to be adapted to tune the characteristics of chitosan and chitosan derivatives for specific delivery applications. Chitosan is prone to chemical and physical modifications, and is very responsive to environmental stimuli such as temperature and pH. These features make chitosan a smart material with great potential for developing multifunctional nanocarrier systems to deliver large varieties of therapeutic agents administrated in multiple ways with reduced side effects.

  8. 2-Aminoimidazole facilitates efficient gene delivery in a low molecular weight poly(amidoamine) dendrimer.

    PubMed

    Wang, Jing; Hu, Xuefeng; Wang, Dongli; Xie, Cao; Lu, Weiyue; Song, Jie; Wang, Ruifeng; Gao, Chunli; Liu, Min

    2018-06-20

    Functional groups have shown great potential in gene delivery. However, a number of the reported functional groups can only overcome one certain physiological barrier, resulting in limited transfection efficiencies. Based on the structure-activity relationships of both imidazolyl and guanidyl, we designed a novel multifunctional group, 2-aminoimidazole (AM), for gene delivery. On modifying with the AM group, the transfection efficiency of low molecular weight poly(amidoamine) (G2) was 200 times greater than the parent dendrimer in vitro. In contrast, the transfection efficiency of G2 showed a decreasing trend when it was grafted with imidazole. Assays revealed that the AM group played multiple roles in gene delivery, including condensing DNA into monodisperse nanoparticles of 80-90 nm in diameter, achieving nearly ten times higher cellular-uptake efficacy, and enhancing the abilities of endosome/lysosome escape and nuclear localization. What's more, AM showed low toxicity. These results demonstrate that the AM group could be a promising tool in non-viral gene delivery.

  9. A multimodal instrument for real-time in situ study of ultrasound and cavitation mediated drug delivery.

    PubMed

    Bian, Shuning; Seth, Anjali; Daly, Dan; Carlisle, Robert; Stride, Eleanor

    2017-03-01

    The development of a multimodal instrument capable of real-time in situ measurements of cavitation activity and effect in tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments is described here. The instrument features an acoustic arm that can expose phantoms to high-intensity focused-ultrasound while measuring cavitation activity and an optical arm that monitors cavitation effect using confocal microscopy. This combination of modalities allows real-time in situ characterisation of drug delivery in tissue and tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments. A representative result, obtained with a tissue mimicking phantom and acoustically activated droplets, is presented here as a demonstration of the instrument's capabilities and potential applications.

  10. Novel mechanism of JNK pathway activation by adenoviral E1A

    PubMed Central

    Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

    2014-01-01

    The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

  11. Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

    PubMed Central

    Ellis, BL; Hirsch, ML; Porter, SN; Samulski, RJ; Porteus, MH

    2016-01-01

    An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy. PMID:22257934

  12. Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation.

    PubMed

    Tsai, Sen-Wei; Tung, Yu-Tang; Chen, Hsiao-Ling; Yang, Shang-Hsun; Liu, Chia-Yi; Lu, Michelle; Pai, Hui-Jing; Lin, Chi-Chen; Chen, Chuan-Mu

    2016-02-01

    Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (p<0.05). And enhancement of the gene expression of the muscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery.

    PubMed

    Hung, Michelle E; Leonard, Joshua N

    2016-01-01

    Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general biophysical properties impact loading and delivery of RNA by EVs, we developed a platform for actively loading engineered cargo RNAs into EVs. In our system, the MS2 bacteriophage coat protein was fused to EV-associated proteins, and the cognate MS2 stem loop was engineered into cargo RNAs. Using this Targeted and Modular EV Loading (TAMEL) approach, we identified a configuration that substantially enhanced cargo RNA loading (up to 6-fold) into EVs. When applied to vesicles expressing the vesicular stomatitis virus glycoprotein (VSVG) - gesicles - we observed a 40-fold enrichment in cargo RNA loading. While active loading of mRNA-length (>1.5 kb) cargo molecules was possible, active loading was much more efficient for smaller (~0.5 kb) RNA molecules. We next leveraged the TAMEL platform to elucidate the limiting steps in EV-mediated delivery of mRNA and protein to prostate cancer cells, as a model system. Overall, most cargo was rapidly degraded in recipient cells, despite high EV-loading efficiencies and substantial EV uptake by recipient cells. While gesicles were efficiently internalized via a VSVG-mediated mechanism, most cargo molecules were rapidly degraded. Thus, in this model system, inefficient endosomal fusion or escape likely represents a limiting barrier to EV-mediated transfer. Altogether, the TAMEL platform enabled a comparative analysis elucidating a key opportunity for enhancing EV-mediated delivery to prostate cancer cells, and this technology should be of

  14. New Type of BACE1 siRNA Delivery to Cells

    PubMed Central

    Jabłkowski, Maciej; Szemraj, Maciej; Oszajca, Katarzyna; Janiszewska, Grażyna; Bartkowiak, Jacek; Szemraj, Janusz

    2014-01-01

    Background Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. Material/Methods An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, β, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. Results Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP – INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. Conclusions Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity. PMID:25491230

  15. Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

    PubMed

    Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

    2016-09-10

    Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.

