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Sample records for adenovirus gene delivery

  1. 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

  2. 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

  3. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  4. Gene Delivery by Subconjunctival Injection of Adenovirus in Rats: A Study of Local Distribution, Transgene Duration and Safety

    PubMed Central

    Lee, Jia Hui; Tsai, Pei-Jhen; Tsai, Han-En; Sheu, Shwu-Jiuan; Lin, Hsiu-Chen; Dusting, Gregory J.; Tai, Ming-Hong; Bee, Youn-Shen

    2015-01-01

    Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, β-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short

  5. Functional characterization of a PEI-CyD-FA-coated adenovirus as delivery vector for gene therapy.

    PubMed

    Yao, Hong; Chen, Shih-Chi; Shen, Zan; Huang, Yun-Chao; Zhu, Xiao; Wang, Xiao-mei; Jiang, Wenqi; Wang, Zi-Feng; Bian, Xiu-Wu; Ling, Eng-Ang; Kung, Hsiang-fu; Lin, Marie C

    2013-01-01

    The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy. PMID:23531212

  6. Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons

    PubMed Central

    Kim, Mi-La; Han, Shengjun; Lee, Sat-Byol; Kim, Jung Hye; Ahn, Hee Kyung

    2010-01-01

    Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression. PMID:21189997

  7. Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure

    PubMed Central

    Wirtz, S; Galle, P; Neurath, M

    1999-01-01

    BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.
METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. β-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.
RESULTS—Intravenous or intraperitoneal injection of AdCMVβGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVβGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVβGal caused a higher β-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with

  8. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

    PubMed Central

    Cristiano, R J; Smith, L C; Kay, M A; Brinkley, B R; Woo, S L

    1993-01-01

    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies. Images Fig. 2 Fig. 4 PMID:8265587

  9. 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.

  10. [Transcatheter delivery of recombinant adenovirus vector containing exogenous aquaporin gene in treatment of Sjögren's syndrome].

    PubMed

    Hong, H E; Jieqiong, Zhang; Yan, Fan; Xiaoshuang, Sun; Yuhao, Zhu

    2016-05-25

    Sjögren's syndrome is a kind of autoimmune disease, whose main clinical symptoms are dry mouth, dry eye and chronic parotid glandular inflammation. The conservative treatments include artificial tears or saliva,oral administration of corticosteroids,and immunosuppressantsl with limited effectiveness. Along with the development of molecular biology, vast attentions are being paid to researches on gene therapy for Sjögren's syndrome, hopefully to bring gospel to patients with Sjögren's syndrome. This article reviews the recent research progresses on transcatheter delivery of recombinant adenovirus vector with aquaporin gene in experimental treatment of Sjögren's syndrome. PMID:27045247

  11. 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.

  12. Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma

    PubMed Central

    2012-01-01

    Background Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model. Methods The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts. Results The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed. Conclusions These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma. PMID:23272637

  13. Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

    SciTech Connect

    Hu Shunying; Chen Yundai; Li Libing; Chen Jinlong; Wu Bin; Zhou, Xiao; Zhi Guang; Li Qingfang; Wang Rongliang; Duan Haifeng; Guo Zikuan; Yang Yuefeng; Xiao Fengjun; Wang Hua; Wang Lisheng

    2009-12-01

    Purpose: Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. Methods and Materials: Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. Results: Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 +- 9.01 vs. 41.94 +- 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 +- 1303 vs. 1667 +- 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 +- 1084 vs. 1566 +- 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. Conclusion: Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

  14. Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways.

    PubMed

    Kinoshita, Nanako; Mizuno, Takeo; Yoshihara, Yoshihiro

    2002-03-01

    Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology. PMID:11923184

  15. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  16. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    SciTech Connect

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig Jameson, J. Larry

    2008-07-04

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the {beta}-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

  17. Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

    PubMed Central

    Damjanovic, Daniela; Zhang, Xizhong; Mu, Jingyu; Fe Medina, Maria; Xing, Zhou

    2008-01-01

    It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern. PMID:18261231

  18. Adenovirus Dodecahedron, as a Drug Delivery Vector

    PubMed Central

    Zochowska, Monika; Paca, Agnieszka; Schoehn, Guy; Andrieu, Jean-Pierre; Chroboczek, Jadwiga; Dublet, Bernard; Szolajska, Ewa

    2009-01-01

    Background Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. Principal Findings Dodecahedron (Dd) structure is preserved at up to about 50°C at pH 7–8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37°C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. Conclusions/Significance Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs. PMID:19440379

  19. Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35

    PubMed Central

    Zhang, Wen-feng; Shao, Hong-wei; Wu, Feng-lin; Xie, Xin; Li, Zhu-Ming; Bo, Hua-Ben; Shen, Han; Wang, Teng; Huang, Shu-lin

    2016-01-01

    Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy. PMID:26972139

  20. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle. PMID:25498212

  1. Neural stem cell-mediated delivery of oncolytic adenovirus.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Qian, Shuo; Spencer, Drew A; Ahmed, Atique U; Lesniak, Maciej S

    2015-01-01

    The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs. PMID:25827347

  2. Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

    PubMed Central

    Wold, William S.M.; Toth, Karoly

    2015-01-01

    Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through the natural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competent adenovirus vectors are safe and have therapeutic activity. PMID:24279313

  3. CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus.

    PubMed

    Merwin, J R; Carmichael, E P; Noell, G S; DeRome, M E; Thomas, W L; Robert, N; Spitalny, G; Chiou, H C

    1995-10-26

    Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer 'tethered' to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene. PMID:7594625

  4. Advances in Gene Delivery Systems

    PubMed Central

    Kamimura, Kenya; Suda, Takeshi; Zhang, Guisheng; Liu, Dexi

    2011-01-01

    The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral- and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives. PMID:22200988

  5. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer.

    PubMed

    Walters, R; Welsh, M

    1999-11-01

    Delivery of a normal copy of CFTR cDNA to airway epithelia may provide a novel treatment for cystic fibrosis lung disease. Unfortunately, current vectors are inefficient because of limited binding to the apical surface of airway epithelia. We recently reported that incorporation of adenovirus in a calcium phosphate coprecipitate (Ad:CaPi) improves adenovirus-mediated gene transfer to airway epithelia in vitro and in vivo. To understand better how coprecipitation improves gene transfer, we tested the hypothesis that incorporation in a CaPi coprecipitate increases the binding of adenovirus to the apical surface of differentiated human airway epithelia. When a Cy3-labelled adenovirus was delivered in a coprecipitate, binding increased 54-fold as compared with adenovirus alone. Moreover, infection by Ad:CaPi was independent of fiber knob-CAR and penton base-integrin interactions. After binding to the cell surface, the virus must enter the cell in order to infect. We hypothesized that Ad:CaPi may stimulate fluid phase endocytosis, thereby facilitating entry. However, we found that neither adenovirus nor Ad:CaPi coprecipitates altered fluid phase endocytosis. Nevertheless, Ad:CaPi preferentially infected cells showing endocytosis. Thus, CaPi coprecipitation improves adenovirus-mediated gene transfer by coating the epithelial surface with a layer of virus which enters cells during the normal process of endocytosis. PMID:10602380

  6. Neural stem cell-mediated delivery of oncolytic adenovirus

    PubMed Central

    Kim, Julius W.; Kane, J. Robert; Young, Jacob S.; Chang, Alan L.; Kanojia, Deepak; Qian, Shuo; Spencer, Drew A.; Ahmed, Atique U.; Lesniak, Maciej S.

    2015-01-01

    The use of stem cells (SCs) as carriers for therapeutic agents has by now progressed to early clinical trials. These clinical trials exploring SC-mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that could provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally-replicative adenoviruses and, then, delivered to the tumor, upon which they may evoke pronounced therapeutic efficacy in the animal (Ahmed et al., 2011; Ahmed et al., 2012; Thaci et al., 2012; Tobias et al., 2013). Here in this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs. PMID:25827347

  7. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  8. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  9. Intraductal Delivery of Adenoviruses Targets Pancreatic Tumors in Transgenic Ela-myc Mice and Orthotopic Xenografts

    PubMed Central

    José, Anabel; Sobrevals, Luciano; Camacho-Sánchez, Juan Miguel; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina

    2013-01-01

    Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p<0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors. PMID:23328228

  10. Influence of coagulation factor x on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors.

    PubMed

    Greig, Jenny A; Buckley, Suzanne Mk; Waddington, Simon N; Parker, Alan L; Bhella, David; Pink, Rebecca; Rahim, Ahad A; Morita, Takashi; Nicklin, Stuart A; McVey, John H; Baker, Andrew H

    2009-10-01

    The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon-containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5. PMID:19603000

  11. Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system.

    PubMed

    Bal, H P; Chroboczek, J; Schoehn, G; Ruigrok, R W; Dewhurst, S

    2000-10-01

    Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino-acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C-terminal fusion with glutathione S-transferase (GST-Ad7PB) and was purified by single-step affinity chromatography. Both GST-Ad7PB and cleaved (GST-free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST-Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C-terminus of this linker peptide. GST-Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD-containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin-mediated uptake of the GST-Ad7PB-FK20-DNA complexes. We propose that Ad7 pentons can be developed into integrin-specific gene delivery agents. PMID:10998069

  12. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors.

    PubMed

    Boviatsis, E J; Chase, M; Wei, M X; Tamiya, T; Hurford, R K; Kowall, N W; Tepper, R I; Breakefield, X O; Chiocca, E A

    1994-02-01

    Three vectors derived from retrovirus, herpes simplex virus type 1 (HSV), and adenovirus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a foreign gene (lacZ from Escherichia coli) into cells was more efficient with either the replication-defective retrovirus vector or the replication-conditional thymidine kinase (TK)-deficient HSV vector than with the replication-defective adenovirus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed three main histopathologic findings: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred into several types of endogenous neural cells, as well as into cells within the tumor; (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector, whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors. PMID:8186298

  13. Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

    NASA Astrophysics Data System (ADS)

    Han, Jianfeng; Zhao, Dong; Zhong, Zhirong; Zhang, Zhirong; Gong, Tao; Sun, Xun

    2010-03-01

    Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

  14. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    PubMed Central

    Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.

    2013-01-01

    Background Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. Methodology/Principal Findings As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. Conclusions/Significance These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers. PMID:23383334

  15. ENHANCEMENT OF ADENOVIRUS DELIVERY AFTER ULTRASOUND-STIMULATED THERAPY IN A CANCER MODEL

    PubMed Central

    Sorace, Anna G.; Warram, Jason M.; Mahoney, Marshall; Zinn, Kurt R.; Hoyt, Kenneth

    2014-01-01

    Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. In vitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for In vivo analysis to quantify Ad infection within the tumor. In vitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p = 0.80). In vivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1 × 106 plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p = 0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues. PMID:24063960

  16. Targeting Motor End Plates for Delivery of Adenoviruses: An Approach to Maximize Uptake and Transduction of Spinal Cord Motor Neurons.

    PubMed

    Tosolini, Andrew Paul; Morris, Renée

    2016-01-01

    Gene therapy can take advantage of the skeletal muscles/motor neurons anatomical relationship to restrict gene expression to the spinal cord ventral horn. Furthermore, recombinant adenoviruses are attractive viral-vectors as they permit spatial and temporal modulation of transgene expression. In the literature, however, several inconsistencies exist with regard to the intramuscular delivery parameters of adenoviruses. The present study is an evaluation of the optimal injection sites on skeletal muscle, time course of expression and mice's age for maximum transgene expression in motor neurons. Targeting motor end plates yielded a 2.5-fold increase in the number of transduced motor neurons compared to injections performed away from this region. Peak adenoviral transgene expression in motor neurons was detected after seven days. Further, greater numbers of transduced motor neurons were found in juvenile (3-7 week old) mice as compared with adults (8+ weeks old). Adenoviral injections produced robust transgene expression in motor neurons and skeletal myofibres. In addition, dendrites of transduced motor neurons were shown to extend well into the white matter where the descending motor pathways are located. These results also provide evidence that intramuscular delivery of adenovirus can be a suitable gene therapy approach to treat spinal cord injury. PMID:27619631

  17. Delivery of oncolytic adenovirus into the nucleus of tumorigenic cells by tumor microparticles for virotherapy.

    PubMed

    Ran, Li; Tan, Xiaohua; Li, Yanchun; Zhang, Huafeng; Ma, Ruihua; Ji, Tiantian; Dong, Wenqian; Tong, Tong; Liu, Yuying; Chen, Degao; Yin, Xiaonan; Liang, Xiaoyu; Tang, Ke; Ma, Jingwei; Zhang, Yi; Cao, Xuetao; Hu, Zhuowei; Qin, Xiaofeng; Huang, Bo

    2016-05-01

    Oncolytic viruses have been utilized for the treatment of various cancers. However, delivery of the viral particles to tumor cells remains a major challenge. Microparticles (MP) are vesicle forms of plasma membrane fragments of 0.1-1 μm in size that are shed by cells. We have previously shown the delivery of chemotherapeutic drugs using tumor cell-derived MPs (T-MP). Here we report that T-MPs can be utilized as a unique carrier system to deliver oncolytic adenoviruses to human tumors, leading to highly efficient cytolysis of tumor cells needed for in vivo treatment efficacy. This T-MP-mediated oncolytic virotherapy approach holds multiple advantages, including: 1) delivery of oncolytic adenovirus by T-MPs is able to avoid the antiviral effect of host antibodies; 2) delivery of oncolytic adenovirus by T-MPs is not limited by virus-specific receptor that mediates the entry of virus into tumor cells; 3) T-MPs are apt at delivering oncolytic adenoviruses to the nucleus of tumor cells as well as to stem-like tumor-repopulating cells for the desired purpose of killing them. These findings highlight a novel oncolytic adenovirus delivery system with highly promising clinical applications. PMID:26950165

  18. Polymeric oncolytic adenovirus for cancer gene therapy

    PubMed Central

    Choi, Joung-Woo; Lee, Young Sook; Yun, Chae-Ok; Kim, Sung Wan

    2015-01-01

    Oncolytic adenovirus (Ad) vectors present a promising modality to treat cancer. Many clinical trials have been done with either naked oncolytic Ad or combination with chemotherapies. However, the systemic injection of oncolytic Ad in clinical applications is restricted due to significant liver toxicity and immunogenicity. To overcome these issues, Ad has been engineered physically or chemically with numerous polymers for shielding the Ad surface, accomplishing extended blood circulation time and reduced immunogenicity as well as hepatotoxicity. In this review, we describe and classify the characteristics of polymer modified oncolytic Ad following each strategy for cancer treatment. Furthermore, this review concludes with the highlights of various polymer-coated Ads and their prospects, and directions for future research. PMID:26453806

  19. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

    PubMed

    Lyons, Mark; Onion, David; Green, Nicky K; Aslan, Kriss; Rajaratnam, Ratna; Bazan-Peregrino, Miriam; Phipps, Sue; Hale, Sarah; Mautner, Vivien; Seymour, Leonard W; Fisher, Kerry D

    2006-07-01

    Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access. PMID:16580883

  20. The Challenge for Gene Therapy: Innate Immune Response to Adenoviruses

    PubMed Central

    Thaci, Bart; Ulasov, Ilya V.; Wainwright, Derek A.; Lesniak, Maciej S.

    2011-01-01

    Adenoviruses are the most commonly used vectors for gene therapy. Despite the promising safety profile demonstrated in clinical trials, the efficacy of using adenoviruses for gene therapy is poor. A major hurdle to adenoviral-mediated gene therapy is the innate immune system. Cell-mediated recognition of viruses via capsid components or nucleic acids has received significant attention, principally thought to be regulated by the toll-like receptors (TLRs). Antiviral innate immune responses are initiated by the infected cell, which activates the interferon (IFN) response to block viral replication, while simultaneously releasing chemokines to attract neutrophils, mononuclear- and natural killer-cells. While the IFN and cellular recruitment pathways are activated and regulated independently of each other, both are required to overcome immune escape mechanisms by adenoviruses. Recent work has shown that the generation of adenoviral vectors lacking specific transcriptionally-active regions decreases immune system activation and increases the chance for immune escape. In this review, we elucidate how adenoviral vector modifications alter the IFN and innate inflammatory pathway response and propose future targets with clinically-translational relevance. PMID:21399236

  1. Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier.

    PubMed Central

    Baker, A; Cotten, M

    1997-01-01

    Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent. PMID:9115362

  2. Novel bat adenoviruses with an extremely large E3 gene.

    PubMed

    Tan, Bing; Yang, Xing-Lou; Ge, Xing-Yi; Peng, Cheng; Zhang, Yun-Zhi; Zhang, Li-Biao; Shi, Zheng-Li

    2016-07-01

    Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs. PMID:27032099

  3. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector.

    PubMed

    Thomas, Michael A; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  4. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector

    PubMed Central

    Thomas, Michael A.; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  5. Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery.

    PubMed

    Rojas, Luis Alfonso; Condezo, Gabriela N; Moreno, Rafael; Fajardo, Carlos Alberto; Arias-Badia, Marcel; San Martín, Carmen; Alemany, Ramon

    2016-09-10

    Recombinant adenoviruses are used as vaccines, gene therapy vectors, and oncolytic viruses. However, the efficacy of such therapies is limited by pre-existing neutralizing antibodies (NAbs), especially when the virus is administered systemically for a wider biodistribution or to reach multiple metastases. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin and maintain the infectivity and replication capacity in presence of NAbs. In pre-immunized mice non-modified viruses are completely neutralized, whereas ABD-modified viruses preserve the ability to transduce target organs, induce oncolysis, or generate immune responses to expressed proteins. Our results indicate that albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus for gene therapy, cancer virotherapy, and vaccination. PMID:27388756

  6. Terplex Gene Delivery System.

    PubMed

    Kim, Sung Wan

    2005-01-01

    Polymeric gene delivery systems have been developed to overcome problems caused by viral carriers. They are low cytotoxic, have no size limit, are convenient in handling, of low cost and reproducible. A Terplex gene delivery system consisting of plasmid DNA, low density lipoprotein and hydropholized poly-L-lysine was designed and characterized. The plasmid DNA, when formulated with stearyl PLL and LDL, forms a stable and hydrophobicity/charge-balanced Terplex system of optimal size for efficient cellular uptake. DNA is still intact after the Terplex formation. This information is expected to be utilized for the development of improved transfection vector for in vivo gene therapy. Terplex DNA complex showed significantly longer retention in the vascular space than naked DNA. This system was used in the augmentation of myocardial transfection at an infarction site with the VEGF gene. PMID:16243067

  7. Terplex gene delivery system.

    PubMed

    Kim, Sung Wan

    2005-01-01

    Polymeric gene delivery systems have been developed to overcome problems caused by viral carriers. They are low cytotoxic, have no size limit, are convenient in handling, of low cost and reproducible. A Terplex gene delivery system consisting of plasmid DNA, low density lipoprotein and hydropholized poly-L-lysine was designed and characterized. The plasmid DNA, when formulated with stearyl PLL and LDL, forms a stable and hydrophobicity/charge-balanced Terplex system of optimal size for efficient cellular uptake. DNA is still intact after the Terplex formation. This information is expected to be utilized for the development of improved transfection vector for in vivo gene therapy. Terplex DNA complex showed significantly longer retention in the vascular space than naked DNA. This system was used in the augmentation of myocardial transfection at an infarction site with the VEGF gene. PMID:16240997

  8. Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene

    PubMed Central

    Zhao, Chen; Wu, Ningning; Deng, Fang; Zhang, Hongmei; Wang, Ning; Zhang, Wenwen; Chen, Xian; Wen, Sheng; Zhang, Junhui; Yin, Liangjun; Liao, Zhan; Zhang, Zhonglin; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Wu, Di; Ye, Jixing; Deng, Youlin; Zhou, Guolin; Luu, Hue H.; Haydon, Rex C.; Si, Weike; He, Tong-Chuan

    2014-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells. PMID:24658746

  9. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

  10. Innate Immunity to Adenovirus

    PubMed Central

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka

    2014-01-01

    Abstract Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate–adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features. PMID:24512150

  11. Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene.

    PubMed Central

    Wang, Y; Krushel, L A; Edelman, G M

    1996-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control

  12. Mutation in fiber of adenovirus serotype 5 gene therapy vector decreases liver tropism

    PubMed Central

    Wang, Zhen; Wang, Baoming; Lou, Junfang; Yan, Jingyi; Gao, Lei; Geng, Ranshen; Yu, Bin

    2014-01-01

    Recombinant adenovirus (Ad) vectors are widely used for both in vitro and in vivo gene transfer. However, intravenous administration of Ad vectors results mainly in hepatocyte transduction and subsequent hepatotoxicity. Coxsackie-adenovirus receptor (CAR) and αvβ integrins, which are functional receptors for the fiber and penton proteins, respectively, are the tropism determinants of Ad type 5 (Ad5). We previously developed a system for rapid construction of fiber-modified Ad5 vectors. We also constructed a fiber-modified Ad5 containing an Arg-Gly-Asp (RGD) motif in the HI-loop and showed that it could enhance anti-tumor effects in vitro and in vivo. Here, we constructed a novel Ad5 vector containing two amino acid mutations in the AB loop of the fiber-modified Ad5 fiber knob and showed that it could significantly reduce liver tropism and increase gene transfer in low-CAR or CAR-deficient cancer cells following intravascular delivery. However, anti-tumor effects of the fiber-mutated Ad5 expressing HSV-TK under control of the hTERT promoter was not found when compared with an unmodified Ad5 vector in cancer lines expressing different levels of CAR, likely due to the activity of the hTERT promoter being lower than that of the CMV promoter. Nevertheless, this study describes an enhanced Ad5 vector for intravascular gene delivery, and further modifications such as changes in the promoter may facilitate the development of this vector for cancer treatment. PMID:25663991

  13. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-01-01

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene). PMID:26782515

  14. [Development and Characterization of a Novel Adenovirus Vector Exhibiting MicroRNA-mediated Suppression of the Leaky Expression of Adenovirus Genes].