    PubMed

    Tong, Yaw-Chong; Chang, Shwu-Fen; Liu, Chia-Yang; Kao, Winston W-Y; Huang, Chong Heng; Liaw, Jiahorng

    2007-11-01

    This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may

  17. Cationic amino acid based lipids as effective nonviral gene delivery vectors for primary cultured neurons.

    PubMed

    Aoshima, Yumiko; Hokama, Ryosuke; Sou, Keitaro; Sarker, Satya Ranjan; Iida, Kabuto; Nakamura, Hideki; Inoue, Takafumi; Takeoka, Shinji

    2013-12-18

    The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

  18. Cationic Amino Acid Based Lipids as Effective Nonviral Gene Delivery Vectors for Primary Cultured Neurons

    PubMed Central

    2013-01-01

    The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl–) and trifluoroacetic acid (TFA–), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA– induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA– showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA– is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity. PMID:24087930

  19. Nanostructure-mediated drug delivery.

    PubMed

    Hughes, Gareth A

    2005-03-01

    Nanotechnology is expected to have an impact on all industries including semiconductors, manufacturing, and biotechnology. Tools that provide the capability to characterize and manipulate materials at the nanoscale level further elucidate nanoscale phenomena and equip researchers and developers with the ability to fabricate novel materials and structures. One of the most promising societal impacts of nanotechnology is in the area of nanomedicine. Personalized health care, rational drug design, and targeted drug delivery are some of the benefits of a nanomedicine-based approach to therapy. This review will focus on the development of nanoscale drug delivery mechanisms. Nanostructured drug carriers allow for the delivery of not only small-molecule drugs but also the delivery of nucleic acids and proteins. Delivery of these molecules to specific areas within the body can be achieved, which will reduce systemic side effects and allow for more efficient use of the drug.

  20. Investigation of the mediating effects of IT governance-value delivery on service quality and ERP performance

    NASA Astrophysics Data System (ADS)

    Tsai, Wen-Hsien; Chou, Yu-Wei; Leu, Jun-Der; Chao Chen, Der; Tsaur, Tsen-Shu

    2015-02-01

    This study aimed to explore the mediating effects of IT governance (ITG)-value delivery in the relationships among the quality of vendor service, the quality of consultant services, ITG-value delivery and enterprise resource planning (ERP) performance. The sampling of this research was acquired from a questionnaire survey concerning ERP implementations in Taiwan. In this survey, 4366 questionnaires were sent to manufacturing and service companies listed in the TOP 5000: The Largest Corporations in Taiwan 2009. The results showed that an ERP system will exhibit a decreased error rate and improved performance if ERP system vendors and consultants provide good service quality. The results also demonstrated that significant relationships exist among the quality of vendor service, the quality of consultant services and value delivery. The contribution of this article is twofold. First, it found that value delivery provides an effective measure of ERP performance under an ITG framework. Second, it provides evidence of the partial mediating effects of value delivery between service quality and ERP performance. In other words, if enterprises want to improve ERP performance, they need to consider factors such as value delivery and the quality of a vendor/consultant's service.

  1. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

    2014-08-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

  2. Advanced drug and gene delivery systems based on functional biodegradable polycarbonates and copolymers.

    PubMed

    Chen, Wei; Meng, Fenghua; Cheng, Ru; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan

    2014-09-28

    Biodegradable polymeric nanocarriers are one of the most promising systems for targeted and controlled drug and gene delivery. They have shown several unique advantages such as excellent biocompatibility, prolonged circulation time, passive tumor targeting via the enhanced permeability and retention (EPR) effect, and degradation in vivo into nontoxic products after completing their tasks. The current biodegradable drug and gene delivery systems exhibit, however, typically low in vivo therapeutic efficacy, due to issues of low loading capacity, inadequate in vivo stability, premature cargo release, poor uptake by target cells, and slow release of therapeutics inside tumor cells. To overcome these problems, a variety of advanced drug and gene delivery systems has recently been designed and developed based on functional biodegradable polycarbonates and copolymers. Notably, polycarbonates and copolymers with diverse functionalities such as hydroxyl, carboxyl, amine, alkene, alkyne, halogen, azido, acryloyl, vinyl sulfone, pyridyldisulfide, and saccharide, could be readily obtained by controlled ring-opening polymerization. In this paper, we give an overview on design concepts and recent developments of functional polycarbonate-based nanocarriers including stimuli-sensitive, photo-crosslinkable, or active targeting polymeric micelles, polymersomes and polyplexes for enhanced drug and gene delivery in vitro and in vivo. These multifunctional biodegradable nanosystems might be eventually developed for safe and efficient cancer chemotherapy and gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Efficient Receptor Mediated siRNA Delivery in Vitro by Folic Acid Targeted Pentablock Copolymer-Based Micelleplexes.

    PubMed

    Lehner, Roman; Liu, Kegang; Wang, Xueya; Hunziker, Patrick

    2017-08-14

    Novel, biocompatible polyplexes, based on the combination of cationic pentablock copolymers with folic acid functionalized copolymers, were designed and developed for target-specific siRNA delivery. The resulting micelleplexes spontaneously formed polymeric micelles with a hydrophobic core surrounded directly by a cationic poly-2-(4-aminobutyl)-oxazole (PABOXA) and subsequently shielded by hydrophilic poly-2-methyl-oxazole (PMOXA) layer. The described micelleplexes form highly stable particles even in complete serum after 24 h compared with the highly cationic polymer PEI, which show aggregate formation in serum containing buffer solution. Targeted siRNA delivery and gene knockdown could be shown using green fluorescent protein (GFP) expressing HeLa cells, resulting in ∼31% and ∼8% suppression of the expression of GFP for targeted and nontargeted micelleplexes, respectively. Comparison studies of folic-receptor positive HeLa cells with normal folic-receptor-negative HEK293 cells revealed involvement of receptor mediated cellular uptake of fluorescently labeled siRNA. The new designed nanocarrier showed no cytotoxicity, having a potential application. The presented concept of shielding a nucleic-acid complexing cationic chains with a stealth layer and combining it with receptor ligand overcomes typical problems with undesired protein and cell interactions in delivery of nucleic acids using polymeric systems, opening new doors for application if RNA inhibition in the organism.

  4. Poly (amidoamine) (PAMAM) dendrimer mediated delivery of drug and pDNA/siRNA for cancer therapy.

    PubMed

    Li, Jun; Liang, Huamin; Liu, Jing; Wang, Ziyuan

    2018-07-30

    Poly (amidoamine) (PAMAM) dendrimers are well-defined, highly branched macromolecules with numerous active amine groups on the surface. Because of their unique properties, PAMAM dendrimers have steadily grown in popularity in drug delivery, gene therapy, medical imaging and diagnostic application. This review focuses on the recent developments on the application in PAMAM dendrimers as effective carriers for drug and gene (pDNA, siRNA) delivery in cancer therapy, including: a) PAMAM for anticancer drug delivery; b) PAMAM and gene therapy; c) PAMAM used in overcoming tumor multidrug resistance; d) PAMAM used for hybrid nanoparticles; and e) PAMAM linked or loaded in other nanoparticles. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Plant transformation via pollen tube-mediated gene transfer

    USDA-ARS?s Scientific Manuscript database

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  6. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    NASA Astrophysics Data System (ADS)

    Zhang, Zubin; Song, Lina; Dong, Jinlai; Guo, Dawei; Du, Xiaolin; Cao, Biyin; Zhang, Yu; Gu, Ning; Mao, Xinliang

    2013-05-01

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  7. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180; Feril, Loreto B., E-mail: ferilism@yahoo.com

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA,more » which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.« less

  8. PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy Treatment.

    PubMed

    Negishi, Yoichi; Ishii, Yuko; Nirasawa, Kei; Sasaki, Eri; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo

    2018-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

  9. Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours

    NASA Astrophysics Data System (ADS)

    Martirosyan, A.; Olesen, M. J.; Fenton, R. A.; Kjems, J.; Howard, K. A.

    2016-06-01

    This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal

  10. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

    PubMed Central

    Seandel, Marco; Butler, Jason M.; Kobayashi, Hideki; Hooper, Andrea T.; White, Ian A.; Zhang, Fan; Vertes, Eva L.; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V.; Hackett, Neil R.; Rabbany, Sina; Boyer, Julie L.; Rafii, Shahin

    2008-01-01

    Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1+ ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1+ ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1+ ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1+ ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1+ ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-κB pathways. Therefore, E4ORF1+ ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis. PMID:19036927

  11. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

    PubMed

    Seandel, Marco; Butler, Jason M; Kobayashi, Hideki; Hooper, Andrea T; White, Ian A; Zhang, Fan; Vertes, Eva L; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V; Hackett, Neil R; Rabbany, Sina; Boyer, Julie L; Rafii, Shahin

    2008-12-09

    Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

  12. p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

    PubMed Central

    Ferrándiz, Nuria; Caraballo, Juan M.; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M. Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M. Jose; Blanco, Rosa; Reyes, Jose C.; Agell, Neus; Delgado, M. Dolores; Dotto, G. Paolo; León, Javier

    2012-01-01

    It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

  13. Adenovirus‑mediated overexpression of cystic fibrosis transmembrane conductance regulator enhances invasiveness and motility of serous ovarian cancer cells.

    PubMed

    Xu, Jiao; Lin, Liangbo; Yong, Min; Dong, Xiaojing; Yu, Tinghe; Hu, Lina

    2016-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the adenosine triphosphate‑binding cassette transporter family, members of which are involved in several types of cancer. Previous studies by our group reported that CFTR was highly expressed in serous ovarian cancer (SOC) tissues, and that knockdown of CFTR suppressed the proliferation of ovarian cancer in vitro and in vivo. Thus, the aim of the present study was to construct a recombinant adenoviral vector for the expression of the human CFTR gene in order to study the role of CFTR overexpression in the malignant invasion and migration of SOC cells in vitro. The present study then focused on the mechanisms of the role of CFTR in the migratory and invasive malignant properties of SOC cells. The CFTR gene was inserted into an adenoviral vector by using the AdEasy system in order to obtain the Ad‑CFTR overexpression vector, which was used to transfect the A2780 SOC cell line. Reverse-transcription polymerase chain reaction, western blot analysis and immunofluorescence were performed to detect the expression and localization of CFTR. Cell invasion and motility of the transfected cells compared with those of control cells were observed using Transwell and wound healing assays. A ~4,700 bp fragment of the CFTR gene was confirmed to be correctly cloned in the adenoviral vector and amplification of Ad‑CFTR was observed in HEK293 cells during package. After 48 h of transfection with Ad‑CFTR, ~90% of A2780 cells were red fluorescence protein‑positive. Immunofluorescence showed that following transfection, CFTR expression was increased and CFTR was located in the cell membrane and cytoplasm. CFTR overexpression was shown to enhance the invasion and motility of A2780 cells in vitro. Furthermore, the effects of CFTR overexpression on the activation c‑Src signaling were observed by western blot analysis. CFTR overexpressing cells showed the lowest activity of phospho‑Src (Tyr530

  14. Lipid nanoparticles as drug/gene delivery systems to the retina.

    PubMed

    del Pozo-Rodríguez, Ana; Delgado, Diego; Gascón, Alicia R; Solinís, Maria Ángeles