    PubMed

    Shimizu, Kahori

    2015-01-01

    Replication-incompetent adenovirus (Ad) vectors have gained attention as gene delivery vehicles. Theoretically, no Ad genes should be expressed following transduction; however, Ad genes are expressed from the vector genome, leading to induction of cellular immunity against Ad proteins and Ad protein-induced toxicity. To suppress the leaky expression of Ad genes, a microRNA (miRNA)-regulated gene expression system was utilized. We developed novel Ad vectors by incorporating targeted sequences of miR-122a or miR-142-3p, which exhibit liver- or spleen-specific expression, respectively, in the 3'-untranslated region (UTR) of the E2A, E4, or pIX genes. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional human embryonic kidney 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold in an miRNA-dependent manner, compared with a conventional Ad vector, by the insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene (Ad-E4-122aT) expressed 1.5- to 34-fold higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver compared with a conventional Ad vector. miR-122a-mediated suppression of E4 gene expression in the liver significantly reduced the hepatotoxicity that an Ad vector causes via both adaptive and non-adaptive immune responses. Ad-E4-122aT would be a promising framework for efficient gene delivery due to its ability to mediate higher and longer-term transgene expression and lower hepatotoxicity than a conventional Ad vector. PMID:26632150

  15. Dendritic cells serve as a “Trojan horse” for oncolytic adenovirus delivery in the treatment of mouse prostate cancer

    PubMed Central

    Li, Zhao-lun; Liang, Xuan; Li, He-cheng; Wang, Zi-ming; Chong, Tie

    2016-01-01

    Aim: Adenovirus-mediated gene therapy is a novel therapeutic approach for the treatment of cancer, in which replication of the virus itself is the anticancer method. However, the success of this novel therapy is limited due to inefficient delivery of the virus to the target sites. In this study, we used dendritic cells (DCs) as carriers for conditionally replicating adenoviruses (CRAds) in targeting prostate carcinoma (PCa). Methods: Four types of CRAds, including Ad-PC (without PCa-specific promoter and a recombinant human tumor necrosis factor, rmhTNF, sequence), Ad-PC-rmhTNF (without PCa-specific promoter), Ad-PPC-NCS (without an rmhTNF sequence) and Ad-PPC-rmhTNF, were constructed. The androgen-insensitive mouse PCa RM-1 cells were co-cultured with CRAd-loading DCs, and the viability of RM-1 cells was examined using MTT assay. The in vivo effects of CRAd-loading DCs on PCa were evaluated in RM-1 xenograft mouse model. Results: Two PCa-specific CRAds (Ad-PPC-NCS, Ad-PPC-rmhTNF) exhibited more potent suppression on the viability of RM-1 cells in vitro than the PCa-non-specific CRAds (Ad-PC, Ad-PC-rmhTNF). In PCa-bearing mice, intravenous injection of the PCa-specific CRAd-loading DCs significantly inhibited the growth of xenografted tumors, extended the survival time, and induced T-cell activation. Additionally, the rmhTNF-containing CRAds exhibited greater tumor killing ability than CRAds without rmhTNF. Conclusion: DCs may be an effective vector for the delivery of CRAds in the treatment of PCa. PMID:27345628

  16. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    PubMed Central

    Wickham, T J; Tzeng, E; Shears, L L; Roelvink, P W; Li, Y; Lee, G M; Brough, D E; Lizonova, A; Kovesdi, I

    1997-01-01

    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor. PMID:9343173

  17. The role of immunosuppression in the efficacy of cancer gene therapy using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Elshami, A A; Kucharczuk, J C; Sterman, D H; Smythe, W R; Hwang, H C; Amin, K M; Litzky, L A; Albelda, S M; Kaiser, L R

    1995-01-01

    OBJECTIVE: To determine whether the immune system limits or improves the therapeutic efficacy of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene in a subcutaneous tumor model. BACKGROUND DATA: Enhanced immune reactions against tumors may be therapeutically useful. However, recent studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. The effect of the immune response on cancer gene therapy with HSVtk gene delivery by an adenovirus vector followed by treatment with ganciclovir is unclear. METHODS: After adenoviral transduction of a Fischer rat syngeneic mesothelioma cell line with the HSVtk gene in vitro, subcutaneous flank tumors were established. The ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Fischer rats, immunodeficient nude rats, and Fischer rats immunosuppressed with cyclosporin. RESULTS: HSVtk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats. CONCLUSION: These results indicate that the immune response against adenovirally transduced cells limits the efficacy of the HSVtk/ganciclovir system and that immunosuppression appears to be a useful adjunct. These findings have important implications for clinical trials using currently available adenovirus vectors as well as for future vector design. PMID:7677460

  18. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases. PMID:26467629

  19. An adenovirus-based vaccine with a double-stranded RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models.

    PubMed

    Scallan, Ciaran D; Tingley, Debora W; Lindbloom, Jonathan D; Toomey, James S; Tucker, Sean N

    2013-01-01

    An oral gene-based avian influenza vaccine would allow rapid development and simplified distribution, but efficacy has previously been difficult to achieve by the oral route. This study assessed protection against avian influenza virus challenge using a chimeric adenovirus vector expressing hemagglutinin and a double-stranded RNA adjuvant. Immunized ferrets and mice were protected upon lethal challenge. Further, ferrets immunized by the peroral route induced cross-clade neutralizing antibodies, and the antibodies were selective against hemagglutinin, not the vector. Similarly, experiments in mice demonstrated selective immune responses against HA with peroral delivery and the ability to circumvent preexisting vector immunity. PMID:23155123

  20. A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

    PubMed Central

    Appleby, CE; Kingston, PA; David, A; Gerdes, CA; Umaña, P; Castro, MG; Lowenstein, PR; Heagerty, AM

    2010-01-01

    Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of β-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases β-galactosidase expression 90-fold in SMC in vitro and ≈40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies. PMID:12907954

  1. Control of adenovirus early gene expression: Posttranscriptional control mediated by both viral and cellular gene products

    SciTech Connect

    Katze, M.G.; Persson, H.; Philipson, L.

    1981-09-01

    An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

  2. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

    PubMed Central

    Caruso, M; Pham-Nguyen, K; Kwong, Y L; Xu, B; Kosai, K I; Finegold, M; Woo, S L; Chen, S H

    1996-01-01

    Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma. Images Fig. 5 PMID:8876130

  3. Vault nanoparticles containing an adenovirus-derived membrane lytic protein facilitate toxin and gene transfer.

    PubMed

    Lai, Cheng-Yu; Wiethoff, Chris M; Kickhoefer, Valerie A; Rome, Leonard H; Nemerow, Glen R

    2009-03-24

    Nonviral methods of gene delivery possess several advantages over that of viral-based vectors, including having increased safety. However, the ability to achieve effective transport of therapeutic molecules across host cell membranes via nonviral methods remains a significant goal. Cell-derived nanoparticles known as vaults have been proposed as novel candidate transfer vehicles for various foreign molecules. Recombinant vault particles enter cells via macropinocytosis or phagocytosis but lack demonstrable membrane penetrating activity. To explore the feasibility of improving vault penetration into target cells, we incorporated the membrane lytic domain of adenovirus protein VI (pVI) into the interior of recombinant vault particles via fusion to the vault poly(ADP-ribose) polymerase (VPARP) interaction domain. The membrane lytic activity of the pVI domain was retained upon incorporation into vault particles. Moreover, internalization of vault-pVI complexes into murine macrophages promoted co-delivery of a soluble ribotoxin or a cDNA plasmid encoding GFP. These findings indicate that vault particles can be modified to enhance cell transfer of selected biomolecules. PMID:19226129

  4. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  5. Homologous Recombination in E3 Genes of Human Adenovirus Species D

    PubMed Central

    Singh, Gurdeep; Robinson, Christopher M.; Dehghan, Shoaleh; Jones, Morris S.; Dyer, David W.; Seto, Donald

    2013-01-01

    Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber. PMID:24027303

  6. NF-κB promotes leaky expression of adenovirus genes in a replication-incompetent adenovirus vector

    PubMed Central

    Machitani, M.; Sakurai, F.; Wakabayashi, K.; Nakatani, K.; Shimizu, K.; Tachibana, M.; Mizuguchi, H.

    2016-01-01

    The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes. PMID:26814140

  7. Multifunctional nanorods for gene delivery

    NASA Astrophysics Data System (ADS)

    Salem, Aliasger K.; Searson, Peter C.; Leong, Kam W.

    2003-10-01

    The goal of gene therapy is to introduce foreign genes into somatic cells to supplement defective genes or provide additional biological functions, and can be achieved using either viral or synthetic non-viral delivery systems. Compared with viral vectors, synthetic gene-delivery systems, such as liposomes and polymers, offer several advantages including ease of production and reduced risk of cytotoxicity and immunogenicity, but their use has been limited by the relatively low transfection efficiency. This problem mainly stems from the difficulty in controlling their properties at the nanoscale. Synthetic inorganic gene carriers have received limited attention in the gene-therapy community, the only notable example being gold nanoparticles with surface-immobilized DNA applied to intradermal genetic immunization by particle bombardment. Here we present a non-viral gene-delivery system based on multisegment bimetallic nanorods that can simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner. This approach allows precise control of composition, size and multifunctionality of the gene-delivery system. Transfection experiments performed in vitro and in vivo provide promising results that suggest potential in genetic vaccination applications.

  8. Adenovirus-induced alterations in host cell gene expression prior to the onset of viral gene expression.

    PubMed

    Granberg, Fredrik; Svensson, Catharina; Pettersson, Ulf; Zhao, Hongxing

    2006-09-15

    In this report, we have studied gene expression profiles in human primary lung fibroblasts (IMR-90) during the very early phase of an adenovirus infection. Eight out of twelve genes with known functions encoded transcription factors linked to two major cellular processes; inhibition of cell growth (ATF3, ATF4, KLF4, KLF6 and ELK3) and immune response (NR4A1 and CEBPB), indicating that the earliest consequences of an adenovirus infection are growth arrest and induction of an immune response. A time course analysis showed that the induction of these immediate-early response genes was transient and suppressed after the onset of the adenovirus early gene expression. PMID:16860366

  9. 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

  10. Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors.

    PubMed

    Okada, Yuka; Okada, Naoki; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki; Kanehira, Makiko; Nishino, Naoko; Takahashi, Koichi; Mizuno, Nobuyasu; Hayakawa, Takao; Mayumi, Tadanori

    2002-03-01

    Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma. PMID:11809531

  11. Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy.

    PubMed

    Choi, Joung-Woo; Park, Ji Won; Na, Youjin; Jung, Soo-Jung; Hwang, June Kyu; Choi, Dongho; Lee, Kyeong Geun; Yun, Chae-Ok

    2015-10-01

    Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy. PMID:26164117

  12. Efficient construction of recombinant adenovirus expression vector of the Qinchuan cattle LYRM1 gene.

    PubMed

    Li, Y K; Fu, C Z; Zhang, Y R; Zan, L S

    2015-01-01

    In this study, we cloned the coding DNA sequence (CDS) region of Qinchuan cattle LYR motif-containing 1 (LYRM1) and constructed a recombinant adenovirus expression vector to examine the function of LYRM1 on the cellular level. Total RNA was extracted from the adipose tissue of Qinchuan cattle, cDNA was obtained by reverse transcription, and polymerase chain reaction was used to amplify the CDS region of the LYRM1 gene. The CDS-containing fragment was inserted into the shuttle vector pAdTrack-CMV to construct pAdTrack-CMV-LYRM1 vector. After linearization of pAdTrack-CMV-LYRM1 and the negative control vector pAdTrack-CMV by restriction endonuclease PmeI, the vectors were transformed into Escherichia coli BJ5183 containing pAdEasy-1 to obtain the recombinant adenovirus vector pAd-LYRM1 and pAd-CMV through homologous recombination. pAd-LYRM1 and pAd-CMV were then digested by PacI and transfected into the 293A cell line. The recombinant adenovirus Ad-LYRM1 and Ad-CMV was obtained at a concentration of 7 x 108 and 1.3 x 109 green fluorescent units/mL, respectively. Preadipocytes derived from Qinchuan cattle were separately infected with Ad-LYRM1 and Ad- CMV. Quantitative real-time polymerase chain reaction demonstrated that the expression of LYRM1 was increased by approximate 28,000-folds after the infection with recombinant adenovirus for 48 h. In conclusion, we successfully cloned the CDS region of the Qinchuan cattle LYRM1 gene, constructed the recombinant adenovirus expression vector, and obtained the adenovirus with high titer, providing valuable materials for studying the function of LYRM1 at the cellular level. PMID:26345880

  13. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  14. 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

  15. In vivo expression of adenovirus-mediated lacZ gene in murine nasal mucosa.

    PubMed

    Arimoto, Yukiko; Nagata, Hiroshi; Isegawa, Naohisa; Kumahara, Keiichiro; Isoyama, Kyoko; Konno, Akiyoshi; Shirasawa, Hiroshi

    2002-09-01

    Adenovirus is a good tool for transferring exogenous genes into various organs because the virus has a wide spectrum of infection. In this report, we demonstrate that a recombinant adenovirus, Ax1CAlacZ, can transfer an exogenous lacZ gene into murine nasal mucosa in vivo. The efficiency of the exogenous gene expression varied for different cell types and was improved by optimizing the method of administration. In the olfactory region, the olfactory epithelia, sustentacular cells and olfactory nerve efficiently expressed lacZ gene transferred by Ax1CAlacZ using either of two administration methods, dripping or injecting. In contrast, in the respiratory region, the respiratory epithelia but not the subepithelial tissues expressed lacZ gene transferred by Ax1CAlacZ, and the efficiency of the gene transfer, which was low when the virus was administered by nasal drops, was improved when the virus was administered by injection. Our study demonstrated that gene transfer mediated by adenovirus is more efficient in the olfactory epithelia than in the respiratory epithelia, and may be applicable to nasal or paranasal diseases such as olfactory epithelial disturbances. PMID:12403125

  16. Targeted delivery of CYP2E1 recombinant adenovirus to malignant melanoma by bone marrow-derived mesenchymal stem cells as vehicles.

    PubMed

    Wang, Jishi; Ma, Dan; Li, Yan; Yang, Yuan; Hu, Xiaoyan; Zhang, Wei; Fang, Qin

    2014-03-01

    The aim of this study was to explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) as intermediate carriers on targeting of P450 gene recombinant adenovirus to malignant melanoma in vitro and in vivo. BMSCs were transduced with pAd5-CMV-CYP2E1 recombinant adenovirus. BMSC migration was detected by Transwell plates in vitro and by superparamagnetic iron oxide particles in vivo. Growth-inhibitory effect and apoptosis were determined by MTT and immunity fluorescence staining. Anticancer effects were examined by a human melanoma nude mouse model in vivo. BMSCs moved toward A375 cells in Transwell plates. Numerous superparamagnetic MSCs labeled with iron oxide were identified in the peripheral areas of the tumor, but were detected in primary organs by Prussian blue staining. BMSC-CYP2E1 cells mediated a bystander killing effect on CYP2E1-negative A375 cells during coculture (IC50 values for A375 cells cocultured with BMSC-EGFP and BMSC-CYP2E1 were 4.08 and 2.68 mmol/l, respectively). Intravenously injecting CYP2E1 recombinant adenovirus-loaded BMSCs in mice with established human melanoma managed to target the tumor site, and BMSCs with forced expression of CYP2E1 inhibited the growth of malignant cells in vivo by activating 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. BMSCs may serve as a platform of P450 gene-directed enzyme prodrug therapy for the delivery of chemotherapeutic prodrugs to tumors. PMID:24413391

  17. Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers

    PubMed Central

    Lecollinet, S.; Gavard, F.; Havenga, M. J. E.; Spiller, O. B.; Lemckert, A.; Goudsmit, J.; Eloit, M.; Richardson, J.

    2006-01-01

    A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses. PMID:16501084

  18. Multiple proteins bind to VA RNA genes of adenovirus type 2.

    PubMed Central

    Van Dyke, M W; Roeder, R G

    1987-01-01

    Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection. Images PMID:3561405

  19. Differential activation of RNA polymerase III-transcribed genes by the polyomavirus enhancer and the adenovirus E1A gene products.

    PubMed Central

    Berger, S L; Folk, W R

    1985-01-01

    We have compared the effect of the polyomavirus cis-acting transcriptional enhancer and the adenovirus trans-acting E1A gene on expression of RNA polymerase III-transcribed genes (the adenovirus VAI gene and a bacterial tRNA gene) using DNA transfection and transient expression assays. The polyomavirus enhancer has little effect upon transcription of the VAI gene by RNA polymerase III in any cell type tested (murine, hamster, or human). In contrast, expression of the E1A gene within adenovirus infected cells stimulates transcription of RNA polymerase III-transcribed genes from co-transfected DNAs. Human 293 cells, which constitutively produce adenovirus E1A gene products, also express high levels of RNA polymerase III transcripts from transfected DNAs. Images PMID:2987823

  20. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

    PubMed Central

    Mittereder, N; March, K L; Trapnell, B C

    1996-01-01

    Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in

  1. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Smythe, W R; Hwang, H C; Elshami, A A; Amin, K M; Eck, S L; Davidson, B L; Wilson, J M; Kaiser, L R; Albelda, S M

    1995-01-01

    OBJECTIVE: The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. BACKGROUND DATA: Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. METHODS: An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. RESULTS: Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. CONCLUSION: These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and

  2. Lipid Nanoparticles for Gene Delivery

    PubMed Central

    Zhao, Yi; Huang, Leaf

    2016-01-01

    Nonviral vectors which offer a safer and versatile alternative to viral vectors have been developed to overcome problems caused by viral carriers. However, their transfection efficacy or level of expression is substantially lower than viral vectors. Among various nonviral gene vectors, lipid nanoparticles are an ideal platform for the incorporation of safety and efficacy into a single delivery system. In this chapter, we highlight current lipidic vectors that have been developed for gene therapy of tumors and other diseases. The pharmacokinetic, toxic behaviors and clinic trials of some successful lipids particles are also presented. PMID:25409602

  3. Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

    PubMed

    Li, Shun; Ou, Mengting; Wang, Guixue; Tang, Liling

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future. PMID:27557721

  4. Regulation of adenovirus transcription by an Ela gene in microinjected Xenopus laevis oocytes

    SciTech Connect

    Jones, N.C.; Richter, J.D.; Weeks, D.L.; Smith, L.D.

    1983-12-01

    The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

  5. Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells

    PubMed Central

    Kaczorowski, Adam; Hammer, Katharina; Liu, Li; Villhauer, Sabine; Nwaeburu, Clifford; Fan, Pei; Zhao, Zhefu; Gladkich, Jury; Groß, Wolfgang; Nettelbeck, Dirk M.; Herr, Ingrid

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies and has poor therapeutic options. We evaluated improved oncolytic adenoviruses (OAds), in which the adenoviral gene E1B19K was deleted or a TRAIL transgene was inserted. Bone marrow mesenchymal stromal cells (MSCs) served as carriers for protected and tumor-specific virus transfers. The infection competence, tumor migration, and oncolysis were measured in cancer stem cell (CSC) models of primary and established tumor cells and in tumor xenografts. All OAds infected and lysed CSCs and prevented colony formation. MSCs migrated into PDA spheroids without impaired homing capacity. Xenotransplantation of non-infected PDA cells mixed with infected tumor cells strongly reduced the tumor volume and the expression of the proliferation marker Ki67 along with a necrotic morphology. Adenoviral capsid protein was detected in tumor xenograft tissue after intravenous injection of infected MSCs, but not in normal tissue, implying tumor-specific migration. Likewise, direct in vivo treatment correlated with a strongly reduced tumor volume, lower expression of Ki67 and CD24, and enhanced activity of caspase 3. These data demonstrate that the improved OAds induced efficient oncolysis with the OAd-TRAIL as most promising candidate for future clinical application. PMID:26824985

  6. Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V.

    PubMed Central

    Baqué, S; Newgard, C B; Gerard, R D; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease). Images Figure 1 Figure 2 Figure 5 PMID:7818463

  7. Ultrasound-Targeted Retroviral Gene Delivery

    NASA Astrophysics Data System (ADS)

    Taylor, Sarah L.; Rahim, Ahad A.; Bush, Nigel L.; Bamber, Jeffrey C.; Porter, Colin D.