    2013-03-01

    This review highlights the application of lipid nanoparticles (Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, or Lipid Drug Conjugates) as effective drug/gene delivery systems for retinal diseases. Most drug products for ocular disease treatment are marketed as eye drop formulations but, due to ocular barriers, the drug concentration in the retina hardly ever turns out to be effective. Up to this date, several delivery systems have been designed to deliver drugs to the retina. Drug delivery strategies may be classified into 3 groups: noninvasive techniques, implants, and colloidal carriers. The best known systems for drug delivery to the posterior eye are intravitreal implants; in fact, some of them are being clinically used. However, their long-term accumulation might impact the patient's vision. On the contrary, colloidal drug delivery systems (microparticles, liposomes, or nanoparticles) can be easily administered in a liquid form. Nanoparticular systems diffuse rapidly and are better internalized in ocular tissues than microparticles. In comparison with liposomes, nanoparticles have a higher loading capacity and are more stable in biological fluids and during storage. In addition, their capacity to adhere to the ocular surface and interact with the endothelium makes these drug delivery systems interesting as new therapeutic tools in ophthalmology. Within the group of nanoparticles, those composed of lipids (Solid Lipid Nanoparticles, Nanostructred Lipid Carriers, and Lipid Drug Conjugates) are more biocompatible, easy to produce at large scale, and they may be autoclaved or sterilized. The present review summarizes scientific results that evidence the potential application of lipid nanoparticles as drug delivery systems for the retina and also as nonviral vectors in gene therapy of retina disorders, although much more effort is still needed before these lipidic systems could be available in the market.

  15. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  16. Neurotrophin gene therapy for sustained neural preservation after deafness.

    PubMed

    Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Hume, Clifford R; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2012-01-01

    The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

  17. Development of a custom biological scaffold for investigating ultrasound-mediated intracellular delivery.

    PubMed

    Bui, Loan; Aleid, Adham; Alassaf, Ahmad; Wilson, Otto C; Raub, Christopher B; Frenkel, Victor

    2017-01-01

    In vitro investigations of ultrasound mediated, intracellular drug and gene delivery (i.e. sonoporation) are typically carried out in cells cultured in standard plastic well plates. This creates conditions that poorly resemble in vivo conditions, as well as generating unwanted ultrasound phenomena that may confound the interpretation of results. Here, we present our results in the development of a biological scaffold for sonoporation studies. The scaffolds were comprised of cellulose fibers coated with chitosan and gelatin. Scaffold formulation was optimized for adherence and proliferation of mouse fibroblasts in terms of the ratio and relative concentration of the two constituents. The scaffolds were also shown to significantly reduce ultrasound reflections compared to the plastic well plates. A custom treatment chamber was designed and built, and the occurrence of acoustic cavitation in the chamber during the ultrasound treatments was detected; a requirement for the process of sonoporation. Finally, experiments were carried out to optimize the ultrasound exposures to minimize cellular damage. Ultrasound exposure was then shown to enable the uptake of 100nm fluorescently labeled polystyrene nanoparticles in suspension into the cells seeded on scaffolds, compared to incubation of cell-seeded scaffolds with nanoparticles alone. These preliminary results set the basis for further development of this platform. They also provide motivation for the development of similar platforms for the controlled investigation of other ultrasound mediated cell and tissue therapies. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  19. Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

    PubMed Central

    Nande, Rounak; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D.; Denvir, Jim; Valluri, Jagan; Duncan, Gary C.; Claudio, Pier Paolo

    2012-01-01

    Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas. PMID:22666387

  20. Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

    PubMed

    Nande, Rounak; Di Benedetto, Altomare; Aimola, Pierpaolo; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D; Denvir, Jim; Valluri, Jagan; Duncan, Gary C; Claudio, Pier Paolo

    2012-01-01

    Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

  1. Strategies to improve drug delivery across the blood-brain barrier.

    PubMed

    de Boer, Albertus G; Gaillard, Pieter J

    2007-01-01

    The blood-brain barrier (BBB), together with the blood-cerebrospinal-fluid barrier, protects and regulates the homeostasis of the brain. However, these barriers also limit the transport of small-molecule and, particularly, biopharmaceutical drugs such as proteins, genes and interference RNA to the brain, thereby limiting the treatment of many brain diseases. As a result, various drug delivery and targeting strategies are currently being developed to enhance the transport and distribution of drugs into the brain. In this review, we discuss briefly the biology and physiology of the BBB as the most important barrier for drug transport to the brain and, in more detail, the possibilities for delivering large-molecule drugs, particularly genes, by receptor-mediated nonviral drug delivery to the (human) brain. In addition, the systemic and intracellular pharmacokinetics of nonviral gene delivery, together with targeted brain imaging, are reviewed briefly.

  2. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    PubMed

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  3. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  4. Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells.

    PubMed

    Huang, Qing-Dong; Zhong, Guo-Xing; Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; Zhu, Wen; Yu, Xiao-Qi

    2011-01-01

    Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. In this report, we designed and synthesized three amphiphilic molecules (L1-L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1-L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.

  5. Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

    PubMed

    Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

    2017-07-01

    Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

  6. Intranasal gene delivery for treating Parkinson's disease: overcoming the blood-brain barrier.

    PubMed

    Aly, Amirah E-E; Waszczak, Barbara L

    2015-01-01

    Developing a disease-modifying gene therapy for Parkinson's disease (PD) has been a high priority for over a decade. However, due to the inability of large biomolecules to cross the blood-brain barrier (BBB), the only means of delivery to the brain has been intracerebral infusion. Intranasal administration offers a non-surgical means of bypassing the BBB to deliver neurotrophic factors, and the genes encoding them, directly to the brain. This review summarizes: i) evidence demonstrating intranasal delivery to the brain of a number of biomolecules having therapeutic potential for various CNS disorders; and ii) evidence demonstrating neuroprotective efficacy of a subset of biomolecules specifically for PD. The intersection of these two spheres represents the area of opportunity for development of new intranasal gene therapies for PD. To that end, our laboratory showed that intranasal administration of glial cell line-derived neurotrophic factor (GDNF), or plasmid DNA nanoparticles encoding GDNF, provides neuroprotection in a rat model of PD, and that the cells transfected by the nanoparticle vector are likely to be pericytes. A number of genes encoding neurotrophic factors have therapeutic potential for PD, but few have been tested by the intranasal route and shown to be neuroprotective in a model of PD. Intranasal delivery provides a largely unexplored, promising approach for development of a non-invasive gene therapy for PD.