    2007-05-01

    This study demonstrates the ability of focused ultrasound to target retroviral gene delivery. Key to our experiments was the use of non-infectious virus particles lacking the envelope protein required for receptor-mediated entry. The novelty of our approach is that spatial control at a distance is exerted upon viral delivery by subsequent exposure to ultrasound, leading to stable gene delivery. The technology is ideally suited to controlling gene delivery in vivo following systemic vector administration. Our data provide a solution to the critical issue of obtaining tissue specificity with retroviral vectors and impart stability of expression to ultrasound-mediated gene delivery.

  8. 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

  9. Development of replication-competent adenovirus for bladder cancer by controlling adenovirus E1a and E4 gene expression with the survivin promoter

    PubMed Central

    Seo, Ho Kyung; Seo, Jeong Bin; Nam, Jae-Kook; Jeong, Kyung-Chae; Shin, Seung-Pil; Kim, In-Hoo; Lee, Sang Don; Lee, Sang-Jin

    2014-01-01

    Survivin is a member of the inhibitors of apoptosis protein family. Here, we examined survivin expression and confirmed abundant survivin expression in bladder cancer cells. This expression pattern indicated that the transcriptional regulatory elements that control survivin expression could be utilized to discriminate cancer from normal cells. We therefore generated a novel adenovirus termed Ad5/35E1apsurvivinE4 with the following characteristics: 1) E1A and E4 protein expression was dependent on survivin promoter activity; 2) the green fluorescence protein gene was inserted into the genome under the control of the CMV promoter; 3) most of the E3 sequences were deleted, but the construct was still capable of expressing the adenovirus death protein with potent cytotoxic effects; and 4) the fiber knob was from serotype 35 adenovirus. As expected from the abundant survivin expression observed in bladder cancer cells, Ad5/35E1apsurvivinE4 replicated better in cancer cells than in normal cells by a factor of 106 to 102. Likewise, Ad5/35E1apsurvivinE4 exerted greater cytotoxic effects on all bladder cancer cell lines tested. Importantly, Ad5/35E1apsurvivinE4 inhibited the growth of Ku7-Luc orthotopic xenografts in nude mice. Taken together, Ad5/35E1apsurvivinE4 indicates that the survivin promoter may be utilized for the development of a replication-competent adenovirus to target bladder cancers. PMID:25015402

  10. 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. PMID:25727710

  11. Vectors for airway gene delivery.

    PubMed

    Davis, Pamela B; Cooper, Mark J

    2007-01-01

    Delivery of genes to the airway epithelium for therapeutic purposes seemed easy at first, because the epithelial cells interface with the environment and are therefore accessible. However, problems encountered were more substantial than were originally expected. Nonviral systems may be preferred for long-term gene expression, for they can be dosed repeatedly. Two nonviral gene transfer systems have been in clinical trials, lipid-mediated gene transfer and DNA nanoparticles. Both have sufficient efficiency to be candidates for correction of the cystic fibrosis defect, and both can be dosed repeatedly. However, lipid-mediated gene transfer in the first generation provokes significant inflammatory toxicity, which may be engineered out by adjustments of the lipids, the plasmid CpG content, or both. Both lipid-mediated gene transfer and DNA nanoparticles in the first generation have short duration of expression, but reengineering of the plasmid DNA to contain mostly eukaryotic sequences may address this problem. Considerable advances in the understanding of the cellular uptake and expression of these agents and in their practical utility have occurred in the last few years; these advances are reviewed here. PMID:17408235

  12. Cationic Bolaamphiphiles for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  13. Targeting lung cancer stem-like cells with TRAIL gene armed oncolytic adenovirus

    PubMed Central

    Yang, Yu; Xu, Haineng; Huang, Weidan; Ding, Miao; Xiao, Jing; Yang, Dongmei; Li, Huaguang; Liu, Xin-Yuan; Chu, Liang

    2015-01-01

    Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors-defined serum-free medium. A549 sphere cells displayed CSC properties, including chemo-resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55-EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55-TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55-TRAIL. In the A549 sphere cells xenograft models, ZD55-TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs. PMID:25683371

  14. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice.

    PubMed

    Ragot, T; Vincent, N; Chafey, P; Vigne, E; Gilgenkrantz, H; Couton, D; Cartaud, J; Briand, P; Kaplan, J C; Perricaudet, M

    1993-02-18

    Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle. PMID:8437625

  15. Genetic organization, size, and complete sequence of early region 3 genes of human adenovirus type 41.

    PubMed Central

    Yeh, H Y; Pieniazek, N; Pieniazek, D; Luftig, R B

    1996-01-01

    The complete nucleotide and predicted amino acid sequences for open reading frames (ORFs) of the human adenovirus type 41 (Ad41) early region 3 (E3) gene have been determined. The sequence of the Ad41 E3 gene (map units 74 to 83.9) consists of 3,373 nucleotides and has one TATA box and two polyadenylation signals (AATAAA). Analysis of the nucleotide sequence reveals that the E3 gene can encode six ORFs, designated RL1 to RL6. These are all expressed at the mRNA level, as determined by reverse transcription-PCR analysis of AD41-infected cell RNA. When compared with known E3 sequences of most other human adenoviruses deposited in GenBank, the sequences of RL1 to RL3 were found to be unique to subgroup F adenoviruses (Ad40 and Ad41). They encode putative proteins of 173 amino acids (19.4 kDa) and 276 amino acids (31.6 kDa) in one reading frame as well as a 59- amino-acid (6.7 kDa) protein in an overlapping reading frame. RL4 encodes a 90-amino-acid protein (10.1 kDa) with 40% homology to the Ad2 E3 10.4-kDa protein, which induces degradation of the epidermal growth factor receptor and functions together with the Ad2 E3 14.5-kDa protein to protect mouse cell lines against lysis. RL5 encodes a protein of 107 amino acid residues (12.3 kDa) and is analogous to the Ad E3 14.5-kDa protein. RL6 codes for a protein of 122 amino acids (14.7 kDa) that is analogous to the Ad2 14.7-kDa protein, which functions to protect Ad-infected cells from tumor necrosis factor-induced cytolysis. This finding of three unique (RL1 to RL3) E3 gene ORFs may explain why subgroup F adenoviruses differ substantially from other human adenoviruses in their host range; i.e., they replicate predominantly in the host's gastrointestinal rather than respiratory tract. A recent phylogenetic study that compared subgroup F Ad40 DNA sequences with representatives of subgroups B (Ad3), C (Ad2), and E (Ad4) reached a similar conclusion about the uniqueness of RL1 and RL2. PMID:8642703

  16. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

    PubMed Central

    Fasbender, A; Lee, J H; Walters, R W; Moninger, T O; Zabner, J; Welsh, M J

    1998-01-01

    Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo. PMID:9649572

  17. Limited but durable changes to cellular gene expression in a model of latent adenovirus infection are reflected in childhood leukemic cell lines.

    PubMed

    Ornelles, D A; Gooding, L R; Dickherber, M L; Policard, M; Garnett-Benson, C

    2016-07-01

    Mucosal lymphocytes support latent infections of species C adenoviruses. Because infected lymphocytes resist re-infection with adenovirus, we sought to identify changes in cellular gene expression that could inhibit the infectious process. The expression of over 30,000 genes was evaluated by microarray in persistently infected B-and T-lymphocytic cells. BBS9, BNIP3, BTG3, CXADR, SLFN11 and SPARCL1 were the only genes differentially expressed between mock and infected B cells. Most of these genes are associated with oncogenesis or cancer progression. Histone deacetylase and DNA methyltransferase inhibitors released the repression of some of these genes. Cellular and viral gene expression was compared among leukemic cell lines following adenovirus infection. Childhood leukemic B-cell lines resist adenovirus infection and also show reduced expression of CXADR and SPARCL. Thus adenovirus induces limited changes to infected B-cell lines that are similar to changes observed in childhood leukemic cell lines. PMID:27085068

  18. Limited but durable changes to cellular gene expression in a model of latent adenovirus infection are reflected in childhood leukemic cell lines

    PubMed Central

    Ornelles, D.A.; Gooding, L.R.; Dickherber, M.L.; Policard, M.; Garnett-Benson, C.

    2016-01-01

    Mucosal lymphocytes support latent infections of species C adenoviruses. Because infected lymphocytes resist re-infection with adenovirus, we sought to identify changes in cellular gene expression that could inhibit the infectious process. The expression of over 30,000 genes was evaluated by microarray in persistently infected B-and T-lymphocytic cells. BBS9, BNIP3, BTG3, CXADR, SLFN11 and SPARCL1 were the only genes differentially expressed between mock and infected B cells. Most of these genes are associated with oncogenesis or cancer progression. Histone deacetylase and DNA methyltransferase inhibitors released the repression of some of these genes. Cellular and viral gene expression was compared among leukemic cell lines following adenovirus infection. Childhood leukemic B-cell lines resist adenovirus infection and also show reduced expression of CXADR and SPARCL. Thus adenovirus induces limited changes to infected B-cell lines that are similar to changes observed in childhood leukemic cell lines. PMID:27085068

  19. Viral and nonviral delivery systems for gene delivery.

    PubMed

    Nayerossadat, Nouri; Maedeh, Talebi; Ali, Palizban Abas

    2012-01-01

    Gene therapy is the process of introducing foreign genomic materials into host cells to elicit a therapeutic benefit. Although initially the main focus of gene therapy was on special genetic disorders, now diverse diseases with different patterns of inheritance and acquired diseases are targets of gene therapy. There are 2 major categories of gene therapy, including germline gene therapy and somatic gene therapy. Although germline gene therapy may have great potential, because it is currently ethically forbidden, it cannot be used; however, to date human gene therapy has been limited to somatic cells. Although numerous viral and nonviral gene delivery systems have been developed in the last 3 decades, no delivery system has been designed that can be applied in gene therapy of all kinds of cell types in vitro and in vivo with no limitation and side effects. In this review we explain about the history of gene therapy, all types of gene delivery systems for germline (nuclei, egg cells, embryonic stem cells, pronuclear, microinjection, sperm cells) and somatic cells by viral [retroviral, adenoviral, adeno association, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex, pox virus, lentivirus, Epstein-Barr virus)] and nonviral systems (physical: Naked DNA, DNA bombardant, electroporation, hydrodynamic, ultrasound, magnetofection) and (chemical: Cationic lipids, different cationic polymers, lipid polymers). In addition to the above-mentioned, advantages, disadvantages, and practical use of each system are discussed. PMID:23210086

  20. Race, genes and preterm delivery.

    PubMed Central

    Fiscella, Kevin

    2005-01-01

    High rates of preterm delivery (PTD) among African Americans are the leading cause of excess infant mortality among African Americans. Failure to fully explain racial disparity in PTD has led to speculation that genetic factors might contribute to this disparity. Current evidence suggests that genetic factors contribute to PTD, but this does not imply that genetic factors contribute to racial disparity in PTD. Environmental factors clearly contribute to PTD. Many of these factors acting over a women's life prior to pregnancy disproportionately affect African Americans and contribute significantly to racial disparity in PTD. Thus, inferring genetic contribution to racial disparity in PTD by attempting to control for environmental factors measured at a single point in time is flawed. There is emerging evidence of gene-environment interactions for PTD, some of which disproportionately affect African Americans. There is also evidence of racial differences in the prevalence of polymorphisms potentially related to PTD. However, to date there is no direct evidence that these differences contribute significantly to racial disparity in PTD. Given the complexity of polygenic conditions such as PTD, the possibility of any single gene contributing substantially to racial disparity in PTD seems remote. PMID:16334498

  1. Cyclodextrins in non-viral gene delivery.

    PubMed

    Lai, Wing-Fu

    2014-01-01

    Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. They consist of (α-1,4)-linked glucose units, and possess a basket-shaped topology with an "inner-outer" amphiphilic character. Over the years, substantial efforts have been undertaken to investigate the possible use of CDs in drug delivery and controlled drug release, yet the potential of CDs in gene delivery has received comparatively less discussion in the literature. In this article, we will first discuss the properties of CDs for gene delivery, followed by a synopsis of the use of CDs in development and modification of non-viral gene carriers. Finally, areas that are noteworthy in CD-based gene delivery will be highlighted for future research. Due to the application prospects of CDs, it is anticipated that CDs will continue to emerge as an important tool for vector development, and will play significant roles in facilitating non-viral gene delivery in the forthcoming decades. PMID:24103652

  2. Adenovirus with p16 gene exerts antitumor effect on laryngeal carcinoma Hep2 cells.

    PubMed

    Yang, Zhengang; Hu, Jingxia; Li, Dajun; Pan, Xinliang

    2016-08-01

    Laryngeal cancer is an uncommon form of cancer. The tumor suppressor P16, known to be mutated or deleted in various types of human tumor, including laryngeal carcinoma, is involved in the formation and development of laryngeal carcinoma. It has been previously reported that the inactivation or loss of P16 is associated with the acquisition of malignant characteristics. The current study hypothesized that restoring wild‑type P16 activity into P16‑null malignant Hep2 cells may exert an antitumor effect. A recombinant adenovirus carrying the P16 gene (Ad‑P16) was used to infect and express high levels of P16 protein in P16‑null Hep2 cells. Cell proliferation and invasion assays and polymerase chain reaction were performed to evaluate the effects of the P16 gene on cell proliferation and the antitumor effect on Hep2 cells. The results demonstrated that the Hep2 cells infected with Ad‑P16 exhibited significantly reduced cell proliferation, invasion and tumor volume compared with untreated or control adenovirus cells. Furthermore, the expression of laryngeal carcinoma‑associated genes, EGFR, survivin and cyclin D1, were measured in Ad‑P16‑infected cells and were significantly reduced compared with control groups. The results of the current study demonstrate that restoring wild‑type P16 activity into P16-null Hep2 cells exerts an antitumor effect. PMID:27277704

  3. Structure and chromosomal localization of the murine coxsackievirus and adenovirus receptor gene.

    PubMed

    Chen, Jin-Wen; Ghosh, Ruma; Finberg, Robert W; Bergelson, Jeffrey M

    2003-04-01

    We analyzed BAC genomic clones encoding the murine coxsackievirus and adenovirus receptor (mCAR). The mCAR gene is situated on the distal portion of murine chromosome 16, and is composed of at least eight exons, with intron-exon boundaries similar to those reported for the human CAR gene. We previously described two cDNAs encoding mCAR isoforms: the extracellular and transmembrane portions of both are encoded by exons 1-6; the cytoplasmic domain of mCAR 1 is encoded by exon 7, whereas mCAR 2 results from an RNA splice linking the proximal portion of exon 7 to an alternative exon 8. RT-PCR analysis of the mCAR RNA 5'-terminus suggests that transcription may begin 141-161 nucleotides upstream of the ATG translational start site. PMID:12823902

  4. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGESBeta

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; et al

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  5. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

  6. Genomic organization and chromosomal localization of the human Coxsackievirus B-adenovirus receptor gene.

    PubMed

    Bowles, K R; Gibson, J; Wu, J; Shaffer, L G; Towbin, J A; Bowles, N E

    1999-10-01

    Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children. Many studies have implicated the enteroviruses and, particularly, the Coxsackievirus-B family as etiologic agents of the acquired forms of these diseases. However, we have shown the group-C adenoviruses to be as commonly detected as enteroviruses in the myocardium of children and adults with these diseases. It has remained something of a conundrum why two such divergent virus families cause these diseases. The recent description of the common human Coxsackievirus B-adenovirus receptor (CAR) offers at least a partial explanation. In order to characterize the CAR gene, we screened a bacterial artificial chromosomal (BAC) library (RPCI11) using a polymerase chain reaction (PCR) product derived from the 3' end of the CAR cDNA sequence. This identified 13 BACs that were further characterized by PCR amplification of seven contiguous regions of the entire cDNA sequence. Eleven of the BACs were determined to encode pseudogenes while the other two BACs (131J5 and 246M1) encoded the presumed functional gene. PCR amplification of a monochromosomal hybrid panel indicated the presence of pseudogenes on chromosomes 15, 18, and 21 while the functional gene is encoded on chromosome 21. Fluorescence in situ hybridization analysis indicated that the gene is located at 21q11.2. DNA sequencing of BACs 131J5 and 246M1 revealed the presence of seven exons. The DNA sequences have been determined for each exon-intron boundary, and putative promoter sequences and transcription initiation sites identified. No consensus polyadenylation signal was identified. PMID:10543405

  7. Unique conditionally replication competent bipartite adenoviruses-cancer terminator viruses (CTV): efficacious reagents for cancer gene therapy.

    PubMed

    Sarkar, Devanand; Su, Zao-Zhong; Fisher, Paul B

    2006-07-01

    The frequent resistance of aggressive cancers to currently available therapies, such as radiotherapy and chemotherapy, mandates development of targeted, nontoxic and more efficacious treatment protocols. Conditionally replication competent adenoviruses (CRCAs) that induce oncolysis by cancer-specific replication are currently being evaluated in clinical trials. However, a single modality approach may not be sufficient to completely eradicate cancer in a patient, because most cancers arise from abnormalities in multiple genetic and signal transduction pathways. The promoter region of rodent progression elevated gene-3 (PEG-3), cloned and characterized in our laboratory, embodies the unique property of increased activity in a broad range of tumor cells, both rodent and human, when compared to normal counterparts. Bipartite adenoviruses were engineered to express the E1A gene, necessary for viral replication, under control of the PEG-3 promoter (PEG-Prom) and simultaneously express a second transgene in the E3 region that encodes an apoptosis-inducing and immunomodulatory cytokine, either immune interferon (IFN-gamma) or melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). These conditionally replication competent bipartite adenoviruses, referred to as cancer terminator viruses (CTVs), facilitated cancer-selective adenovirus replication, robust transgene expression and apoptosis induction with complete eradication of both primary and distant (metastatic) human cancers xenotransplanted in athymic nude mice. These findings suggest that CTVs might prove efficacious for the therapy of primary and advanced neoplastic diseases. PMID:16861924

  8. Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

    PubMed Central

    Khalil, IA; Kogure, K; Futaki, S; Hama, S; Akita, H; Ueno, M; Kishida, H; Kudoh, M; Mishina, Y; Kataoka, K; Yamada, M; Harashima, H

    2007-01-01

    This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies. PMID:17268535

  9. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer With Recombinant Adenovirus in Nonhuman Primates

    PubMed Central

    Fontanellas, Antonio; Hervás-Stubbs, Sandra; Mauleón, Itsaso; Dubrot, Juan; Mancheño, Uxua; Collantes, María; Sampedro, Ana; Unzu, Carmen; Alfaro, Carlos; Palazón, Asis; Smerdou, Cristian; Benito, Alberto; Prieto, Jesús; Peñuelas, Iván; Melero, Ignacio

    2010-01-01

    Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector. PMID:20087317

  10. Delivery of genes into the CF airway.

    PubMed

    Gill, Deborah R; Hyde, Stephen C

    2014-10-01

    Gene therapy was suggested as a potential treatment for cystic fibrosis (CF), even before the identification of the CFTR gene. Initial enthusiasm has been tempered as it became apparent that reintroduction of the CFTR gene into the cells of the lung is more difficult than anticipated. Here, we review the major gene delivery vectors evaluated clinically, and suggest that advances in either plasmid DNA design and/or hybrid lentivirus biology may finally facilitate lung gene transfer with efficiencies sufficient for CF gene therapy to offer clinical benefit. PMID:25015239

  11. Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome.

    PubMed

    Shimizu, Kahori; Sakurai, Fuminori; Tomita, Kyoko; Nagamoto, Yasuhito; Nakamura, Shin-Ichiro; Katayama, Kazufumi; Tachibana, Masashi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2014-01-01

    Leaky expression of adenovirus (Ad) genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3'-untranslated region (UTR) of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses. PMID:26015975

  12. Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer.

    PubMed

    Rajendran, Simon; O'Hanlon, Deirdre; Morrissey, David; O'Donovan, Tracey; O'Sullivan, Gerald C; Tangney, Mark

    2011-04-01

    Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in

  13. 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. PMID:25903647

  14. Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene.

    PubMed

    Liu, Elizabeth B; Ferreyra, Leonardo; Fischer, Stephen L; Pavan, Jorge V; Nates, Silvia V; Hudson, Nolan Ryan; Tirado, Damaris; Dyer, David W; Chodosh, James; Seto, Donald; Jones, Morris S

    2011-01-01

    In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event. PMID:21915339

  15. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    PubMed Central

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing

    2014-01-01

    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers. PMID:24473833

  16. [Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A].

    PubMed

    Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Xu, Yi; Jia, Run-Qing; Bo, Hong; Dong, Jie; Zhou, Jian-Fang; Shu, Yue-Long; Zhang, Zhi-Qing

    2009-03-01

    Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector. PMID:19678564

  17. Magnetic nanoparticles for gene and drug delivery

    PubMed Central

    McBain, Stuart C; Yiu, Humphrey HP; Dobson, Jon

    2008-01-01

    Investigations of magnetic micro- and nanoparticles for targeted drug delivery began over 30 years ago. Since that time, major progress has been made in particle design and synthesis techniques, however, very few clinical trials have taken place. Here we review advances in magnetic nanoparticle design, in vitro and animal experiments with magnetic nanoparticle-based drug and gene delivery, and clinical trials of drug targeting. PMID:18686777

  18. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    PubMed Central

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

  19. Genomic and bioinformatics analysis of HAdV-7, a human adenovirus of species B1 that causes acute respiratory disease: implications for vector development in human gene therapy.

    PubMed

    Purkayastha, Anjan; Su, Jing; Carlisle, Steve; Tibbetts, Clark; Seto, Donald

    2005-02-01

    Human adenovirus serotype 7 (HAdV-7) is a reemerging pathogen identified in acute respiratory disease (ARD), particularly in epidemics affecting basic military trainee populations of otherwise healthy young adults. The genome has been sequenced and annotated (GenBank accession no. ). Comparative genomics and bioinformatics analyses of the HAdV-7 genome sequence provide insight into its natural history and phylogenetic relationships. A putative origin of HAdV-7 from a chimpanzee host is observed. This has implications within the current biotechnological interest of using chimpanzee adenoviruses as vectors for human gene therapy and DNA vaccine delivery. Rapid genome sequencing and analyses of this species B1 member provide an example of exploiting accurate low-pass DNA sequencing technology in pathogen characterization and epidemic outbreak surveillance through the identification, validation, and application of unique pathogen genome signatures. PMID:15661145

  20. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

    PubMed Central

    Wickham, T J; Segal, D M; Roelvink, P W; Carrion, M E; Lizonova, A; Lee, G M; Kovesdi, I

    1996-01-01

    A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer. PMID:8794324

  1. Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells.

    PubMed

    Hosoya, Noriaki; Miura, Toshiyuki; Kawana-Tachikawa, Ai; Koibuchi, Tomohiko; Shioda, Tatsuo; Odawara, Takashi; Nakamura, Tetsuya; Kitamura, Yoshihiro; Kano, Munehide; Kato, Atsushi; Hasegawa, Mamoru; Nagai, Yoshiyuki; Iwamoto, Aikichi

    2008-03-01

    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients. PMID:18205221

  2. Platelet-Derived Growth Factor Gene Delivery Stimulates ex Vivo Gingival Repair

    PubMed Central

    ANUSAKSATHIEN, ORASA; WEBB, SARAH A.; JIN, QI-MING; GIANNOBILE, WILLIAM V.

    2008-01-01

    Destruction of tooth support due to the chronic inflammatory disease periodontitis is a major cause of tooth loss. There are limitations with available treatment options to tissue engineer soft tissue periodontal defects. The exogenous application of growth factors (GFs) such as platelet-derived growth factor (PDGF) has shown promise to enhance oral and periodontal tissue regeneration. However, the topical administration of GFs has not led to clinically significant improvements in tissue regeneration because of problems in maintaining therapeutic protein levels at the defect site. The utilization of PDGF gene transfer may circumvent many of the limitations with protein delivery to soft tissue wounds. The objective of this study was to test the effect of PDGF-A and PDGF-B gene transfer to human gingival fibroblasts (HGFs) on ex vivo repair in three-dimensional collagen lattices. HGFs were transduced with adenovirus encoding PDGF-A and PDGF-B genes. Defect fill of bilayer collagen gels was measured by image analysis of cell repopulation into the gingival defects. The modulation of gene expression at the defect site and periphery was measured by RT-PCR during a 10-day time course after gene delivery. The results demonstrated that PDGF-B gene transfer stimulated potent (>4-fold) increases in cell repopulation and defect fill above that of PDGF-A and corresponding controls. PDGF-A and PDGF-B gene expression was maintained for at least 10 days. PDGF gene transfer upregulated the expression of phosphatidylinosital 3-kinase and integrin α5 subunit at 5 days after adenovirus transduction. These results suggest that PDGF gene transfer has potential for periodontal soft tissue-engineering applications. PMID:13678451

  3. 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

  4. Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method

    PubMed Central

    2011-01-01

    Conventional parenteral injection of vaccines is limited in its ability to induce locally-produced immune responses in the respiratory tract, and has logistical disadvantages in widespread vaccine administration. Recent studies suggest that intranasal delivery or vaccination in the respiratory tract with recombinant viral vectors can enhance immunogenicity and protection against respiratory diseases such as influenza and tuberculosis, and can offer more broad-based generalized protection by eliciting durable mucosal immune responses. Controlled aerosolization is a method to minimize vaccine particle size and ensure delivery to the lower respiratory tract. Here, we characterize the dynamics of aerosolization and show the effects of vaccine concentration on particle size, vector viability, and the actual delivered dose of an aerosolized adenoviral vector. In addition, we demonstrate that aerosol delivery of a recombinant adenoviral vaccine encoding H1N1 hemagglutinin is immunogenic and protects ferrets against homologous viral challenge. Overall, aerosol delivery offers comparable protection to intramuscular injection, and represents an attractive vaccine delivery method for broad-based immunization campaigns. PMID:22103776

  5. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    PubMed Central

    Lee, Sang-Soo; George Priya Doss, C.; Yagihara, Shin; Kim, Do-Young

    2014-01-01

    Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD) blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole. PMID:25250340

  6. Immune Recognition of Gene Transfer Vectors: Focus on Adenovirus as a Paradigm

    PubMed Central

    Aldhamen, Yasser Ali; Seregin, Sergey S.; Amalfitano, Andrea

    2011-01-01

    Recombinant Adenovirus (Ad) based vectors have been utilized extensively as a gene transfer platform in multiple pre-clinical and clinical applications. These applications are numerous, and inclusive of both gene therapy and vaccine based approaches to human or animal diseases. The widespread utilization of these vectors in both animal models, as well as numerous human clinical trials (Ad-based vectors surpass all other gene transfer vectors relative to numbers of patients treated, as well as number of clinical trials overall), has shed light on how this virus vector interacts with both the innate and adaptive immune systems. The ability to generate and administer large amounts of this vector likely contributes not only to their ability to allow for highly efficient gene transfer, but also their elicitation of host immune responses to the vector and/or the transgene the vector expresses in vivo. These facts, coupled with utilization of several models that allow for full detection of these responses has predicted several observations made in human trials, an important point as lack of similar capabilities by other vector systems may prevent detection of such responses until only after human trials are initiated. Finally, induction of innate or adaptive immune responses by Ad vectors may be detrimental in one setting (i.e., gene therapy) and be entirely beneficial in another (i.e., prophylactic or therapeutic vaccine based applications). Herein, we review the current understanding of innate and adaptive immune responses to Ad vectors, as well some recent advances that attempt to capitalize on this understanding so as to further broaden the safe and efficient use of Ad-based gene transfer therapies in general. PMID:22566830

  7. Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector

    PubMed Central

    Shayakhmetov, Dmitry M.; Papayannopoulou, Thalia; Stamatoyannopoulos, George; Lieber, André

    2000-01-01

    Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and αv integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34+ cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34+ cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an αv integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34+ cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34+ cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34+ cells expressing αv integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34+ cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34+ cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34+ c-Kit+ cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34+ c-Kit+ cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells. PMID:10684271

  8. "Programmed packaging" for gene delivery.

    PubMed

    Hyodo, M; Sakurai, Y; Akita, H; Harashima, H

    2014-11-10

    We report on the development of a multifunctional envelope-type nano device (MEND) based on our packaging concept "Programmed packaging" to control not only intracellular trafficking but also the biodistribution of encapsulated compounds such as nucleic acids/proteins/peptides. Our strategy for achieving this is based on molecular mechanisms of cell biology such as endocytosis, vesicular trafficking, etc. In this review, we summarize the concept of programmed packaging and discuss some of our recent successful examples of using MENDs. Systematic evolution of ligands by exponential enrichment (SELEX) was applied as a new methodology for identifying a new ligand toward cell or mitochondria. The delivery of siRNA to tumors and the tumor vasculature was achieved using pH sensitive lipid (YSK05), which was newly designed and optimized under in vivo conditions. The efficient delivery of pDNA to immune cells such as dendritic cells has also been developed using the KALA ligand, which can be a breakthrough technology for DNA vaccine. Finally, ss-cleavable and pH-activated lipid-like surfactant (ssPalm) which is a lipid like material with pH-activatable and SS-cleavable properties is also introduced as a proof of our concept. PMID:24780263

  9. Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers.

    PubMed

    Hoyos, Valentina; Del Bufalo, Francesca; Yagyu, Shigeki; Ando, Miki; Dotti, Gianpietro; Suzuki, Masataka; Bouchier-Hayes, Lisa; Alemany, Ramon; Brenner, Malcolm K

    2015-09-01

    Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC. PMID:26084970

  10. Modified montmorillonite as vector for gene delivery.

    PubMed

    Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

    2006-06-01

    Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future. PMID:16488006

  11. Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

    PubMed Central

    Tran, P L; Weinbach, J; Opolon, P; Linares-Cruz, G; Reynes, J P; Grégoire, A; Kremer, E; Durand, H; Perricaudet, M

    1997-01-01

    A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis. PMID:9045862

  12. Development and assessment of human adenovirus type 11 as a gene transfer vector.

    PubMed

    Stone, Daniel; Ni, Shaoheng; Li, Zong-Yi; Gaggar, Anuj; DiPaolo, Nelson; Feng, Qinghua; Sandig, Volker; Lieber, André

    2005-04-01

    Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism. PMID:15795294

  13. Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression

    PubMed Central

    Stilwell, Jackie L.; McCarty, Douglas M.; Negishi, Atsuko; Superfine, Richard; Samulski, R. Jude

    2003-01-01

    Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone. PMID:14610209

  14. [Adenovirus-delivered BMI-1 shRNA].

    PubMed

    Chen, Zhen-Ping; Chen, Xiao-Li; Zhen, Jie

    2009-10-01

    Recently, some plasmid vectors that direct transcription of small hairpin RNAs have been developed, which are processed into functional siRNAs by cellular enzymes. Although these vectors possess certain advantages over synthesized siRNA, many disadvantages exist, including low and variable transfection efficiency. This study was aimed to establish an adenoviral siRNA delivery system without above-mentioned disadvantages on the basis of commercially available vectors. A vector was designed to target the human polycomb gene BMI-1. The pAd-BMI-1shRNA-CMV-GFP vector was produced by cloning a 300 bp U6-BMI-1 cassette from the pGE1BMI-1shRNA plasmid and a CMV-GFP cassette from pAdTrack CMV in pShutter vector. The adenovirus was produced from the 293A packaging cell line and then infected K562 cells. The mRNA and protein levels of Bmi-1 were detected by real time-PCR and Western blot respectively. The results showed that the adenovirus carrying the BMI-1shRNA was successfully produced. After being transfected with the adenovirus, the K562 cells dramatically down-regulated BMI-1 expression, whereas the adenoviruses carrying control shRNA had no effect on BMI-1 expression. It is concluded that the adenoviruses are efficient vectors for delivery of siRNA into mammalian cells and may become a candidate vector carrying siRNA drugs for gene therapy. PMID:19840467

  15. SOCS-1 gene delivery cooperates with cisplatin plus pemetrexed to exhibit preclinical antitumor activity against malignant pleural mesothelioma.

    PubMed

    Iwahori, Kota; Serada, Satoshi; Fujimoto, Minoru; Ripley, Barry; Nomura, Shintaro; Mizuguchi, Hiroyuki; Shimada, Kazuki; Takahashi, Tsuyoshi; Kawase, Ichiro; Kishimoto, Tadamitsu; Naka, Tetsuji

    2013-01-15

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO-4, H28 and H226) with adenovirus-expressing SOCS-1 vector to examine the effect of SOCS-1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS-1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF-κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS-1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS-1 regulated NF-κB and STAT3 signaling to induce apoptosis in MESO-4 and H226 cells. Furthermore, SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF-κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS-1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed. PMID:22532243

  16. Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia.

    PubMed

    Walters, R W; van't Hof, W; Yi, S M; Schroth, M K; Zabner, J; Crystal, R G; Welsh, M J

    2001-08-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  17. Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

    PubMed Central

    Walters, Robert W.; van't Hof, Wouter; Yi, Su Min P.; Schroth, Mary K.; Zabner, Joseph; Crystal, Ronald G.; Welsh, Michael J.

    2001-01-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl− transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  18. Adenovirus-mediated delivery of herpes simplex virus thymidine kinase administration improves outcome of recurrent high-grade glioma

    PubMed Central

    Liu, Cang; Gu, Zheng; Chen, Shizhang; Guo, Ying; Fan, Zhong; Wang, Xiao; Chen, Jianfei; Zhao, Yanyan; Zhou, Jianfeng; Wang, Jisheng; Ma, Ding; Li, Ning

    2016-01-01

    Background This randomized, open-label, multicenter, phase II clinical trial was conducted to assess the anti-tumor efficacy and safety of replication-deficient adenovirus mutant thymidine kinase (ADV-TK) in combination with ganciclovir administration in patients with recurrent high-grade glioma (HGG). Patients and Methods 53 patients with recurrent HGG were randomly allocated to receive intra-arterial cerebral infusion of ADV-TK or conventional treatments. The primary end point was 6-month progression-free survival (PFS-6). Secondary end points included progression-free survival (PFS), overall survival (OS), safety, and clinical benefit. This trial is registered with Clinicaltrials.gov, NCT00870181. Results In ADV-TK group, PFS-6 was 54.5%, the median PFS was 29.6 weeks, the median OS was 45.4 weeks, and better survivals were achieved when compared with control group. The one-year PFS and OS were 22.7% and 44.6% in ADV-TK group respectively, and clinical benefit was 68.2%. There are 2 patients alive for more than 4 years without progression in ADV-TK group. In the subgroup of glioblastoma received ADV-TK, PFS-6 was 71.4%, median PFS was 34.9 weeks, median OS was 45.7 weeks respectively, much better than those in control group. The one-year PFS and OS were 35.7% and 50.0% in ADV-TK group respectively. ADV-TK/ganciclovir gene therapy was well tolerated, and no treatment-related severe adverse events were noted. Conclusion Our study demonstrated a notable improvement of PFS-6, PFS and OS in ADV-TK treated group, and the efficacy and safety appear to be comparable to other reported treatments used for recurrent HGG. ADV-TK gene therapy is therefore a valuable therapeutic option for recurrent HGG. PMID:26716896

  19. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  20. Canine adenovirus downstream processing protocol.

    PubMed

    Puig, Meritxell; Piedra, Jose; Miravet, Susana; Segura, María Mercedes

    2014-01-01

    Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %. PMID:24132487

  1. Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract.

    PubMed

    Terao, R; Honda, K; Hatano, E; Uehara, T; Yamamoto, M; Yamaoka, Y

    1998-09-01

    Proliferative cholangitis (PC) associated with hepatolithiasis develops the stricture of main bile ducts, and is the main cause of residual and/or recurrent stones after repeated treatments for hepatolithiasis. The aim of this study was to inhibit PC using the cytostatic gene therapy with direct adenovirus-mediated retinoblastoma (Rb) gene transfer to the biliary tract. PC was induced by introducing a fine nylon thread into the bile duct in a rat model. The adenovirus vector encoding a nonphosphorylatable, constitutively active form of retinoblastoma gene product (AdRb) was administered directly into the biliary tract. The adenovirus vector encoding beta-galactosidase (AdlacZ) was also given as a control. The bile duct wall thickness and 5'-bromodeoxyuridine (BrdU) labeling index were compared among uninfected, AdlacZ-infected, and AdRb-infected PC rats. The Rb expression in the bile duct was detected using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical study. AdRb-infected bile ducts showed inhibition of the epithelial and fibrous tissue proliferation and the peribiliary gland hyperplasia, resulting in a significant reduction of wall thickness compared with uninfected and AdlacZ-infected ones. The BrdU labeling index was 4.87% +/- 3.06% in the AdRb-infected bile ducts, while those of uninfected and AdlacZ-infected ones were 15.48% +/- 4.61% and 11.72% +/- 1.23%, respectively (P < .05). In conclusion, our cytostatic gene therapy approach using direct Rb gene transfer into the biliary tract suppressed PC in a rat model and may offer an effective therapeutic option for reducing recurrences following treatments against hepatolithiasis. PMID:9731547

  2. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

    NASA Astrophysics Data System (ADS)

    Fields, Rachel Jennifer

    The lung is an important target for gene and drug therapy of many diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), tubuerculosis (TB) and lung cancer. In fact, the pulmonary route has been employed as a means of delivering drugs for centuries, dating back 4000 years to India where inhaled vapors were used for medicinal purpose. Currently, pulmonary administration of small, hydrophobic drugs leads to rapid local and systemic absorption. However, delivery of large biomacromolecules, such as therapeutic genes, has not yet been accomplished. Here, I test the hypothesis that a rationally engineered nanoparticle (NP) vector can improve delivery of large biomacromolecules. . In this dissertation I tested this hypothesis using a hybrid NP delivery system consisting of a blend of poly(lactic-co-glycolic acid) (PLGA) and a poly(beta-amino ester) (PBAE), a cationic polymer that is particularly useful for delivery of nucleic acids.. PBAE/PLGA nanoparticles (15% PBAE) loaded with plasmid DNA were surface modified with cell-penetrating peptides (CPPs) via a PEGylated phospholipid linker. This optimized NP formulation was able to induce substantial intracellular uptake and transfect lung epithelial cells in vitro while imparting minimal cellular toxicity. In order to determine the most effective method to deliver these NPs to the lung I used fluorescently labeled particles to study the biodistribution of particles after administration to the lung of mice via various administration routes. I determined that the intranasal route was most effective. I further investigated this route and determined that an average of 37.1 +/- 15.1 % of lung cells had NP association after 4hrs. I also investigated the association of particles with different lung cell types like macrophages and alveolar epithelial cells and determined that our best particle formulations associated with approximately 80% of both of these cell types. To demonstrate the ability of the

  3. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.

    PubMed Central

    Yang, Y; Li, Q; Ertl, H C; Wilson, J M

    1995-01-01

    Recombinant adenoviruses are an attractive vehicle for gene therapy to the lung in the treatment of cystic fibrosis (CF). First-generation viruses deleted of E1a and E1b transduce genes into airway epithelial cells in vivo; however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. In this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive transfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. Our studies indicate that major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes are activated in response to newly synthesized antigens, leading to destruction of virus infected cells and loss of transgene expression. Major histocompatibility complex class II-associated presentation of exogenous viral antigens activates CD4+ T-helper (TH) cells of the TH1 subset and, to a lesser extent, of the TH2 subset. CD4+ cell-mediated responses are insufficient in the absence of cytotoxic T cells to completely eliminate transgene containing cells; however, they contribute to the formation of neutralizing antibodies in the airway which block subsequent adenovirus-mediated gene transfer. Definition of immunological barriers to gene therapy of cystic fibrosis should facilitate the design of rational strategies to overcome them. PMID:7884845

  4. Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer.

    PubMed

    Freytag, Svend O; Barton, Kenneth N; Brown, Stephen L; Narra, Vinod; Zhang, Yingshu; Tyson, Don; Nall, Colleen; Lu, Mei; Ajlouni, Munther; Movsas, Benjamin; Kim, Jae Ho

    2007-09-01

    In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer. PMID:17551507

  5. Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

    PubMed

    Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

    2016-01-01

    Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage. PMID:26558992

  6. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer.

    PubMed

    Pickles, R J; McCarty, D; Matsui, H; Hart, P J; Randell, S H; Boucher, R C

    1998-07-01

    Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium. PMID:9621064

  7. Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer

    PubMed Central

    Pickles, Raymond J.; McCarty, Douglas; Matsui, Hirotoshi; Hart, Pádraig J.; Randell, Scott H.; Boucher, Richard C.

    1998-01-01

    Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of αvβ3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium. PMID:9621064

  8. Genomic and Bioinformatics Analysis of HAdV-4, a Human Adenovirus Causing Acute Respiratory Disease: Implications for Gene Therapy and Vaccine Vector Development

    PubMed Central

    Purkayastha, Anjan; Ditty, Susan E.; Su, Jing; McGraw, John; Hadfield, Ted L.; Tibbetts, Clark; Seto, Donald

    2005-01-01

    Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD. PMID:15681456

  9. Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development.

    PubMed

    Purkayastha, Anjan; Ditty, Susan E; Su, Jing; McGraw, John; Hadfield, Ted L; Tibbetts, Clark; Seto, Donald

    2005-02-01

    Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD. PMID:15681456

  10. An intestinal Trojan horse for gene delivery

    NASA Astrophysics Data System (ADS)

    Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun

    2015-02-01

    The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases.

  11. An intestinal Trojan horse for gene delivery.

    PubMed

    Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun

    2015-03-14

    The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases. PMID:25619169

  12. Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

    PubMed Central

    Teramoto, S.; Bartlett, J. S.; McCarty, D.; Xiao, X.; Samulski, R. J.; Boucher, R. C.

    1998-01-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  13. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

    PubMed

    Teramoto, S; Bartlett, J S; McCarty, D; Xiao, X; Samulski, R J; Boucher, R C

    1998-11-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  14. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

    NASA Astrophysics Data System (ADS)

    Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

    2000-04-01

    Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

  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. Lipid Nanoparticles for Ocular Gene Delivery

    PubMed Central

    Wang, Yuhong; Rajala, Ammaji; Rajala, Raju V. S.