  7. A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

    PubMed

    Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

    2009-08-01

    Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

  8. Design and Fabrication of N-Alkyl-Polyethylenimine-Stabilized Iron Oxide Nanoclusters for Gene Delivery

    PubMed Central

    Liu, Gang; Wang, Zhiyong; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

    2013-01-01

    With the rapid development of nanotechnology, inorganic magnetic nanoparticles, especially iron oxide nanoparticles (IOs), have emerged as great vehicles for biomedical diagnostic and therapeutic applications. In order to rationally design IO-based gene delivery nanovectors, surface modification is essential and determines the loading and release of the gene of interest. Here we highlight the basic concepts and applications of nonviral gene delivery vehicles based on low molecular weight N-alkyl polyethylenimine-stabilized IOs. The experimental protocols related to these topics are described in this chapter. PMID:22568910

  9. The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF(165).

    PubMed

    Keeney, Michael; van den Beucken, Jeroen J J P; van der Kraan, Peter M; Jansen, John A; Pandit, Abhay

    2010-04-01

    Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165). 2009 Elsevier Ltd. All rights reserved.

  10. Water insoluble and soluble lipids for gene delivery.

    PubMed

    Mahato, Ram I

    2005-04-05

    Among various synthetic gene carriers currently in use, liposomes composed of cationic lipids and co-lipids remain the most efficient transfection reagents. Physicochemical properties of lipid/plasmid complexes, such as cationic lipid structure, cationic lipid to co-lipid ratio, charge ratio, particle size and zeta potential have significant influence on gene expression and biodistribution. However, most cationic lipids are toxic and cationic liposomes/plasmid complexes do not disperse well inside the target tissues because of their large particle size. To overcome the problems associated with cationic lipids, we designed water soluble lipopolymers for gene delivery to various cells and tissues. This review provides a critical discussion on how the components of water insoluble and soluble lipids affect their transfection efficiency and biodistribution of lipid/plasmid complexes.

  11. Virus-mimetic polyplex particles for systemic and inflammation-specific targeted delivery of large genetic contents.

    PubMed

    Kang, S; Lu, K; Leelawattanachai, J; Hu, X; Park, S; Park, T; Min, I M; Jin, M M

    2013-11-01

    Systemic and target-specific delivery of large genetic contents has been difficult to achieve. Although viruses effortlessly deliver kilobase-long genome into cells, its clinical use has been hindered by serious safety concerns and the mismatch between native tropisms and desired targets. Nonviral vectors, in contrast, are limited by low gene transfer efficiency and inherent cytotoxicity. Here we devised virus-mimetic polyplex particles (VMPs) based on electrostatic self-assembly among polyanionic peptide (PAP), cationic polymer polyethyleneimine (PEI) and nucleic acids. We fused PAP to the engineered ligand-binding domain of integrin αLβ2 to target intercellular adhesion molecule-1 (ICAM-1), an inducible marker of inflammation. Fully assembled VMPs packaged large genetic contents, bound specifically to target molecules, elicited receptor-mediated endocytosis and escaped endosomal pathway, resembling intracellular delivery processes of viruses. Unlike conventional PEI-mediated transfection, molecular interaction-dependent gene delivery of VMPs was unaffected by the presence of serum and achieved higher efficiency without toxicity. By targeting overexpressed ICAM-1, VMPs delivered genes specifically to inflamed endothelial cells and macrophages both in vitro and in vivo. Simplicity and versatility of the platform and inflammation-specific delivery may open up opportunities for multifaceted gene therapy that can be translated into the clinic and treat a broad range of debilitating immune and inflammatory diseases.

  12. Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound.

    PubMed

    Stavarache, Mihaela A; Petersen, Nicholas; Jurgens, Eric M; Milstein, Elizabeth R; Rosenfeld, Zachary B; Ballon, Douglas J; Kaplitt, Michael G

    2018-04-27

    OBJECTIVE Surgical infusion of gene therapy vectors has provided opportunities for biological manipulation of specific brain circuits in both animal models and human patients. Transient focal opening of the blood-brain barrier (BBB) by MR-guided focused ultrasound (MRgFUS) raises the possibility of noninvasive CNS gene therapy to target precise brain regions. However, variable efficiency and short follow-up of studies to date, along with recent suggestions of the potential for immune reactions following MRgFUS BBB disruption, all raise questions regarding the viability of this approach for clinical translation. The objective of the current study was to evaluate the efficiency, safety, and long-term stability of MRgFUS-mediated noninvasive gene therapy in the mammalian brain. METHODS Focused ultrasound under the control of MRI, in combination with microbubbles consisting of albumin-coated gas microspheres, was applied to rat striatum, followed by intravenous infusion of an adeno-associated virus serotype 1/2 (AAV1/2) vector expressing green fluorescent protein (GFP) as a marker. Following recovery, animals were followed from several hours up to 15 months. Immunostaining for GFP quantified transduction efficiency and stability of expression. Quantification of neuronal markers was used to determine histological safety over time, while inflammatory markers were examined for evidence of immune responses. RESULTS Transitory disruption of the BBB by MRgFUS resulted in efficient delivery of the AAV1/2 vector to the targeted rodent striatum, with 50%-75% of striatal neurons transduced on average. GFP transgene expression appeared to be stable over extended periods of time, from 2 weeks to 6 months, with evidence of ongoing stable expression as long as 16 months in a smaller cohort of animals. No evidence of substantial toxicity, tissue injury, or neuronal loss was observed. While transient inflammation from BBB disruption alone was noted for the first few days, consistent

  13. Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

    PubMed

    Kim, Ha; Sin, Jeong-Im

    2012-11-01

    Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.

  14. Type I IFN gene delivery suppresses regulatory T cells within tumors.

    PubMed

    Hashimoto, H; Ueda, R; Narumi, K; Heike, Y; Yoshida, T; Aoki, K

    2014-12-01

    Type I interferon (IFN) is a pleiotropic cytokine regulating the cancer cell death and immune response. IFN-α can, as we have also reported, effectively induce an antitumor immunity by the activation of tumor-specific T cells and maturation of dendritic cells in various animal models. Unknown, however, is how the type I IFN alters the immunotolerant microenvironment in the tumors. Here, we found that intratumoral IFN-α gene transfer significantly decreased the frequency of regulatory T cells (Tregs) per CD4(+) T cells in tumors. The concentration of a Treg-inhibitory cytokine, interleukin (IL)-6, was correlated with the IFN-α expression level in tumors, and intratumoral CD11c(+) cells produced IL-6 in response to IFN-α stimulation. To confirm the role of IL-6 in the suppression of Tregs in tumors, an anti-IL-6 receptor antibody was administered in IFN-α-treated mice. The antibody increased the frequency of Tregs in the tumors, and attenuated systemic tumor-specific immunity induced by IFN-α. Furthermore, the IFN-α-mediated IL-6 production increased the frequency of Th17 cells in the tumors, which may be one of the mechanisms for the reduction of Tregs. The study demonstrated that IFN-α gene delivery creates an environment strongly supporting the enhancement of antitumor immunity through the suppression of Tregs.

  15. Design and fabrication of N-alkyl-polyethylenimine-stabilized iron oxide nanoclusters for gene delivery.

    PubMed

    Liu, Gang; Wang, Zhiyong; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

    2012-01-01

    With the rapid development of nanotechnology, inorganic magnetic nanoparticles, especially iron oxide nanoparticles (IOs), have emerged as great vehicles for biomedical diagnostic and therapeutic applications. In order to rationally design IO-based gene delivery nanovectors, surface modification is essential and determines the loading and release of the gene of interest. Here we highlight the basic concepts and applications of nonviral gene delivery vehicles based on low molecular weight N-alkyl polyethylenimine-stabilized IOs. The experimental protocols related to these topics are described in this chapter. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    PubMed

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5–based Constructs

    PubMed Central

    Alonso-Padilla, Julio; Papp, Tibor; Kaján, Győző L; Benkő, Mária; Havenga, Menzo; Lemckert, Angelique; Harrach, Balázs; Baker, Andrew H

    2016-01-01

    Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes. PMID:26478249

  18. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    PubMed

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  19. Virally mediated gene manipulation in the adult CNS

    PubMed Central

    Edry, Efrat; Lamprecht, Raphael; Wagner, Shlomo; Rosenblum, Kobi

    2011-01-01

    Understanding how the CNS functions poses one of the greatest challenges in modern life science and medicine. Studying the brain is especially challenging because of its complexity, the heterogeneity of its cellular composition, and the substantial changes it undergoes throughout its life-span. The complexity of adult brain neural networks results also from the diversity of properties and functions of neuronal cells, governed, inter alia, by temporally and spatially differential expression of proteins in mammalian brain cell populations. Hence, research into the biology of CNS activity and its implications to human and animal behavior must use novel scientific tools. One source of such tools is the field of molecular genetics—recently utilized more and more frequently in neuroscience research. Transgenic approaches in general, and gene targeting in rodents have become fundamental tools for elucidating gene function in the CNS. Although spectacular progress has been achieved over recent decades by using these approaches, it is important to note that they face a number of restrictions. One of the main challenges is presented by the temporal and spatial regulation of introduced genetic manipulations. Viral vectors provide an alternative approach to temporally regulated, localized delivery of genetic modifications into neurons. In this review we describe available technologies for gene transfer into the adult mammalian CNS that use both viral and non-viral tools. We discuss viral vectors frequently used in neuroscience, with emphasis on lentiviral vector (LV) systems. We consider adverse effects of LVs, and the use of LVs for temporally and spatially controllable manipulations. Especially, we highlight the significance of viral vector-mediated genetic manipulations in studying learning and memory processes, and how they may be effectively used to separate out the various phases of learning: acquisition, consolidation, retrieval, and maintenance. PMID:22207836

  20. Hydrodynamic gene delivery in human skin using a hollow microneedle device.

    PubMed

    Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

    2017-11-10

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Cytosolic co-delivery of miRNA-34a and docetaxel with core-shell nanocarriers via caveolae-mediated pathway for the treatment of metastatic breast cancer

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Yang, Xin; Lv, Yaqi; Xin, Xiaofei; Qin, Chao; Han, Xiaopeng; Yang, Lei; He, Wei; Yin, Lifang

    2017-04-01

    Co-delivery of microRNAs and chemotherapeutic drugs into tumor cells is an attractive strategy for synergetic breast cancer therapy due to their complementary mechanisms. In this work, a core-shell nanocarrier coated by cationic albumin was developed to simultaneously deliver miRNA-34a and docetaxel (DTX) into breast cancer cells for improved therapeutic effect. The co-delivery nanocarriers showed a spherical morphology with an average particle size of 183.9 nm, and they efficiently protected miRNA-34a from degradation by RNase and serum. Importantly, the nanocarriers entered the cytosol via a caveolae-mediated pathway without entrapment in endosomes/lysosomes, thus improving the utilization of the cargo. In vitro, the co-delivery nanocarriers suppressed the expression of anti-apoptosis gene Bcl-2 at both transcription and protein levels, inhibited tumor cell migration and efficiently induced cell apoptosis and cytotoxicity. In vivo, the co-delivery nanocarriers prolonged the blood circulation of DTX, enhanced tumor accumulation of the cargo and significantly inhibited tumor growth and metastasis in 4T1-tumor bearing mice models. Taken together, the present nanocarrier co-loading with DTX and miRNA-34a is a new nanoplatform for the combination of insoluble drugs and gene/protein drugs and provides a promising strategy for the treatment of metastatic breast cancer.