    2015-01-01

    Lipids contain hydrocarbons and are the building blocks of cells. Lipids can naturally form themselves into nano-films and nano-structures, micelles, reverse micelles, and liposomes. Micelles or reverse micelles are monolayer structures, whereas liposomes are bilayer structures. Liposomes have been recognized as carriers for drug delivery. Solid lipid nanoparticles and lipoplex (liposome-polycation-DNA complex), also called lipid nanoparticles, are currently used to deliver drugs and genes to ocular tissues. A solid lipid nanoparticle (SLN) is typically spherical, and possesses a solid lipid core matrix that can solubilize lipophilic molecules. The lipid nanoparticle, called the liposome protamine/DNA lipoplex (LPD), is electrostatically assembled from cationic liposomes and an anionic protamine-DNA complex. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers. SLNs are extensively used to deliver drugs to the cornea. LPD nanoparticles are used to target the retina. Age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy are the most common retinal diseases in humans. There have also been promising results achieved recently with LPD nanoparticles to deliver functional genes and micro RNA to treat retinal diseases. Here, we review recent advances in ocular drug and gene delivery employing lipid nanoparticles. PMID:26062170

  17. Liposomes for Use in Gene Delivery

    PubMed Central

    Balazs, Daniel A.; Godbey, WT.

    2011-01-01

    Liposomes have a wide array of uses that have been continuously expanded and improved upon since first being observed to self-assemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Liposomes are often used to deliver a molecular cargo such as DNA for therapeutic benefit. The lipids used to form such lipoplexes can be cationic, anionic, neutral, or a mixture thereof. Herein physical packing parameters and specific lipids used for gene delivery will be discussed, with lipids classified according to overall charge. PMID:21490748

  18. The Ad5 [E1-, E2b-]-based vector: a new and versatile gene delivery platform

    NASA Astrophysics Data System (ADS)

    Jones, Frank R.; Gabitzsch, Elizabeth S.; Balint, Joseph P.

    2015-05-01

    Based upon advances in gene sequencing and construction, it is now possible to identify specific genes or sequences thereof for gene delivery applications. Recombinant adenovirus serotype-5 (Ad5) viral vectors have been utilized in the settings of gene therapy, vaccination, and immunotherapy but have encountered clinical challenges because they are recognized as foreign entities to the host. This recognition leads to an immunologic clearance of the vector that contains the inserted gene of interest and prevents effective immunization(s). We have reported on a new Ad5-based viral vector technology that can be utilized as an immunization modality to induce immune responses even in the presence of Ad5 vector immunity. We have reported successful immunization and immunotherapy results to infectious diseases and cancers. This improved recombinant viral platform (Ad5 [E1-, E2b-]) can now be utilized in the development of multiple vaccines and immunotherapies.

  19. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

    PubMed Central

    Askanas, V; McFerrin, J; Baqué, S; Alvarez, R B; Sarkozi, E; Engel, W K

    1996-01-01

    As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8577761

  20. Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure.

    PubMed

    Seth, P

    1994-12-15

    Adenoviruses have been previously shown to enhance the delivery of many ligands including proteins and plasmid DNAs to the cells. The key biochemical step during this process is the ability of adenovirus to disrupt (lyse) the endosome membrane releasing the co-internalized virus and the other ligands into the cytosol (Seth et al, 1986, In: Adenovirus attachment and entry into cells, pp 191-195, American Society for Microbiology, Washington, D.C.). To understand the role of the adenovirus proteins involved in the endosome lysis, it is further shown here that empty capsids of adenovirus also possess this membrane vesicle lytic activity; though the activity is about 5-times lower than the adenovirus. Incubation of adenovirus with low concentration of ionic detergent or brief exposure to 45 degrees C destroyed this lytic activity without affecting the adenovirus binding to cell surface receptor, suggesting the lytic activity of adenovirus to be of enzymatic nature. However, exposing adenovirus to conditions that can disrupt adenovirus capsid structure such as heating at 65 degrees C, treating with 0.5% SDS, treating with different proteases, dialyzing against no glycerol buffer, treating with 6 M urea or with 10% pyridine, and sonication destroyed the adenovirus-associated lytic activity. Results suggest the requirement of an intact capsid structure for adenovirus-mediated lysis of the endosome. PMID:7802664

  1. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells. PMID:26280081

  2. Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: Toward realistic long-term neurological gene therapy for chronic diseases

    PubMed Central

    Thomas, Clare E.; Schiedner, Gudrun; Kochanek, Stefan; Castro, Maria G.; Löwenstein, Pedro R.

    2000-01-01

    Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8+ cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders. PMID:10840055

  3. Engineering Biomaterial Systems to Enhance Viral Vector Gene Delivery

    PubMed Central

    Jang, Jae-Hyung; Schaffer, David V; Shea, Lonnie D

    2011-01-01

    Integrating viral gene delivery with engineered biomaterials is a promising strategy to overcome a number of challenges associated with virus-mediated gene delivery, including inefficient delivery to specific cell types, limited tropism, spread of vectors to distant sites, and immune responses. Viral vectors can be combined with biomaterials either through encapsulation within the material or immobilization onto a material surface. Subsequent biomaterial-based delivery can increase the vector's residence time within the target site, thereby potentially providing localized delivery, enhancing transduction, and extending the duration of gene expression. Alternatively, physical or chemical modification of viral vectors with biomaterials can be employed to modulate the tropism of viruses or reduce inflammatory and immune responses, both of which may benefit transduction. This review describes strategies to promote viral gene delivery technologies using biomaterials, potentially providing opportunities for numerous applications of gene therapy to inherited or acquired disorders, infectious disease, and regenerative medicine. PMID:21629221

  4. Retrograde Ductal Administration of the Adenovirus-mediated NDRG2 Gene Leads to Improved Sialaden Hypofunction in Estrogen-deficient Rats

    PubMed Central

    Li, Yan; Liu, Changhao; Hou, Wugang; Li, Yang; Ma, Ji; Lin, Kaifeng; Situ, Zhenqiang; Xiong, Lize; Li, Shaoqing; Yao, Libo

    2014-01-01

    One of the most common oral manifestations of menopause is xerostomia. Oral dryness can profoundly affect quality of life and interfere with basic daily functions, such as chewing, deglutition, and speaking. Although the feeling of oral dryness can be ameliorated after estrogen supplementation, the side effects of estrogen greatly restrict its application. We previously found that N-myc downstream-regulated gene 2 (NDRG2) is involved in estrogen-mediated ion and fluid transport in a cell-based model. In the present study, we used an ovariectomized rat model to mimic xerostomia in menopausal women and constructed two adenovirus vectors bearing NDRG2 to validate their therapeutic potential. Ovariectomized rats exhibited severe sialaden hypofunction, including decreased saliva secretion and ion reabsorption as well as increased water intake. Immunohistochemistry revealed that the expression of NDRG2 and Na+ reabsorption-related Na+/K+-ATPase and epithelial sodium channels (EnaC) decreased in ovariectomized rat salivary glands. We further showed that the localized delivery of NDRG2 improved the dysfunction of Na+ and Cl− reabsorption. In addition, the saliva flow rate and water drinking recovered to normal. This study elucidates the mechanism of estrogen deficiency-mediated xerostomia or sialaden hypofunction and provides a promising strategy for therapeutic intervention. PMID:24343104

  5. Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

    PubMed

    Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

    2016-01-01

    Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy. PMID:26909111

  6. Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma

    PubMed Central

    Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

    2016-01-01

    Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy. PMID:26909111

  7. Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

    PubMed Central

    Weiss, R S; Lee, S S; Prasad, B V; Javier, R T

    1997-01-01

    An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties. PMID:9032316

  8. Induction of CD8(+) T cell responses and protective efficacy following microneedle-mediated delivery of a live adenovirus-vectored malaria vaccine.

    PubMed

    Pearson, Frances E; O'Mahony, Conor; Moore, Anne C; Hill, Adrian V S

    2015-06-22

    There is an urgent need for improvements in vaccine delivery technologies. This is particularly pertinent for vaccination programmes within regions of limited resources, such as those required for adequate provision for disposal of used needles. Microneedles are micron-sized structures that penetrate the stratum corneum of the skin, creating temporary conduits for the needle-free delivery of drugs or vaccines. Here, we aimed to investigate immunity induced by the recombinant simian adenovirus-vectored vaccine ChAd63.ME-TRAP; currently undergoing clinical assessment as a candidate malaria vaccine, when delivered percutaneously by silicon microneedle arrays. In mice, we demonstrate that microneedle-mediated delivery of ChAd63.ME-TRAP induced similar numbers of transgene-specific CD8(+) T cells compared to intradermal (ID) administration with needle-and-syringe, following a single immunisation and after a ChAd63/MVA heterologous prime-boost schedule. When mice immunised with ChAd63/MVA were challenged with live Plasmodium berghei sporozoites, microneedle-mediated ChAd63.ME-TRAP priming demonstrated equivalent protective efficacy as did ID immunisation. Furthermore, responses following ChAd63/MVA immunisation correlated with a specific design parameter of the array used ('total array volume'). The level of transgene expression at the immunisation site and skin-draining lymph node (dLN) was also linked to total array volume. These findings have implications for defining silicon microneedle array design for use with live, vectored vaccines. PMID:25839104

  9. Production and clinical development of nanoparticles for gene delivery

    PubMed Central

    Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2016-01-01

    Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected. PMID:27088105

  10. 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

  11. Microneedles As a Delivery System for Gene Therapy.

    PubMed

    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

  12. Recent progress in nanomaterials for gene delivery applications.

    PubMed

    Keles, Erhan; Song, Yang; Du, Dan; Dong, Wen-Ji; Lin, Yuehe

    2016-08-16

    Nanotechnology-based gene delivery is the division of nanomedicine concerned with the synthesis, characterization, and functionalization of nanomaterials to be used in targeted-gene delivery applications. Nanomaterial-based gene delivery systems hold great promise for curing fatal inherited and acquired diseases, including neurological disorders, cancer, cardiovascular diseases, and acquired immunodeficiency syndrome (AIDS). However, their use in clinical applications is still controversial. To date, the Food and Drug Administration (FDA) has not approved any gene delivery system because of the unknown long-term toxicity and the low gene transfection efficiency of nanomaterials in vivo. Compared to viral vectors, nonviral gene delivery vectors are characterized by a low preexisting immunogenicity, which is important for preventing a severe immune response. In addition, nonviral vectors provide higher loading capacity and ease of fabrication. For these reasons, this review article focuses on applications of nonviral gene delivery systems, including those based on lipids, polymers, graphene, and other inorganic nanoparticles, and discusses recent advances in nanomaterials for gene therapy. Methods of synthesizing these nanomaterials are briefly described from a materials science perspective. Also, challenges, critical issues, and concerns about the in vivo applications of nanomaterial-based gene delivery systems are discussed. It should be noted that this article is not a comprehensive review of the literature. PMID:27480033

  13. Adenovirus vector induced Innate Immune responses: Impact upon efficacy and toxicity in gene therapy and vaccine applications

    PubMed Central

    Hartman, Zachary C.; Appledorn, Daniel M.; Amalfitano, Andrea

    2013-01-01

    Extensively characterized, modified, and employed for a variety of purposes, Adenovirus (Ad) vectors are generally regarded as having great potential by many applied virologists who wish to manipulate and use viral biology to achieve beneficial clinical outcomes. Despite widespread functional prominence and utility, (i.e.: Ad based clinical trials have begun to progress to critical Phase III levels, it has recently become apparent that investigations regarding the innate immune response to Ads may reveal not only reasons behind previous failures, but also reveal novel insights that will allow for safer, more efficacious uses of this important gene transfer platform. Insights gained by the exploration of Ad induced innate immune responses will likely be most important to the fields of vaccine development, since Ad based vaccines are highly acknowledged as one of the more promising vaccine platforms in development today. Adenovirus is currently known to interact with several different extracellular, intracellular, and membrane bound innate immune sensing systems. Past and recent studies involving manipulation of the Ad infectious cycle as well as use of different mutants have shed light on some of the initiation mechanisms underlying Ad induced immune responses. More recent studies using microarray based analyses, genetically modified cell lines and/or mouse mutants, and advanced generation Ad vectors have revealed important new insights into the scope and mechanism of this cellular defensive response. This review is an attempt to synthesize these studies, update Ad biologists to the current knowledge surrounding these increasingly important issues, as well point areas where future research should be directed. It should also serve as a sobering reality to researchers exploring the use of any gene transfer vector, as to the complexities potentially involved when contemplating use of such vectors for human applications. PMID:18036698

  14. Getting the most from gene delivery by repeated DNA transfections

    NASA Astrophysics Data System (ADS)

    Montani, Maura; Marchini, Cristina; Badillo Pazmay, Gretta Veronica; Andreani, Cristina; Bartolacci, Caterina; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2015-06-01

    Intracellular delivery of reporter genes causes cells to be luminescent or fluorescent, this condition being of tremendous relevance in applied physics research. Potential applications range from the study of spatial distribution and dynamics of plasma membrane and cytosolic proteins up to the rational design of nanocarriers for gene therapy. Since efficiency of gene delivery is the main limit in most biophysical studies, versatile methods that can maximize gene expression are urgently needed. Here, we describe a robust methodology based on repeated gene delivery in mammalian cells. We find this procedure to be much more efficient than the more traditional route of gene delivery making it possible to get high-quality data without affecting cell viability. Implications for biophysical investigations are discussed.

  15. In Vivo Gene Delivery by Nonviral Vectors: Overcoming Hurdles?

    PubMed Central

    Zhang, Yuan; Satterlee, Andrew; Huang, Leaf

    2012-01-01

    The promise of cancer gene therapeutics is hampered by difficulties in the in vivo delivery to the targeted tumor cells, and systemic delivery remains to be the biggest challenge to be overcome. Here, we concentrate on systemic in vivo gene delivery for cancer therapy using nonviral vectors. In this review, we summarize the existing delivery barriers together with the requirements and strategies to overcome these problems. We will also introduce the current progress in the design of nonviral vectors, and briefly discuss their safety issues. PMID:22525514

  16. Large numbers of random point and cluster mutations within the adenovirus VA I gene allow characterization of sequences required for efficient transcription.

    PubMed Central

    Snouwaert, J; Bunick, D; Hutchison, C; Fowlkes, D M

    1987-01-01

    We have isolated clones with well over 100 randomly dispersed point mutations distributed throughout the 5' half of chemically synthesized adenovirus type 2 VA I genes. In addition, we have isolated clusters of mutations targeted to the regions corresponding to the A and B block consensus sequences of eukaryotic tRNA and adenovirus VA genes. In vitro analyses of these constructs have allowed us to survey in detail the importance of DNA sequence to transcriptional efficiency. Our analyses demonstrate that certain constructs with radically substituted A block regions can be transcribed efficiently. In contrast, there is little tolerance for variation in the sequence within the B block region. We propose that the B block sequence should be R-G-A/T-T-C-R-A-N-N-C for optimal transcriptional efficiency of the VA I gene in mammalian cells. PMID:3671085

  17. Development of lentiviral vectors for antiangiogenic gene delivery.

    PubMed

    Shichinohe, T; Bochner, B H; Mizutani, K; Nishida, M; Hegerich-Gilliam, S; Naldini, L; Kasahara, N

    2001-11-01

    Growth and metastasis of malignant tumors requires angiogenesis. Inhibition of tumor-induced angiogenesis may represent an effective cytostatic strategy. We have constructed recombinant self-inactivating lentiviral vectors expressing angiostatin and endostatin, and have tested their antiangiogenic activities. As VSV-G-pseudotyped lentiviral vectors showed low relative transduction titers on bovine aortic and human umbilical vein endothelial cells, it was difficult to achieve significant inhibition of endothelial cell growth by lentivirus-mediated antiangiogenic gene transfer directly to endothelial cells without concomitant vector-associated cytotoxicity. However, lentivirus vectors could efficiently and stably transduce T24 human bladder cancer cells that are relatively resistant to adenovirus infection due to loss of coxsackievirus-adenovirus receptor expression. Long-term expression and secretion of angiostatin and endostatin from lentivirus-transduced T24 cells resulted in significant inhibition of cellular proliferation on coculture with endothelial cells. This report represents the first use of lentivirus-based vectors to deliver the antiangiogenic factors, angiostatin and endostatin, and suggests the potential utility of antiangiogenic gene therapy with lentiviral vectors for the treatment of cancer. PMID:11773978

  18. Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

    PubMed Central

    Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D.; Chorro, Laurent; Carlin, Leo M.; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S.

    2013-01-01

    Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs. PMID:23386724

  19. Adenovirus E1A gene induction of susceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells.

    PubMed Central

    Cook, J L; May, D L; Lewis, A M; Walker, T A

    1987-01-01

    Rodent cells immortalized by the E1A gene of nononcogenic adenoviruses are susceptible to lysis by natural killer (NK) cells and activated macrophages. This cytolysis-susceptible phenotype may contribute to the rejection of adenovirus-transformed cells by immunocompetent animals. Such increased cytolytic susceptibility has also been observed with infected rodent cells. This infection model provided a means to study the role of E1A gene products in induction of cytolytic susceptibility without cell selection during transformation. Deletion mutations outside of the E1A gene had no effect on adenovirus type 2 (Ad2) or Ad5 induction of cytolytic susceptibility in infected hamster cells, while E1A-minus mutant viruses could not induce this phenotype. E1A mutant viruses that induced expression of either E1A 12S or 13S mRNA in infected cells were competent to induce cytolytic susceptibility. Furthermore, there was a correlation between the accumulation of E1A gene products in Ad5-infected cells and the level of susceptibility of such target cells to lysis by NK cells. The results of coinfection studies indicated that the E1A gene products of highly oncogenic Ad12 could not complement the lack of induction of cytolytic susceptibility by E1A-minus Ad5 virus in infected cells and also could not block induction of this infected-cell phenotype by Ad5. These data suggest that expression of the E1A gene of nononcogenic adenoviruses may cause the elimination of infected cells by the immunologically nonspecific host inflammatory cell response prior to cellular transformation. The lack of induction of this cytolysis-susceptible phenotype by Ad12 E1A may result in an increased persistence of Ad12-infected cells in vivo and may lead to an increased Ad12-transformed cell burden for the host. Images PMID:2959793

  20. Breast cancer gene therapy using an adenovirus encoding human IL-2 under control of mammaglobin promoter/enhancer sequences.

    PubMed

    Chaurasiya, S; Hew, P; Crosley, P; Sharon, D; Potts, K; Agopsowicz, K; Long, M; Shi, C; Hitt, M M

    2016-06-01

    Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful. PMID:27151235

  1. In vivo gene delivery by cationic tetraamino fullerene.

    PubMed

    Maeda-Mamiya, Rui; Noiri, Eisei; Isobe, Hiroyuki; Nakanishi, Waka; Okamoto, Koji; Doi, Kent; Sugaya, Takeshi; Izumi, Tetsuro; Homma, Tatsuya; Nakamura, Eiichi

    2010-03-23

    Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene. PMID:20194788

  2. Improving gene transfer in human renal carcinoma cells: Utilization of adenovirus vectors containing chimeric type 5 and type 35 fiber proteins

    PubMed Central

    ACHARYA, BISHNU; TERAO, SHUJI; SUZUKI, TORU; NAOE, MICHIO; HAMADA, KATSUYUKI; MIZUGUCHI, HIROYUKI; GOTOH, AKINOBU

    2010-01-01

    The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally low due to the down-regulated expression of Coxsackie and adenovirus receptor (CAR) in target cells. By contrast, the infectivity of adenovirus serotype 35 vectors depends on the binding rate to CD46 receptor, independent of CAR. In this study, we examined whether an adenovirus vector containing chimeric type 5 and type 35 fiber proteins (Ad5/F35) increases transduction efficiency compared to Ad5 vector in human renal carcinoma cells in vitro. The expression of CAR was much lower in the human renal carcinoma cells than in control HEK293 cells. By contrast, the expression of CD46 was similar and perhaps at a higher level in the human renal carcinoma cells than in the HEK293 cells. The transduction efficacy of Ad5/F35 vector was dramatically higher compared to that of Ad5 in human renal carcinoma cells, and was correlated to the expression of CD46. Thus, Ad5/35 vector may be useful for the development of novel gene therapy approaches to renal cell carcinoma. PMID:22993573

  3. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  4. Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

    PubMed Central

    Bhat, R A; Metz, B; Thimmappaya, B

    1983-01-01

    The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes. Images PMID:6656762

  5. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    SciTech Connect

    Mu, Haixi; Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu; Li, Lili; Ren, Guosheng; Xu, Yongzhu; Zhou, Xiangyang; Xiang, Tingxiu

    2015-03-15

    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

  6. In vivo gene delivery of XIAP protects against myocardial apoptosis and infarction following ischemia/reperfusion in conscious rabbits

    PubMed Central

    Kim, Song-Jung; Kuklov, Alex; Crystal, George J.

    2011-01-01

    Aims We tested the hypothesis that an in vivo gene delivery of the pro-survival protein XIAP (X-chromosome linked inhibitor of apoptosis protein) protects against myocardial apoptosis and infarction following ischemia/reperfusion. Main Methods Nineteen rabbits were chronically instrumented with an hydraulic occluder placed around the circumflex coronary artery. Adenovirus harboring XIAP (Ad.XIAP; 1×1010 pfu/ml) or β-galactosidase (5×109 pfu/ml), as a control, was constructed and transfected into the heart using a catheter place into the left ventricle accompanied by cross-clamping. 1-2 weeks after gene delivery, myocardial ischemia was induced by a 30-min occlusion followed by reperfusion for four days. Protein expression was determined by Western blot and Apoptosis (% of myocytes) was quantified by TUNEL staining. Key Findings Myocardial infarct size, expressed as a fraction of the area at risk, was reduced in Ad.XIAP (n=5) compared to control (n=7) rabbits (21±3% vs. 30±2%, p<0.05). Apoptosis was reduced in Ad.XIAP rabbits compared to control rabbits (2.96±0.68% vs. 8.98±1.84%, p<0.01). This was associated with an approximate 60% decrease in the cleaved caspase-3 level in Ad.XIAP rabbits compared to control rabbits. Significances The current findings demonstrate that overexpression of XIAP via in vivo delivery in an adenovirus can reduce both myocardial apoptosis and infarction following ischemia/reperfusion, at least in part, due to the ability of XIAP to inhibit caspase-3. These findings confirm previous work suggesting a link between myocardial apoptosis and infarction i.e., anti-apoptotic therapy was effective in reducing myocardial infarct size. PMID:21277870

  7. 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.

  8. Central Nervous System Delivery of Helper-Dependent Canine Adenovirus Corrects Neuropathology and Behavior in Mucopolysaccharidosis Type VII Mice

    PubMed Central

    Ariza, Lorena; Giménez-Llort, Lydia; Cubizolle, Aurélie; Pagès, Gemma; García-Lareu, Belén; Serratrice, Nicolas; Cots, Dan; Thwaite, Rosemary; Chillón, Miguel; Kremer, Eric J.

    2014-01-01

    Abstract Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing β-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in β-glucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of β-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease. PMID:24299455

  9. Theranostic agents for intracellular gene delivery with spatiotemporal imaging

    PubMed Central

    Knipe, Jennifer M.; Peters, Jonathan T.; Peppas, Nicholas A.

    2013-01-01

    Gene therapy is the modification of gene expression to treat a disease. However, efficient intracellular delivery and monitoring of gene therapeutic agents is an ongoing challenge. Use of theranostic agents with suitable targeted, controlled delivery and imaging modalities has the potential to greatly advance gene therapy. Inorganic nanoparticles including magnetic nanoparticles, gold nanoparticles, and quantum dots have been shown to be effective theranostic agents for the delivery and spatiotemporal tracking of oligonucleotides in vitro and even a few cases in vivo. Major concerns remain to be addressed including cytotoxicity, particularly of quantum dots; effective dosage of nanoparticles for optimal theranostic effect; development of real-time in vivo imaging; and further improvement of gene therapy efficacy. PMID:23606894

  10. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population

    PubMed Central

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment. PMID:27485384

  11. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

    PubMed

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-09-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment. PMID:27485384

  12. Adenoviral Delivery of the EMX2 Gene Suppresses Growth in Human Gastric Cancer

    PubMed Central

    Li, Jie; Mo, Minli; Chen, Zhao; Chen, Zhe; Sheng, Qing; Mu, Hang; Zhang, Fang; Zhang, Yi; Zhi, Xiu-Yi; Li, Hui; He, Biao; Zhou, Hai-Meng

    2012-01-01

    Background EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. Results In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. Conclusion Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer. PMID:23029345

  13. Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion

    PubMed Central

    Prost, Sandrine; Sheahan, Sharon; Rannie, Dominic; Harrison, David J.

    2001-01-01

    This study evaluates the utility of Cre-expressing adenovirus for deletion of floxed genes in primary cells using primary murine hepatocytes. Adenovirus infection was very efficient, even at very low MOI (>95% infection at a MOI of 6) and did not reduce viability. High level LacZ expression was cytotoxic to hepatocytes but Cre expression had no effect on viability. Cre-mediated recombination was completed within a timespan that permits experimentation during primary culture (>95% recombination after 24 h), independently of the number of floxed alleles per cell. Recombination did not induce p53 or produce cytological nuclear abnormalities (even in polyploid cells). Contrary to expectation, deletion of DNA ligase 1 did not alter cell cycle progression, although Cre expression hastens entry to S phase from G1, independently of the presence of floxed sequences. We conclude that adenovirus-mediated deletion of floxed alleles in primary cells is a straightforward and highly efficient tool for conducting preliminary studies of conditional gene targeting. Primary cells have advantages of differentiation, relative purity and ease of experimentation within controlled conditions, while avoiding confounding problems encountered in vivo (i.e. target cell specificity, kinetics and level of recombination, and elicitation of inflammatory and immune responses). This system could help identify important phenotypic effects and design and interpret in vivo studies. PMID:11504888

  14. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  15. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene

    PubMed Central

    De Kozak, Y; Thillaye-Goldenberg, B; Naud, M -C; Viana Da Costa, A; Auriault, C; Verwaerde, C

    2002-01-01

    Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-γ and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis. PMID:12390308

  17. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  18. Utilising inorganic nanocarriers for gene delivery.

    PubMed

    Loh, Xian Jun; Lee, Tung-Chun; Dou, Qingqing; Deen, G Roshan

    2016-01-01

    The delivery of genetic materials into cells to elicit cellular responses has been extensively studied by biomaterials scientists globally. Many materials such as lipids, peptides, viruses, synthetically modified cationic polymers and certain inorganic nanomaterials could be used to complex the negatively charged plasmids and deliver the formed package into cells. The recent literature on the delivery of genetic materials utilising inorganic nanoparticles is carefully examined in this review. We have picked out the most relevant references and concisely summarised the findings with illustrated examples. We further propose alternative approaches and suggest future pathways towards the practical use of multifunctional nanocarriers. PMID:26484365

  19. Adenovirus-mediated artificial MicroRNAs targeting matrix or nucleoprotein genes protect mice against lethal influenza virus challenge.

    PubMed

    Zhang, H; Tang, X; Zhu, C; Song, Y; Yin, J; Xu, J; Ertl, H C J; Zhou, D

    2015-08-01

    Influenza virus (IV) infection is a major public health problem, causing millions of cases of severe illness and as many as 500 000 deaths each year worldwide. Given the limitations of current prevention or treatment of acute influenza, novel therapies are needed. RNA interference (RNAi) through microRNAs (miRNA) is an emerging technology that can suppress virus replication in vitro and in vivo. Here, we describe a novel strategy for the treatment of infuenza based on RNAi delivered by a replication-defective adenovirus (Ad) vector, derived from chimpanzee serotype 68 (AdC68). Our results showed that artificial miRNAs (amiRNAs) specifically targeting conserved regions of the IV genome could effectively inhibit virus replication in human embryonic kidney 293 cells. Moreover, our results demonstrated that prophylactic treatment with AdC68 expressing amiRNAs directed against M1, M2 or nucleoprotein genes of IV completely protected mice from homologous A/PR8 virus challenge and partially protected the mice from heterologous influenza A virus strains such as H9N2 and H5N1. Collectively, our data demonstrate that amiRNAs targeting the conserved regions of influenza A virus delivered by Ad vectors should be pursued as a novel strategy for prophylaxis of IV infection in humans and animals. PMID:25835311

  20. Nanoscale structure of protamine/DNA complexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Motta, Simona; Brocca, Paola; Del Favero, Elena; Rondelli, Valeria; Cantù, Laura; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2013-02-01

    Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

  1. 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

  2. Efficacy of helper-dependent adenovirus vector-mediated gene therapy in murine glycogen storage disease type Ia.

    PubMed

    Koeberl, Dwight D; Sun, B; Bird, A; Chen, Y T; Oka, K; Chan, L

    2007-07-01

    Genetic deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, also known as von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia and growth retardation. We tested whether helper-dependent adenovirus (HDAd)-mediated hepatic delivery of G6Pase would lead to prolonged survival and sustained correction of the metabolic abnormalities in G6Pase knockout (KO) mice, a model for a severe form of GSD-Ia. An HDAd vector encoding G6Pase was administered intravenously (2 or 5 x 10(12)vector particles/kg) to 2-week-old (w.o.) G6Pase-KO mice. Following HDAd vector administration survival was prolonged to a median of 7 months, in contrast to untreated affected mice that did not survive past 3 weeks of age. G6Pase levels increased more than tenfold between 3 days and 28 weeks after HDAd injection (P < 0.03). The weights of untreated 2 w.o. G6Pase-KO mice were approximately half those of their unaffected littermates, and treatment stimulated their growth to the size of wild-type mice. Severe hypoglycemia and hypercholesterolemia, which are hallmarks of GSD-Ia both in humans and in mice, were also restored to normalcy by the treatment. Glycogen accumulation in the liver was markedly reduced. The efficacy of HDAd-G6Pase treatment in reversing the physiological and biochemical abnormalities associated with GSD-Ia in affected G6Pase-KO mice justifies further preclinical evaluation in murine and canine models of GSD-Ia. PMID:17505475

  3. Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

    PubMed Central

    Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

    2015-01-01

    SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

  4. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

    PubMed

    Gilbert, R; Nalbantoglu, J; Howell, J M; Davies, L; Fletcher, S; Amalfitano, A; Petrof, B J; Kamen, A; Massie, B; Karpati, G

    2001-09-20

    Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer. PMID:11560768

  5. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  6. Engineered nonviral nanocarriers for intracellular gene delivery applications.

    PubMed

    Ojea-Jiménez, Isaac; Tort, Olivia; Lorenzo, Julia; Puntes, Victor F

    2012-10-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully. PMID:22972254

  7. Smart Polymeric Nanoparticles for Cancer Gene Delivery

    PubMed Central

    2015-01-01

    The massive amount of human genetic information already available has accelerated the identification of target genes, making gene and nucleic acid therapy the next generation of medicine. Nanoparticle (NP)-based anticancer gene therapy treatment has received significant interest in this evolving field. Recent advances in vector technology have improved gene transfection efficiencies of nonviral vectors to a level similar to viruses. This review serves as an introduction to surface modifications of NPs based on polymeric structural improvements and target moieties. A discussion regarding the future perspective of multifunctional NPs in cancer therapy is also included. PMID:25531409

  8. Modulation of Treg function improves adenovirus vector-mediated gene expression in the airway.

    PubMed

    Nagai, Y; Limberis, M P; Zhang, H

    2014-02-01

    Virus vector-mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the cystic fibrosis transmembrane conductance regulator gene. The pathological proinflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limits the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (T(reg)) and to improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway. PMID:24385144

  9. 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

  10. The Effect of Adenovirus-Mediated Gene Expression of FHIT in Small Cell Lung Cancer Cells

    PubMed Central

    Zandi, Roza; Xu, Kai; Poulsen, Hans S.; Roth, Jack A.; Ji, Lin

    2012-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in combination with the mutant p53-reactivating molecule, PRIMA-1Met/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1Met/APR-246, a synergistic cell growth inhibition was achieved. PMID:22085272

  11. Effects of capsid-modified oncolytic adenoviruses and their combinations with gemcitabine or silica gel on pancreatic cancer.

    PubMed

    Kangasniemi, Lotta; Parviainen, Suvi; Pisto, Tommi; Koskinen, Mika; Jokinen, Mika; Kiviluoto, Tuula; Cerullo, Vincenzo; Jalonen, Harry; Koski, Anniina; Kangasniemi, Anna; Kanerva, Anna; Pesonen, Sari; Hemminki, Akseli

    2012-07-01

    Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach. PMID:21834073

  12. Surface modification of nonviral nanocarriers for enhanced gene delivery.

    PubMed

    Fortier, Charles; Durocher, Yves; De Crescenzo, Gregory

    2014-01-01

    Biomedical nanotechnology has given a new lease of life to gene therapy with the ever-developing and ever-diversifying nonviral gene delivery nanocarriers. These are designed to pass a series of barriers in order to bring their nucleic acid cargo to the right subcellular location of particular cells. For a given application, each barrier has its dedicated strategy, which translates into a physicochemical, biological and temporal identity of the nanocarrier surface. Different strategies have thus been explored to implement adequate surface identities on nanocarriers over time for systemic delivery. In that context, this review will mainly focus on organic nanocarriers, for which these strategies will be described and discussed. PMID:24354815

  13. Tissue Inhibitor of Metalloproteinase-2 Gene Delivery Ameliorates Post-Infarction Cardiac Remodeling

    PubMed Central

    Ramani, Ravi; Nilles, Kathleen; Gibson, Gregory; Burkhead, Benjamin; Mathier, Michael; McNamara, Dennis; McTiernan, Charles F.

    2011-01-01

    Hypothesis Adenoviral-mediated (AdV-T2) overexpression of TIMP-2 would blunt ventricular remodeling and improve survival in a murine model of chronic ischemic injury. Methods Male mice (n=124) aged 10–14 weeks underwent either 1) left coronary artery ligation to induce myocardial infarction (MI group, n=36), 2) myocardial injection of 6×1010 viral particles of AdV-T2 immediately post-MI (MI+T2 group, n=30), 3) myocardial injection of 6×1010 viral particles of a control adenovirus (MI+Ct, n=38), or 4) received no intervention (controls, n=20). On post-MI day 7, surviving mice (n=79) underwent echocardiographic, immunohistochemical and biochemical analysis. Results In infarcted animals, the MI+T2 group demonstrated improved survival (p< 0.02), better preservation of developed pressure and ventricular diameter (p<0.04), and the lowest expression and activity of MMP-2 and MMP-9 (P<0.04) compared with MI and MI+Ct groups.. All infarcted hearts displayed significantly increased inflammatory cell infiltration (p<0.04 versus control, MI, or MI+T2), with infiltration highest in the MI+Ct group and lowest in the MI+T2 group (p<0.04). Conclusions Adenoviral mediated myocardial delivery of the TIMP-2 gene improves post-MI survival and limits adverse remodeling in a murine model of myocardial infarction. PMID:21348952

  14. Adenovirus in a synthetic membrane wrapper: an example of hybrid vigor?

    PubMed

    Thompson, David H

    2008-05-01

    Nucleic acid delivery applications require the development of carrier systems that are effective, selective, and non-toxic. Many different viral and non-viral approaches, including the use of retroviruses, adenoviruses, liposomes, and dendrimers, have been investigated. Unfortunately, issues still remain with regard to the safety and efficiency of these delivery vehicles. In this Perspective, the challenges of designing a stable vector that is capable of effective gene therapy are highlighted. Progress in the area is also presented, including the work of Kostarelos and co-workers appearing in this issue of ACS Nano, in which they describe a novel delivery vehicle that consists of lipid envelopes encasing viral nanoparticles. PMID:19206477

  15. Stimulation of cerebral angiogenesis by gene delivery.

    PubMed

    Tang, Yaohui; Li, Yaning; Lin, Xiaojie; Miao, Peng; Wang, Yongting; Yang, Guo-Yuan

    2014-01-01

    Angiogenesis, an important process for long term neurological recovery, could be induced by ischemic brain injury. In this chapter, we describe a system to deliver adeno-associated viral (AAV) vector-mediated gene therapy for ischemic stroke. This includes the methods to construct, produce, and purify an AAV vector expressing target gene and an approach to quantify the number of microvessels and capillary density with synchrotron radiation angiography (SRA) imaging. PMID:24510875

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

    PubMed

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

    2013-01-01

    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. PMID:23676471

  17. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma.

    PubMed

    Kievit, Forrest M; Veiseh, Omid; Fang, Chen; Bhattarai, Narayan; Lee, Donghoon; Ellenbogen, Richard G; Zhang, Miqin

    2010-08-24

    Glioma accounts for 80% of brain tumors and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG), and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA-bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers. PMID:20731441

  18. Chlorotoxin Labeled Magnetic Nanovectors for Targeted Gene Delivery to Glioma

    PubMed Central

    Kievit, Forrest M.; Veiseh, Omid; Fang, Chen; Bhattarai, Narayan; Lee, Donghoon; Ellenbogen, Richard G.; Zhang, Miqin

    2010-01-01

    Glioma accounts for 80% of brain tumors, and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG) and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site, but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers. PMID:20731441

  19. 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

  20. Nanoparticle-motivated gene delivery for ophthalmic application.

    PubMed

    Mitra, Rajendra Narayan; Zheng, Min; Han, Zongchao

    2016-01-01

    Ophthalmic gene therapy is an intellectual and intentional manipulation of desired gene expression into the specific cells of an eye for the treatment of ophthalmic (ocular) genetic dystrophies and pathological conditions. Exogenous nucleic acids such as DNA, small interfering RNA, micro RNA, and so on, are used for the purpose of managing expression of the desired therapeutic proteins in ocular tissues. The delivery of unprotected nucleic acids into the cells is limited because of exogenous and endogenous degradation modalities. Nanotechnology, a promising and sophisticated cutting edge tool, works as a protective shelter for these therapeutic nucleic acids. They can be safely delivered to the required cells in order to modulate anticipated protein expression. To this end, nanotechnology is seen as a potential and promising strategy in the field of ocular gene delivery. This review focused on current nanotechnology modalities and other promising nonviral strategies being used to deliver therapeutic genes in order to treat various devastating ocular diseases. PMID:26109528

  1. Gene delivery to the heart by magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Li, Wenzhong; Nesselmann, Catharina; Zhou, Zhaohui; Ong, Lee-Lee; Öri, Ferenc; Tang, Guping; Kaminski, Alexander; Lützow, Karola; Lendlein, Andreas; Liebold, Andreas; Stamm, Christof; Wang, John; Steinhoff, Gustav; Ma, Nan

    2007-04-01

    Gene delivery with non-viral gene vectors to the cardiovascular system suffers from low transfection efficiency. In this study, magnetic fields were investigated to assist cardiovascular gene delivery via magnetic nanobeads both in vitro and in vivo. The magnetic field was provided with a 1120 mT Nd-Fe-B permanent magnet while complexes of poly-ethyleneimine (PEI) and various DNA plasmids were conjugated with magnetic nanobeads (MNB) using a Sulfo-NHS-LC-Biotin linker. In vitro results showed that transfection in two cell lines was 30-80-fold higher in magnetically conjugated MNB/PEI/DNA complexes than transfection from PEI/DNA complexes alone. Similarly, in vivo results using mouse models showed 72 h after injection observable gene expression in the heart with conjugated MNB/PEI/DNA complexes, but barely with PEI/DNA complexes alone.

  2. Toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human FSH receptor restores folliculogenesis in FSHR(−/−) FORKO mice

    PubMed Central

    Ghadami, M.; El-Demerdash, E.; Salama, S.A.; Binhazim, A.A.; Archibong, A.E.; Chen, X.; Ballard, B.R.; Sairam, M.R.; Al-Hendy, A.

    2010-01-01

    A homozygous missense mutation, C566T, in the follicle stimulation hormone receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called follitropin receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intra-ovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6–10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26 ± 4 follicles/ovary in treated group with 8 ± 2 follicles at the antral stage compared with only 5 ± 2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2–3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring. PMID:20086006

  3. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.

    PubMed

    Gordon, J W

    2001-04-01

    The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10(10) infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 x 10(8) particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adbeta-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5-7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low. PMID:11319918

  4. Strategies on the nuclear-targeted delivery of genes

    PubMed Central

    Yao, Jing; Fan, Ying; Li, Yuanke; Huang, Leaf

    2016-01-01

    To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus. PMID:23964565

  5. A Versatile Gene Delivery System for Efficient and Tumor Specific Gene Manipulation in vivo

    PubMed Central

    Wang, Wei; Dong, Bingning; Ittmann, Michael M.; Yang, Feng

    2016-01-01

    The Replication-Competent Avian Sarcoma-leukosis virus long-terminal repeat with splice acceptor (RCAS)-Tumor Virus A (TVA) gene delivery system has been created based on the fact that avian sarcoma leukosis virus subgroup A only infects cells expressing its receptor, TVA. This system has been successfully applied to create various mouse models for human cancers. Here we briefly discuss the advantages and the potential caveats of using this RCAS-TVA gene delivery system in cancer research. We also introduce and discuss how our newly designed RCAS-based gene delivery system (RCI-Oncogene, for RCAS-Cre-IRES-Oncogene) allows concise and efficient manipulation of gene expression in tumors in vivo, and how this system can be used to rapidly study the biological function of gene(s) and/or the collaborative actions of multiple genes in regulating tumor initiation, progression and/or metastasis. PMID:27376150

  6. 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

  7. 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

  8. Cell targeted gene delivery system based on modified pectin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus pectin modified with various amine groups have been studied for its potential as a novel non-viral gene delivery carrier. The modified cationic pectin was able to condense DNA and mediate transfection in a cell type specific manner. The modified pectin seems to be a promising carrier, attra...

  9. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    PubMed Central

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211

  10. Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy.

    PubMed

    Kim, You-Kyoung; Xing, Lei; Chen, Bao-An; Xu, Fengguo; Jiang, Hu-Lin; Zhang, Can

    2014-11-01

    The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD-SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results demonstrate that SD-SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy. PMID:24983766

  11. Autoregulation of Adenovirus Type 5 Early Gene Expression II. Effect of Temperature-Sensitive Early Mutations on Virus RNA Accumulation

    PubMed Central

    Carter, T. H.; Blanton, R. A.