  2. scAAV-Mediated IL-1Ra gene delivery for the Treatment of Osteoarthritis: Test of Efficacy in an Equine Model.

    PubMed

    Watson Levings, Rachael; Smith, Andrew D; Broome, Ted A; Rice, Brett L; Gibbs, Eric P; Myara, D Alex; Hyddmark, Viktoria; Nasri, Elham; Zarezadeh, Ali; Levings, Padraic P; Lu, Yuan; Dacanay, E Anthony; Foremny, Gregory B; Evans, Christopher H; Morton, Alison J; Winter, Mathew; Dark, Michael J; Nickerson, David M; Colahan, Patrick T; Ghivizzani, Steven Craig

    2018-06-05

    We are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of IL-1Ra, and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene-transfer on a scale proportional to the human knee, a frequent site of OA incidence, we focused our studies on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, we previously identified a functional ceiling dose of ~5 x 1012 viral genomes, which elevated the steady state levels of eqIL-1Ra in synovial fluids by more than 40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, we examined the functional capacity of scAAV.IL-1Ra gene-delivery in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several-fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by > 400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and ~25% improvement in total joint pathology by both MRI and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively our studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

  3. Viral Vectors for Gene Delivery to the Central Nervous System

    PubMed Central

    Lentz, Thomas B.; Gray, Steven J.; Samulski, R. Jude

    2011-01-01

    The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features. PMID:22001604

  4. Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

    PubMed

    Priya, S S; Rekha, M R; Sharma, Chandra P

    2014-02-15

    Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET.

    PubMed

    Watanabe, Yukiko; Horie, Sachiko; Funaki, Yoshihito; Kikuchi, Youhei; Yamazaki, Hiromichi; Ishii, Keizo; Mori, Shiro; Vassaux, Georges; Kodama, Tetsuya

    2010-06-01

    The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging. Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy). Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice. The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The

  6. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

    PubMed

    Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

    2005-05-01

    Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

  7. Challenges in carrier-mediated intracellular delivery: moving beyond endosomal barriers.

    PubMed

    Stewart, Martin P; Lorenz, Anna; Dahlman, James; Sahay, Gaurav

    2016-05-01

    The deployment of molecular to microscale carriers for intracellular delivery has tremendous potential for biology and medicine, especially for in vivo therapies. The field remains limited, however, by a poor understanding of how carriers gain access to the cell interior. In this review, we provide an overview of the different types of carriers, their speculated modes of entry, putative pathways of vesicular transport, and sites of endosomal escape. We compare this alongside pertinent examples from the cell biology of how viruses, bacteria, and their effectors enter cells and escape endosomal confinement. We anticipate insights into the mechanisms of cellular entry and endosomal escape will benefit future research efforts on effective carrier-mediated intracellular delivery. WIREs Nanomed Nanobiotechnol 2016, 8:465-478. doi: 10.1002/wnan.1377 For further resources related to this article, please visit the WIREs website. © 2015 Wiley Periodicals, Inc.

  8. Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

    PubMed

    Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

    2014-06-01

    Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

  9. Controlled Striatal DOPA Production From a Gene Delivery System in a Rodent Model of Parkinson's Disease.

    PubMed

    Cederfjäll, Erik; Broom, Lauren; Kirik, Deniz

    2015-05-01

    Conventional symptomatic treatment for Parkinson's disease (PD) with long-term L-3,4-dihydroxyphenylalanine (DOPA) is complicated with development of drug-induced side effects. In vivo viral vector-mediated gene expression encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) provides a drug delivery strategy of DOPA with distinct advantages over pharmacotherapy. Since the brain alterations made with current gene transfer techniques are irreversible, the therapeutic approaches taken to the clinic should preferably be controllable to match the needs of each individual during the course of their disease. We used a recently described tunable gene expression system based on the use of destabilized dihydrofolate reductase (DD) and generated a N-terminally coupled GCH1 enzyme (DD-GCH1) while the TH enzyme was constitutively expressed, packaged in adeno-associated viral (AAV) vectors. Expression of DD-GCH1 was regulated by the activating ligand trimethoprim (TMP) that crosses the blood-brain barrier. We show that the resulting intervention provides a TMP-dose-dependent regulation of DOPA synthesis that is closely linked to the magnitude of functional effects. Our data constitutes the first proof of principle for controlled reconstitution of dopamine capacity in the brain and suggests that such next-generation gene therapy strategies are now mature for preclinical development toward use in patients with PD.

  10. The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

    PubMed

    Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

    2011-05-01

    Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

    NASA Astrophysics Data System (ADS)

    Wu, Lijuan; Wu, Changlin; Liu, Guangwan; Liao, Nannan; Zhao, Fang; Yang, Xuxia; Qu, Hongyuan; Peng, Bo; Chen, Li; Yang, Guang

    2016-12-01

    siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, 13C NMR (CP/MAS), UV-vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV-vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