    1978-01-01

    The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5°C by wild-type (WT) adenovirus type 5 and a temperature-sensitive “early” mutant, H5ts125 (ts125), were compared by hybridization of unlabeled RNA in solution to the 3H-labeled l strand of Ad5 DNA HindIII restriction endonuclease fragment A. In the presence of 1-β-d-arabinofuranosylcytosine, Al RNA accumulated in WT-infected cells for 9 h and then decreased in concentration to 6% of the 9-h concentration by 18 h. In ts125-infected cells, Al RNA accumulated for 12 h and then remained at the same concentration for at least 6 h thereafter. The concentrations of virus RNA from the four early transcription regions of the genome were measured at 15 h in cells infected at 40.5°C in the presence of 1-β-d-arabinofuranosylcytosine by: (i) ts125 and WT; (ii) two other ts early mutants, ts107 and ts149; and (iii) a revertant of ts125. The revertant and ts149, a mutant from a different complementation group than ts125, both accumulated all early virus cytoplasmic RNA species in amounts similar to, or less than, WT. However, both ts125 and ts107, independently isolated mutations in the 72,000-molecular-weight (72K) DNA-binding protein gene, accumulated cytoplasmic early RNA in excess of that found in WT infection. This pattern of RNA accumulation with the mutants and WT virus was the same in the nuclei as in the cytoplasm at 40.5°C. At 32°C, however, the abundance of nuclear virus RNA from all four early regions was the same in cells infected by either ts125 or WT. Differences in the relative abundance of nuclear RNA from the four early regions were observed in cells infected at 40.5 and 32°C, but were not dependent upon the infecting virus genotype. These results are consistent with autoregulation of early gene expression by the 72K protein and support the hypothesis that the 72K protein either decreases the rate of early virus transcription or increases the rate of virus

  12. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    PubMed Central

    Gahéry-Ségard, H; Molinier-Frenkel, V; Le Boulaire, C; Saulnier, P; Opolon, P; Lengagne, R; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A; Schatz, C; Courtney, M; Le Chevalier, T; Tursz, T; Guillet, J G; Farace, F

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein. PMID:9410899

  13. Application of Ferriferous Oxide Modified by Chitosan in Gene Delivery

    PubMed Central

    Kuang, Yu; Yuan, Tun; Zhang, Zhongwei; Li, Mingyuan; Yang, Yuan

    2012-01-01

    New approaches to improve the traditional gene carriers are still required. Here we explore Fe3O4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field. Two magnetic Fe3O4 nanoparticles coated with chitosan (CTS) and polyethylene glycol (PEG) were synthesized by means of controlled chemical coprecipitation. Plasmid pEGFP was encapsulated as a reported gene. The ferriferous oxide complexes were approximately spherical; surface charge of CTS-Fe3O4 and PEG-Fe3O4 was about 20 mv and 0 mv, respectively. The controlled release of DNA from the CTS-Fe3O4 nanoparticles was observed. Concurrently, a desired Fe3O4 concentration of less than 2 mM was verified as safe by means of a cytotoxicity test in vitro. Presence of the permanent magnetic field significantly increased the transfection efficiency. Furthermore, the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test. The novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo. PMID:23326667

  14. Canine adenovirus based rabies vaccines.

    PubMed

    Tordo, N; Foumier, A; Jallet, C; Szelechowski, M; Klonjkowski, B; Eloit, M

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control. PMID:18634509

  15. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  16. Conceptual and technical aspects of transfection and gene delivery.

    PubMed

    Kaestner, Lars; Scholz, Anke; Lipp, Peter

    2015-03-15

    Genetically modified animals are state of the art in biomedical research as gene therapy is a promising perspective in the attempt to cure hereditary diseases. Both approaches have in common that modified or corrected genetic information must be transferred into cells in general or into particular cell types of an organism. Here we give an overview of established and emerging methods of transfection and gene delivery and provide conceptual and technical advantages and drawbacks of their particular use. Additionally, based on a flow chart, we compiled a rough guideline to choose a gene transfer method for a particular field of application. PMID:25677659

  17. Design of Microbubbles for Gene/Drug Delivery.

    PubMed

    Bettinger, Thierry; Tranquart, François

    2016-01-01

    The role of ultrasound contrast agents (UCA) initially designed for diagnosis has evolved towards a therapeutic use. Ultrasound (US) for triggered drug delivery has many advantages. In particular, it enables a high spatial control of drug release, thus potentially allowing activation of drug delivery only in the targeted region, and not in surrounding healthy tissue. Moreover, UCA imaging can also be used firstly to precisely locate the target region to, and then used to monitor the drug delivery process by tracking the location of release occurrence. All these features make UCA and ultrasound attractive means to mediate drug delivery. The three main potential clinical indications for drug/gene US delivery are (i) the cardiovascular system, (ii) the central nervous system for small molecule delivery, and (iii) tumor therapy using cytotoxic drugs. Although promising results have been achieved in preclinical studies in various animal models, still very few examples of clinical use have been reported. In this chapter will be addressed the aspects pertaining to UCA formulation (chemical composition, mode of preparation, analytical methods…) and the requirement for a potential translation into the clinic following approval by regulatory authorities. PMID:26486339

  18. The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity.

    PubMed Central

    Wong, H K; Ziff, E B

    1996-01-01

    We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level. PMID:8523545

  19. The relevance of coagulation factor X protection of adenoviruses in human sera

    PubMed Central

    Duffy, M R; Doszpoly, A; Turner, G; Nicklin, S A; Baker, A H

    2016-01-01

    Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5–FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad–FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy. PMID:27014840

  20. Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

    PubMed Central

    Jouneau, Luc; Bourge, Mickael; Bouet-Cararo, Coraline; Bonneau, Michel; Zientara, Stephan; Klonjkowski, Bernard; Schwartz-Cornil, Isabelle

    2012-01-01

    Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b+ -type and CD103+ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b+ -type DCs was far higher and broader than in the CD103+ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b+ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b+ DC type is more responsive to CAV2 than the CD103+ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness. PMID:23300693

  1. Canine recombinant adenovirus vector induces an immunogenicity-related gene expression profile in skin-migrated CD11b⁺ -type DCs.

    PubMed

    Contreras, Vanessa; Urien, Céline; Jouneau, Luc; Bourge, Mickael; Bouet-Cararo, Coraline; Bonneau, Michel; Zientara, Stephan; Klonjkowski, Bernard; Schwartz-Cornil, Isabelle

    2012-01-01

    Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b(+) -type and CD103(+) -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b(+) -type DCs was far higher and broader than in the CD103(+) -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b(+) -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b(+) DC type is more responsive to CAV2 than the CD103(+) DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness. PMID:23300693

  2. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  3. Identification and gene mapping of a 14,700-molecular-weight protein encoded by region E3 of group C adenoviruses.

    PubMed Central

    Tollefson, A E; Wold, W S

    1988-01-01

    Early region E3 of adenovirus type 5 should encode at least nine proteins as judged by the DNA sequence and the spliced structures of the known mRNAs. Only two E3 proteins have been proved to exist, a glycoprotein (gp19K) and an 11,600-molecular-weight protein (11.6K protein). Here we describe an abundant 14.7K protein coded by a gene in the extreme 3' portion of E3. To identify this 14.7K protein, we constructed a bacterial vector which synthesized a TrpE-14.7K fusion protein, then we prepared antiserum against the fusion protein. This antiserum immunoprecipitated the 14.7K protein from cells infected with adenovirus types 5 and 2, as well as with a variety of E3 deletion mutants. Synthesis of the 14.7K protein correlated precisely with the presence or absence of the 14.7K gene and with the synthesis of the mRNA (mRNA h) which encodes the 14.7K protein. The 14.7K protein appeared as a triplet on immunoprecipitation gels and Western blots (immunoblots). Images PMID:3275435

  4. 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:25485106

  5. Facile Noninvasive Retinal Gene Delivery Enabled by Penetratin.

    PubMed

    Liu, Chang; Jiang, Kuan; Tai, Lingyu; Liu, Yu; Wei, Gang; Lu, Weiyue; Pan, Weisan

    2016-08-01

    Gene delivery to the posterior segment of the eye is severely hindered by the impermeability of defensive barriers; therefore, in clinical settings, genomic medicines are mainly administered by intravitreal injection. We previously found that penetratin could transport the covalently conjugated fluorophore to the fundus oculi by topical instillation. In this study, gene delivery systems enabled by penetratin were designed based on electrostatic binding to target the retina via a noninvasive administration route and prepared with red fluorescent protein plasmid (pRFP) and/or poly(amidoamine) dendrimer of low molecular weight (G3 PAMAM). Formulation optimization, structure confirmation, and characterization were subsequently conducted. Penetratin alone showed limited ability to condense the plasmid but had powerful uptake and transfection by corneal and conjunctival cells. G3 PAMAM was nontoxic to the ocular cells, and when introduced into the penetratin-incorporated complex, the plasmid was condensed more compactly. Therefore, further improved cellular uptake and transfection were observed. After being instilled in the conjunctival sac of rats, the intact complexes penetrated rapidly from the ocular surface into the fundus and resided in the retina for more than 8 h, which resulted in efficient expression of RFP in the posterior segment. Intraocular distribution of the complexes suggested that the plasmids were absorbed into the eyes through a noncorneal pathway during which penetratin played a crucial role. This study provides a facile and friendly approach for intraocular gene delivery and is an important step toward the development of noninvasive gene therapy for posterior segment diseases. PMID:27400087

  6. Uteroplacental Adenovirus Vascular Endothelial Growth Factor Gene Therapy Increases Fetal Growth Velocity in Growth-Restricted Sheep Pregnancies

    PubMed Central

    Wallace, Jacqueline M.; Aitken, Raymond P.; Milne, John S.; Mehta, Vedanta; Martin, John F.; Zachary, Ian C.; Peebles, Donald M.; David, Anna L.

    2014-01-01

    Abstract Fetal growth restriction (FGR) occurs in ∼8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterized by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad)-mediated overexpression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from midgestation correlates with reduced lamb birthweight near term. Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high intake (n=45) or control intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomized midgestation to receive bilateral UtA injections of 5×1011 particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14), or control saline (n=13). Fetal growth/well-being were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularization within the perivascular adventitia, and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry, and qRT-PCR, respectively. Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3–4 weeks postinjection (p=0.016–0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) after Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter-to-abdominal circumference and brain-to-liver weight ratios). No effects were observed on UBF or neovascularization; however, Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal-to-placental weight ratio) and FLT1/KDR mRNA expression were

  7. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  8. Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob

    PubMed Central

    Krasnykh, Victor; Dmitriev, Igor; Mikheeva, Galina; Miller, C. Ryan; Belousova, Natalya; Curiel, David T.

    1998-01-01

    The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies. PMID:9499035

  9. Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob.

    PubMed

    Krasnykh, V; Dmitriev, I; Mikheeva, G; Miller, C R; Belousova, N; Curiel, D T

    1998-03-01

    The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies. PMID:9499035

  10. Targeted gene knockout by direct delivery of ZFN proteins

    PubMed Central

    Gaj, Thomas; Guo, Jing; Kato, Yoshio; Sirk, Shannon J.; Barbas, Carlos F.

    2012-01-01

    Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in a variety of mammalian cell types with minimal off-target effects. PMID:22751204

  11. 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. PMID:25513958

  12. 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

  13. Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

    PubMed Central

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  14. Transient expression of proteins by hydrodynamic gene delivery in mice.

    PubMed

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  15. Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

    PubMed Central

    Yoshida, K; Narita, M; Fujinaga, K

    1989-01-01

    Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor. Images PMID:2532319

  16. Neogenesis and proliferation of {beta}-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    SciTech Connect

    Tokui, Yae . E-mail: ytokui@imed2.med.osaka-u.ac.jp; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro |

    2006-12-01

    Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of {beta}-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a {beta}-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of {beta}-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of {beta}-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted {beta}-cell differentiation (mainly from duct cells) and proliferation of pre-existing {beta}-cells, resulting in an increase of the {beta}-cell mass that improved glucose tolerance in diabetic mice.

  17. Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo

    PubMed Central

    Watson, Rachael S; Gouze, Elvire; Levings, Padraic P; Bush, Marsha L; Kay, Jesse D; Jorgensen, Marda S; Dacanay, E Anthony; Reith, John W; Wright, Thomas W; Ghivizzani, Steven C

    2013-01-01

    To understand the cellular and molecular events contributing to arthrofibrosis, we used an adenovirus to deliver and overexpress transforming growth factor-beta 1 (TGF-β1) cDNA (Ad.TGF-β1) in the knee joints of immunocompromised rats. Following delivery, animals were killed periodically, and joint tissues were examined macroscopically and histologically. PCR-array was used to assay alterations in expression patterns of extracellular matrix (ECM)-associated genes. By days 5 and 10, TGF-β1 induced an increase in knee diameter and complete encasement of joints in dense scar-like tissue, locking joints at 90° of flexion. Histologically, massive proliferation of synovial fibroblasts was seen, followed by their differentiation into myofibroblasts. The fibrotic tissue displaced the normal architecture of the joint capsule and fused with articular cartilage. RNA expression profiles showed high levels of transcription of numerous MMPs, matricellular and ECM proteins. By day 30, the phenotype of the fibrotic tissue had undergone chondrometaplasia, indicated by cellular morphology, matrix composition and >100-fold increases in expression of collagen type II and cartilage link protein. Pre-labeling of articular cells by injection with recombinant lentivirus containing eGFP cDNA showed fibrotic/cartilaginous tissues appeared to arise almost entirely from local proliferation and differentiation of resident fibroblasts. Altogether, these results indicate that TGF-β1 is a potent inducer of arthrofibrosis, and illustrate the proliferative potential and plasticity of articular fibroblasts. They suggest the mechanisms causing arthrofibrosis share many aspects with tumorigenesis. PMID:20697373

  18. Rationale for the selection of an aerosol delivery system for gene delivery.

    PubMed

    Lentz, Yvonne K; Anchordoquy, Thomas J; Lengsfeld, Corinne S

    2006-01-01

    Genetic therapeutics show great promise toward the treatment of illnesses associated with the lungs; however, current methods of delivery such as jet and ultrasonic nebulization decrease the activity and effectiveness of these treatments. Extremely low transfection rates exhibited by non-complexed plasmid DNA in these nebulizers have been primarily attributed to poor translocation and loss of molecular integrity as a consequence of shear-induced degradation. Current research focusing on methods to increase transfection rates via the pulmonary delivery route has largely concentrated on the incorporation of carbon dioxide in the air stream to increase breath depth as well as the addition of cationic agents that condense DNA into compact, ordered complexes. The purpose of this study was to examine the impact of several classic as well as the latest atomization devices on the structure of non-complexed DNA. Various sizes of plasmid and cosmid DNA were processed through an electrostatic spray, ultrasonic nebulizer, vibrating mesh nebulizer, and jet nebulizer. Results varied dramatically based upon atomization device as well as DNA size. This may explain the inefficiency experienced by genetic therapeutics during pulmonary delivery. More importantly, this suggests that the selection of an atomization device should consider DNA size in order to achieve optimal gene delivery to the lungs. PMID:17034312

  19. 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

  20. Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2).

    PubMed

    Tang, M; Harp, J A; Wesley, R D

    2002-11-01

    Immunization with recombinant adenoviral vaccine that induces potent immunity has been applied to many infectious diseases. We report here developing a recombinant adenoviral vaccine encoding the HA gene from swine H3N2 influenza virus (SIV). Two replication-defective recombinant adenoviruses were generated: (1) rAd-HA: recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus, and (2) rAd-vector: a control recombinant adenovirus containing adenovirus and transfer plasmids without a foreign HA gene. Mice given rAd-HA developed high titers of neutralizing and hemagglutination inhibition antibodies to SIV in comparison to mice inoculated with rAd-vector or PBS as early as 2 weeks after immunization, and these antibodies were substantially increased in the mice given rAd-HA within the next 3 weeks following the first dose. However, these antibodies were not able to neutralize the virus, A/HK/68 (H3N2), used for challenge. Nonetheless mice immunized with one or two doses of rAd-HA were protected from lethal challenge with heterologous virus, A/HK/1/68 (H3N2). A statistically significant ( P < 0.03) difference between survival rates of rAd-HA mice vs. rAd-vector or PBS mice was observed. PMID:12417948

  1. Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression

    PubMed Central

    Lu, YiQing; Kucharski, Thomas J.; Gamache, Isabelle; Blanchette, Paola; Branton, Philip E.

    2014-01-01

    ABSTRACT Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4−) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4− virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCE Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and

  2. Hydrogels: a journey from diapers to gene delivery.

    PubMed

    Chawla, Pooja; Srivastava, Alok Ranjan; Pandey, Priyanka; Chawla, Viney

    2014-02-01

    Hydrogels are the biomaterials comprising network of natural or synthetic polymers capable of absorbing large amount of water. Hydrogels are "Smart Gels" or "Intelligent Gels" which can be made to respond to the various environmental conditions like temperature, pH, magnetic/electric field, ionic strength, inflammation, external stress etc. There are numerous potential applications of hydrogels in modern day life ranging from a diaper to gene delivery. This review succinctly describes the classification, properties and preparation methods along with numerous diverse applications of hydrogels like agricultural hydrogels, hydrogel for drug delivery, sensing, dental adhesives, wound healing and tissue regeneration, diet aid and gastric retention and in tissue engineering etc. Hydrogels can be regarded as highly valuable biomaterials for human-beings. PMID:24387710

  3. 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

  4. Direct Gene Therapy for Bone Regeneration: Gene Delivery, Animal Models, and Outcome Measures

    PubMed Central

    Pelled, Gadi; Ben-Arav, Ayelet; Hock, Colleen; Reynolds, David G.; Yazici, Cemal; Zilberman, Yoram; Gazit, Zulma; Awad, Hani; Gazit, Dan

    2010-01-01

    While various problems with bone healing remain, the greatest clinical change is the absence of an effective approach to manage large segmental defects in limbs and craniofacial bones caused by trauma or cancer. Thus, nontraditional forms of medicine, such as gene therapy, have been investigated as a potential solution. The use of osteogenic genes has shown great potential in bone regeneration and fracture healing. Several methods for gene delivery to the fracture site have been described. The majority of them include a cellular component as the carrying vector, an approach known as cell-mediated gene therapy. Yet, the complexity involved with cell isolation and culture emphasizes the advantages of direct gene delivery as an alternative strategy. Here we review the various approaches of direct gene delivery for bone repair, the choice of animal models, and the various outcome measures required to evaluate the efficiency and safety of each technique. Special emphasis is given to noninvasive, quantitative, in vivo monitoring of gene expression and biodistribution in live animals. Research efforts should aim at inducing a transient, localized osteogenic gene expression within a fracture site to generate an effective therapeutic approach that would eventually lead to clinical use. PMID:20143927

  5. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis.

    PubMed Central

    Babiss, L E; Ginsberg, H S

    1984-01-01

    To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or

  6. Mutation of the fiber shaft heparan sulphate binding site of a 5/3 chimeric adenovirus reduces liver tropism.

    PubMed

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology. PMID:23585829

  7. Mutation of the Fiber Shaft Heparan Sulphate Binding Site of a 5/3 Chimeric Adenovirus Reduces Liver Tropism

    PubMed Central

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology. PMID:23585829

  8. Use of Polymer Micro-Structures for Drug & Gene Delivery

    NASA Astrophysics Data System (ADS)

    Chu, Ben

    2005-03-01

    The design of polymer microstructures, including polyelectrolyte-surfactant complex formation, plays an important role in the protection and controlled release of drugs & DNA fragments. Two examples are presented: one for drug release and one for gene delivery. Non-viral gene therapy is a challenging problem that has not yet met much success even though numerous attempts have been made. The gene delivery illustration aims to present one specific approach on how DNA fragments can be delivered to a cell by using an electro-spun scaffold as a carrier, i.e., to consider how DNA fragments can be trapped into a scaffold for subsequent release and transfection. Our scheme is to capture the DNA fragments by taking advantage of the DNA coil-to-globule transition and to encapsulate the condensed DNA globule by using block copolymers. The supra-molecular capsule can then be incorporated into a nano-structured biodegradable polymer scaffold by means of electro-spinning. Subsequent DNA release to cells that adhere to the scaffolds was measured by using fluorescence microscopy.AcknowledgementsFinancial Support:National Science Foundation, Polymers Program (DMR9984102 & Creativity Extension Award), Center for Biotechnology at Stony Brook, ITG Grant, and NIH SBIR Grant to STAR.Main contributors include Professors Benjamin S. Hsiao and Michael Hadjiargyrou, Drs. Dufei Fang, Dehai Liang and Kwangsok Kim, Ms. K. Luu and Mr. J. Chiu.

  9. Platelet-adenovirus vs. inert particles interaction: effect on aggregation and the role of platelet membrane receptors.