  12. Dendrimer-coupled sonophoresis-mediated transdermal drug-delivery system for diclofenac.

    PubMed

    Huang, Bin; Dong, Wei-Jiang; Yang, Gao-Yi; Wang, Wei; Ji, Cong-Hua; Zhou, Fei-Ni

    2015-01-01

    The purpose of the present study was to develop a novel transdermal drug-delivery system comprising a polyamidoamine dendrimer coupled with sonophoresis to enhance the permeation of diclofenac (DF) through the skin. The novel transdermal drug-delivery system was developed by using a statistical Plackett-Burman design. Hairless male Wistar rat skin was used for the DF-permeation study. Coupling media concentration, ultrasound-application time, duty cycle, distance from probe to skin, and a third-generation polyamidoamine-dendrimer concentration were selected as independent variables, while in vitro drug release was selected as a dependent variable. Independent variables were found to be statistically significant (P<0.05). DF gel without dendrimer and ultrasound treatment to skin (passive delivery, run 13) showed 56.69 µg/cm(2) cumulative drug permeated through the skin, while the DF-dendrimer gel without sonophoresis treatment (run 14) showed 257.3 µg/cm(2) cumulative drug permeated through the skin after 24 hours. However, when the same gel was applied to sonophoresis-treated skin, drastic permeation enhancement was observed. In the case of run 3, the cumulative drug that permeated through the skin was 935.21 µg/cm(2). It was concluded that dendrimer-coupled sonophoresis-mediated transdermal drug delivery system has the potential to enhance the permeation of DF through the skin.

  13. Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian

    2014-06-01

    Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

  14. The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

    PubMed

    Li, D; Kong, Y; Yu, H; Lehtinen, A; Huang, H; Shen, F; Min, L; Zhou, J; Tang, G; Wang, Q

    2008-04-01

    A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

  15. Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

    PubMed

    Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

    2015-12-28

    Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery.

    PubMed

    Molla, Mijanur R; Böser, Alexander; Rana, Akshita; Schwarz, Karina; Levkin, Pavel A

    2018-04-18

    Efficient delivery of nucleic acids into cells is of great interest in the field of cell biology and gene therapy. Despite a lot of research, transfection efficiency and structural diversity of gene-delivery vectors are still limited. A better understanding of the structure-function relationship of gene delivery vectors is also essential for the design of novel and intelligent delivery vectors, efficient in "difficult-to-transfect" cells and in vivo clinical applications. Most of the existing strategies for the synthesis of gene-delivery vectors require multiple steps and lengthy procedures. Here, we demonstrate a facile, three-component one-pot synthesis of a combinatorial library of 288 structurally diverse lipid-like molecules termed "lipidoids" via a thiolactone ring opening reaction. This strategy introduces the possibility to synthesize lipidoids with hydrophobic tails containing both unsaturated bonds and reducible disulfide groups. The whole synthesis and purification are convenient, extremely fast, and can be accomplished within a few hours. Screening of the produced lipidoids using HEK293T cells without addition of helper lipids resulted in identification of highly stable liposomes demonstrating ∼95% transfection efficiency with low toxicity.

  17. Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

    PubMed Central

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-01-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

  18. Targeted Gene Transfer to the Brain via the Delivery of Brain-Penetrating DNA Nanoparticles with Focused Ultrasound

    PubMed Central

    Mead, Brian P.; Mastorakos, Panagiotis; Suk, Jung Soo; Klibanov, Alexander L.; Hanes, Justin; Price, Richard J.

    2016-01-01

    Gene therapy holds promise for the treatment of many pathologies of the central nervous system (CNS), including brain tumors and neurodegenerative diseases. However, the delivery of systemically administered gene carriers to the CNS is hindered by both the blood-brain barrier (BBB) and the nanoporous and electrostatically charged brain extracelluar matrix (ECM), which acts as a steric and adhesive barrier. We have previously shown that these physiological barriers may be overcome by, respectively, opening the BBB with MR image-guided focused ultrasound (FUS) and microbubbles and using highly compact “brain penetrating” nanoparticles (BPN) coated with a dense polyethylene glycol corona that prevents adhesion to ECM components. Here, we tested whether this combined approach could be utilized to deliver systemically administered DNA-bearing BPN (DNA-BPN) across the BBB and mediate localized, robust, and sustained transgene expression in the rat brain. Systemically administered DNA-BPN delivered through the BBB with FUS led to dose-dependent transgene expression only in the FUS-treated region that was evident as early as 24 h post administration and lasted for at least 28 days. In the FUS-treated region ~42% of all cells, including neurons and astrocytes, were transfected, while less than 6% were transfected in the contralateral non-FUS treated hemisphere. Importantly, this was achieved without any sign of toxicity or astrocyte activation. We conclude that the image-guided delivery of DNA-BPN with FUS and microbubbles constitutes a safe and non-invasive strategy for targeted gene therapy to the brain. PMID:26732553

  19. Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery.

    PubMed

    Yang, Xinchao; Zhao, Nana; Xu, Fu-Jian

    2014-06-07

    Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

  20. Gene therapy to develop a genetically engineered cardiac pacemaker.

    PubMed

    Glenn, Christopher M; Pogwizd, Steven M

    2003-01-01

    While cardiac pacemakers are frequently used for the treatment of bradydysrhythmias (from diseases of the cardiac conduction system), their use is still limited by complications that can be life-threatening and expensive. Genetic engineering approaches offer an opportunity to modulate cellular automaticity in a manner that could have significant therapeutic potential. It is well known that ventricular myocytes exhibit a more negative diastolic potential than do pacemaker cells, in large part because of the inward rectifying potassium current/K1 (which pacemaker cells lack). Taking advantage of these intrinsic electrophysiological differences, a biological pacemaker has recently been developed by Miake et al (Nature 2002; 419:132-133) using adenoviral gene transfer approaches. By isolating the gene responsible for/K1 (the Kir2.1 gene), mutating it to make it a dysfunctional channel (a dominant-negative), inserting the mutated gene into an adenoviral vector, and delivering the virus to the hearts of guinea pigs, the investigators were able to successfully convert some ventricular myocytes to pacemaker cells. While issues of safety and long-term efficacy need to be further established, the results of these experiments provide proof of principle that gene transfer offers great promise for treatment of electrophysiological disorders including conduction system disease.