    PubMed

    Gupalo, Elena; Kuk, Cynthia; Qadura, Mohammad; Buriachkovskaia, Liudmila; Othman, Maha

    2013-01-01

    Platelets are involved in host defense via clearance of bacteria from the circulation, interaction with virus particles, and uptake of various size particulates. There is a growing interest in micro- and nanoparticles for drug delivery and there is evidence that the properties of these particles critically influence their interaction and uptake by various tissues and cells including platelets. Virus mediated gene therapy applications are still challenged by the resultant thrombocytopenia and the mechanism(s) of platelet-foreign particles interaction remains unclear. We studied the specifics of platelet interaction with an active biological agent (adenovirus) and inert latex microspheres (MS) and investigated the role of platelet proteins in this interaction. We show that activated and not resting platelets internalize MS, without influencing platelet aggregation. In contrast, adenovirus induces and potentiates ADP-induced platelet aggregation and results in rapid expression of P-selectin. Platelets then internalize adenovirus and viral particles appear inside the open canalicular system. Inhibition of platelet αIIbβ3, GPIbα, and P-selectin decreases both platelet aggregation and internalization of MS. Inhibition of αIIbβ3 and αVβ3 does not abolish adenovirus platelet internalization and adenovirus-induced platelet activation is maintained. Our study demonstrates that platelets react differentially with foreign particles and that αIIbβ3 is a key player in platelet engulfing of foreign particles but not in mediating adenovirus internalization. Other platelet candidate molecules remain to be investigated as potential targets for management of adenovirus-induced thrombocytopenia. PMID:22812520

  10. Liver lobe and strain difference in gene expression after hydrodynamics-based gene delivery in mice.

    PubMed

    Nakamura, Shingo; Maehara, Tadaaki; Watanabe, Satoshi; Ishihara, Masayuki; Sato, Masahiro

    2015-01-01

    Hydrodynamics-based gene delivery (HGD) is a widely recognized technique for delivering exogenous DNA with high efficiency to murine hepatocytes. In this study, we investigated stimulation of exogenous DNA uptake and expression using a commercially available reagent for HGD. We also examined which mouse strain and mouse liver lobe would achieve the best gene delivery performance. Mice were injected with a solution containing reporter plasmid DNA or DNA and a gene delivery reagent. One day after the HGD procedure, liver samples were isolated and subjected to biochemical and histochemical analyses. The reporter plasmid DNA showed the strongest signal when the DNA was dissolved in TransIT-EE Hydrodynamic Delivery Solution (Takara Bio Inc., Shiga, Japan). Evaluation of transgene expression in each hepatic lobe in ICR, C57BL/6N, Balb/cA, and B6C3F1 mice showed that ICR mice exhibited the best gene transfer and that the right median lobe had the highest level of transgene expression. These findings suggest the importance of choice in mouse strains and liver lobes when performing gene-based manipulations of the liver. PMID:25153456

  11. 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

  12. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

  13. Arginine-Rich Polyplexes for Gene Delivery to Neuronal Cells

    PubMed Central

    Morris, Viola B.; Labhasetwar, Vinod

    2015-01-01

    Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood-brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery. PMID:26000961

  14. Hepatic gene delivery system electrostatically assembled with glycyrrhizin.

    PubMed

    Kurosaki, Tomoaki; Kawanabe, Saki; Kodama, Yukinobu; Fumoto, Shintaro; Nishida, Koyo; Nakagawa, Hiroo; Higuchi, Norihide; Nakamura, Tadahiro; Kitahara, Takashi; Sasaki, Hitoshi

    2014-05-01

    In this study, a novel liver-targeted gene delivery vector was developed by electrostatically coating the cationic complex of pDNA and polyethylenimine (PEI) with glycyrrhizin (GL). The ternary complex, pDNA/PEI/GL, had approximately 100 nm stable particles with a negative charge surface. pDNA/PEI/GL showed high gene expression comparable to that of the complex of pDNA and PEI (pDNA/PEI) in human hepatoma cell line HepG2 without cytotoxicity and agglutination. After intravenous injection of pDNA/PEI/GL into mice, the highest gene expression was observed in the liver. pDNA/PEI/GL showed significantly higher gene expression in parenchymal cells than in nonparenchymal cells. On the basis of these results, we evaluated the pharmacological activity of the ternary complex including the pDNA encoding insulin (pCMV-Ins). The pCMV-Ins/PEI/GL decreased blood glucose concentrations 24 h after its intravenous administration to mice. The ternary complex of pDNA, PEI, and GL may be a promising liver-targeted gene vector. PMID:24673596

  15. Replication-competent human adenovirus 11p vectors can propagate in Vero cells.

    PubMed

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-01

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. PMID:27176913

  16. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  17. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  18. Modified pectin-based carrier for gene delivery: Cellular barriers in gene delivery course

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of biodegradable and biocompatible polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential liga...

  19. Ultrasound mediated delivery of drugs and genes to solid tumors

    PubMed Central

    Frenkel, Victor

    2008-01-01

    It has long been shown that therapeutic ultrasound can be used effectively to ablate solid tumors, and a variety of cancers are presently being treated in the clinic using these types of ultrasound exposures. There is, however, an ever-increasing body of preclinical literature that demonstrates how ultrasound energy can also be used non-destructively for increasing the efficacy of drugs and genes for improving cancer treatment. In this review, a summary of the most important ultrasound mechanisms will be given with a detailed description of how each one can be employed for a variety of applications. This includes the manner by which acoustic energy deposition can be used to create changes in tissue permeability for enhancing the delivery of conventional agents, as well as for deploying and activating drugs and genes via specially tailored vehicles and formulations. PMID:18474406

  20. Fenton-treated functionalized diamond nanoparticles as gene delivery system.

    PubMed

    Martín, Roberto; Alvaro, Mercedes; Herance, José Raúl; García, Hermenegildo

    2010-01-26

    When raw diamond nanoparticles (Dnp, 7 nm average particle size) obtained from detonation are submitted to harsh Fenton-treatment, the resulting material becomes free of amorphous soot matter and the process maintains the crystallinity, reduces the particle size (4 nm average particle size), increases the surface OH population, and increases water solubility. All these changes are beneficial for subsequent Dnp covalent functionalization and for the ability of Dnp to cross cell membranes. Fenton-treated Dnps have been functionalized with thionine and the resulting sample has been observed in HeLa cell nuclei. A triethylammonium-functionalized Dnp pairs electrostatically with a plasmid having the green fluorescent protein gene and acts as gene delivery system permitting the plasmid to cross HeLa cell membrane, something that does not occur for the plasmid alone without assistance of polycationic Dnp. PMID:20047335

  1. 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

    2015-03-01

    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. PMID:25634573

  2. Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors

    PubMed Central

    Greish, Khaled; Frandsen, Jordan; Scharff, Stephanie; Gustafson, Joshua; Cappello, Joseph; Li, Daqing; O’Malley, Bert W.; Ghandehari, Hamidreza

    2010-01-01

    Background Adenoviral directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. Challenges with this approach however are transient gene expression and dissemination of viruses to distant organs. Methods We used recombinant silk-elastinlike protein copolymer (SELP) matrices for intratumoral delivery of adenoviruses containing both thymidine kinase-1, and luciferase genes in a nude mice model of JHU-022 head and neck tumor. Hydrogels made from two SELP analogues (47K and 815K) with similar silk to elastinlike block ratios but different block lengths were studied for intratumoral viral delivery. Tumor bearing mice were followed up for tumor progression and luciferase gene expression concomitantly for five weeks. Polymer’s safety was evaluated through body weight change, blood count, liver and kidney functions in addition to gross and microscopic histological examination. Results SELP 815K analogues efficiently controlled the duration and extent of transfection in tumors for up to 5 weeks with no detectable spread to the liver. About five-fold greater reduction in tumor volume was obtained with matrix-mediated delivery compared to intra-tumoral injection of adenoviruses in saline. SELP matrix proved safe in all injected mice compared to control group. Conclusion SELP- controlled gene delivery approach could potentially improve the anticancer activity of virus-mediated gene therapy while limiting viral spread to normal organs. PMID:20603862

  3. Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

    PubMed

    Iqbal Ahmed, C M; Johnson, D E; Demers, G W; Engler, H; Howe, J A; Wills, K N; Wen, S F; Shinoda, J; Beltran, J; Nodelman, M; Machemer, T; Maneval, D C; Nagabhushan, T L; Sugarman, B J

    2001-10-01

    A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells. PMID:11687902

  4. An Orthotopic Bladder Cancer Model for Gene Delivery Studies

    PubMed Central

    Kasman, Laura; Voelkel-Johnson, Christina

    2013-01-01

    Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications. PMID:24326612

  5. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery.

    PubMed

    Huang, Shixian; Shao, Kun; Kuang, Yuyang; Liu, Yang; Li, Jianfeng; An, Sai; Guo, Yubo; Ma, Haojun; He, Xi; Jiang, Chen

    2013-07-01

    A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery. PMID:23562171

  6. Cross-linked polyethylenimine-tripolyphosphate nanoparticles for gene delivery.

    PubMed

    Huang, Xianzhang; Shen, Sujing; Zhang, Zhanfeng; Zhuang, Junhua

    2014-01-01

    The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site-enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery. PMID:25342902

  7. Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.

    PubMed Central

    Lutz, P; Kedinger, C

    1996-01-01

    The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus. PMID:8627656

  8. Use of macrophages to target therapeutic adenovirus to human prostate tumors.

    PubMed

    Muthana, Munitta; Giannoudis, Athina; Scott, Simon D; Fang, Hsin-Yu; Coffelt, Seth B; Morrow, Fiona J; Murdoch, Craig; Burton, Julian; Cross, Neil; Burke, Bernard; Mistry, Roshna; Hamdy, Freddie; Brown, Nicola J; Georgopoulos, Lindsay; Hoskin, Peter; Essand, Magnus; Lewis, Claire E; Maitland, Norman J

    2011-03-01

    New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells. PMID:21233334

  9. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  10. Sucrose ester based cationic liposomes as effective non-viral gene vectors for gene delivery.

    PubMed

    Zhao, Yinan; Zhu, Jie; Zhou, Hengjun; Guo, Xin; Tian, Tian; Cui, Shaohui; Zhen, Yuhong; Zhang, Shubiao; Xu, Yuhong

    2016-09-01

    As sucrose esters (SEs) are natural and biodegradable excipients with excellent drug dissolution and drug absorption/permeation in controlled release systems, we firstly incorporated SE into liposomes for gene delivery in this article. A peptide-based lipid (CDO14), Gemini-based quaternary ammonium-based lipid (CTA14), and mono-head quaternary ammonium lipid (CPA14), and SE as helper lipid, were prepared into liposomes which could enhance the interactions between liposomes and pDNA. Most importantly, the liposomes with helper lipid SE showed higher transfection and lower cytotoxicity than those without SE in Hela and A549 cells. It was also found that the transfection efficiency increased with the increase of SE content. The selected liposome, CDO14/SE, was able to deliver siRNA against luciferase for silencing gene in lung tumors of mice, with little in vivo toxicity. The results convincingly demonstrated SEs could be highly desirable candidates for gene delivery systems. PMID:27232309

  11. Gene delivery systems for gene therapy in tissue engineering and central nervous system applications.

    PubMed

    Giordano, C; Causa, F; Bianco, F; Perale, G; Netti, P A; Ambrosio, L; Cigada, A

    2008-12-01

    The present review aims to describe the potential applications of gene delivery systems to tissue engineering and central nervous system diseases. Some key experimental work has been done with interesting results, but the subject is far from being fully explored. The combined approach of gene therapy and material science has a huge potential to improve the therapeutic approaches now available for a wide range of medical applications. Focus is given to this multidisciplinary strategy in neurodegenerative pathologies, where the use of polymeric matrices as gene carriers might make a crucial difference. PMID:19115193

  12. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  13. Simian adenovirus type 35 has a recombinant genome comprising human and simian adenovirus sequences, which predicts its potential emergence as a human respiratory pathogen

    PubMed Central

    Dehghan, Shoaleh; Seto, Jason; Jones, Morris S.; Dyer, David W.; Chodosh, James; Seto, Donald

    2013-01-01

    Emergent human and simian adenoviruses (HAdVs) may arise from genome recombination. Computational analysis of SAdV type 35 reveals a genome comprising a chassis with elements mostly from two simian adenoviruses, SAdV-B21 and -B27, and regions of high sequence similarity shared with HAdV-B21 and HAdV-B16. Although recombination direction cannot be determined, the presence of these regions suggests prior infections of humans by an ancestor of SAdV-B35, and/or vice versa. Absence of this virus in humans may reflect non-optimal conditions for zoonosis. The presence of both a critical viral replication element found in HAdV genomes and genes that are highly similar to ones in HAdVs suggest the potential to establish in a human host. This allows a prediction that this virus may be a nascent human respiratory pathogen. The recombination potential of human and simian adenovirus genomes should be considered in the use of SAdVs as vectors for gene delivery in humans. PMID:24210123

  14. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants.

    PubMed

    Railey, J F; Wu, G J

    1988-03-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner. PMID:3367906

  15. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants.

    PubMed Central

    Railey, J F; Wu, G J

    1988-01-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner. Images PMID:3367906

  16. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: Fine mapping with linker-scanning mutants

    SciTech Connect

    Railey, J.F.; Wu, G.J.

    1988-03-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type genes using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 292 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner.

  17. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

  18. Cutting edge: recombinant adenoviruses induce CD8 T cell responses to an inserted protein whose expression is limited to nonimmune cells.

    PubMed

    Prasad, S A; Norbury, C C; Chen, W; Bennink, J R; Yewdell, J W

    2001-04-15

    CD8 T cells (T(CD8+)) play a crucial role in immunity to viruses. Current understanding of activation of naive T cells entails Ag presentation by professional APCs (pAPCs). What happens, however, when viruses evolve to avoid infecting pAPCs? We have studied the consequences of this strategy by generating recombinant adenoviruses that express influenza A virus nucleoprotein under the control of tissue-specific promoters. We show that the immunogenicity of such viruses requires their delivery to organs capable of expressing nucleoprotein. This indicates that infection of pAPCs is not required for adenoviruses to elicit a T(CD8+) response, probably due to a cross-priming via pAPCs. While this bodes well for recombinant adenoviruses as vaccines, it dims their prospects as gene therapy vectors. PMID:11290753

  19. 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

  20. Magnetofection: a reproducible method for gene delivery to melanoma cells.

    PubMed

    Prosen, Lara; Prijic, Sara; Music, Branka; Lavrencak, Jaka; Cemazar, Maja; Sersa, Gregor

    2013-01-01

    Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in some proof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueous medium, were tested after varying different parameters of the synthesis procedure. The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting in a reproducible and efficient magnetofection. To achieve high gene expression is important, however, the pH of PEI water solution for SPIONs-PAA functionalization, which should be in the alkaline range. PMID:23862136

  1. AAV Hybrid Serotypes: Improved Vectors for Gene Delivery

    PubMed Central

    Choi, Vivian W.; McCarty, Douglas M.; Samulski, R. Jude

    2006-01-01

    In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus. PMID:15975007

  2. Linear-dendritic block copolymer for drug and gene delivery.

    PubMed

    Fan, Xiaohui; Zhao, Yanli; Xu, Wei; Li, Lingbing

    2016-05-01

    Dendrimers as a new class of polymeric materials have a highly ordered branched structure, exact molecular weight, multivalency and available internal cavities, which make them extensively used in biology and drug-delivery. Concurrent with the development of dendrimers, much more attention is drawn to a novel block copolymer which combines linear chains with dendritic macromolecules, the linear-dendritic block copolymer (LDBC). Because of the different solubility of the contrasting regions, the amphiphilic LDBCs could self-assemble to form aggregates with special core-shell structures which exhibit excellent properties different from traditional micelles, such as lower critical micelle concentration, prolonged circulation in the bloodstream, better biocompatibility, and lower toxicity. The present review briefly describes the type of LDBC, the self-assembly behavior in solution, and the application in delivery system including the application as drug carriers and gene vectors. The interactions between block copolymers and drugs are also summarized to better understand the release mechanism of drugs from the linear-dendritic block copolymers. PMID:26952501

  3. Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through two interspecies recombination events

    PubMed Central

    Dehghan, Shoaleh; Seto, Jason; Liu, Elizabeth B.; Walsh, Michael P.; Dyer, David W.; Chodosh, James; Seto, Donald

    2013-01-01

    Computational analysis of human adenovirus type 4 (HAdV-E4), a pathogen that is the only HAdV member of species E, provides insights into its zoonotic origin and molecular adaptation. Its genome encodes a domain of the major capsid protein, hexon, from HAdV-B16 recombined into the genome chassis of a simian adenovirus. Genomes of two recent field strains provide a clue to its adaptation to the new host: recombination of a NF-I binding site motif, which is required for efficient viral replication, from another HAdV genome. This motif is absent in the chimpanzee adenoviruses and the HAdV-E4 prototype, but is conserved amongst other HAdVs. This is the first report of an interspecies recombination event for HAdVs, and the first documentation of a lateral partial gene transfer from a chimpanzee AdV. The potential for such recombination events are important when considering chimpanzee adenoviruses as candidate gene delivery vectors for human patients. PMID:23763770

  4. Multifunctional nanoparticles for drug/gene delivery in nanomedicine

    NASA Astrophysics Data System (ADS)

    Seale, Mary-Margaret; Zemlyanov, Dimitry; Cooper, Christy L.; Haglund, Emily; Prow, Tarl W.; Reece, Lisa M.; Leary, James F.

    2007-02-01

    Multifunctional nanoparticles hold great promise for drug/gene delivery. Multilayered nanoparticles can act as nanomedical systems with on-board "molecular programming" to accomplish complex multi-step tasks. For example, the targeting process has only begun when the nanosystem has found the correct diseased cell of interest. Then it must pass the cell membrane and avoid enzymatic destruction within the endosomes of the cell. Since the nanosystem is only about one millionth the volume of a human cell, for it to have therapeutic efficacy with its contained package, it must deliver that drug or gene to the appropriate site within the living cell. The successive de-layering of these nanosystems in a controlled fashion allows the system to accomplish operations that would be difficult or impossible to do with even complex single molecules. In addition, portions of the nanosystem may be protected from premature degradation or mistargeting to non-diseased cells. All of these problems remain major obstacles to successful drug delivery with a minimum of deleterious side effects to the patient. This paper describes some of the many components involved in the design of a general platform technology for nanomedical systems. The feasibility of most of these components has been demonstrated by our group and others. But the integration of these interacting sub-components remains a challenge. We highlight four components of this process as examples. Each subcomponent has its own sublevels of complexity. But good nanomedical systems have to be designed/engineered as a full nanomedical system, recognizing the need for the other components.

  5. Design of novel polysaccharidic nanostructures for gene delivery

    NASA Astrophysics Data System (ADS)

    de la Fuente, M.; Seijo, B.; Alonso, M. J.

    2008-02-01

    The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10 kDa (HAO) and chitosan 125 (CS) or 10-12 (CSO) kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230 nm, a positive zeta potential of +10 to +32 mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK 293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In conclusion, HA:CS nanoparticles are promising carriers for gene delivery.

  6. Electrosonic ejector microarray for drug and gene delivery.

    PubMed

    Zarnitsyn, Vladimir G; Meacham, J Mark; Varady, Mark J; Hao, Chunhai; Degertekin, F Levent; Fedorov, Andrei G

    2008-04-01

    We report on development and experimental characterization of a novel cell manipulation device-the electrosonic ejector microarray-which establishes a pathway for drug and/or gene delivery with control of biophysical action on the length scale of an individual cell. The device comprises a piezoelectric transducer for ultrasound wave generation, a reservoir for storing the sample mixture and a set of acoustic horn structures that form a nozzle array for focused application of mechanical energy. The nozzles are micromachined in silicon or plastic using simple and economical batch fabrication processes. When the device is driven at a particular resonant frequency of the acoustic horn structures, the sample mixture of cells and desired transfection agents/molecules suspended in culture medium is ejected from orifices located at the nozzle tips. During sample ejection, focused mechanical forces (pressure and shear) are generated on a microsecond time scale (dictated by nozzle size/geometry and ejection velocity) resulting in identical "active" microenvironments for each ejected cell. This process enables a number of cellular bioeffects, from uptake of small molecules and gene delivery/transfection to cell lysis. Specifically, we demonstrate successful calcein uptake and transfection of DNA plasmid encoding green fluorescent protein (GFP) into human malignant glioma cells (cell line LN443) using electrosonic microarrays with 36, 45 and 50 mum diameter nozzle orifices and operating at ultrasound frequencies between 0.91 and 0.98 MHz. Our results suggest that efficacy and the extent of bioeffects are mainly controlled by nozzle orifice size and the localized intensity of the applied acoustic field. PMID:17994280

  7. Prevention of autoimmune recurrence and rejection by adenovirus-mediated CTLA4Ig gene transfer to the pancreatic graft in BB rat.

    PubMed

    Uchikoshi, F; Yang, Z D; Rostami, S; Yokoi, Y; Capocci, P; Barker, C F; Naji, A

    1999-03-01

    Type 1 diabetes is the result of a selective destruction of pancreatic islets by autoreactive T-cells. Therefore, in the context of islet or pancreas transplantation, newly transplanted beta-cells are threatened by both recurrent autoimmune and alloimmune responses in recipients with type 1 diabetes. In the present study, using spontaneously diabetic BB rats, we demonstrate that whereas isolated islets are susceptible to autoimmune recurrence and rejection, pancreaticoduodenal grafts are resistant to these biological processes. This resistance is mediated by lymphohematopoietic cells transplanted with the graft, since inactivation of these passenger cells by irradiation uniformly rendered the pancreaticoduodenal grafts susceptible to recurrent autoimmunity. We further studied the impact of local immunomodulation on autoimmune recurrence and rejection by ex vivo adenovirus-mediated CTLA4Ig gene transfer to pancreaticoduodenal grafts. Syngeneic DR-BB pancreaticoduodenal grafts transduced with AdmCTLA4Ig were rescued from recurrent autoimmunity. In fully histoincompatible LEW-->BB transplants, in which rejection and recurrence should be able to act synergistically, AdmCTLA4Ig transduced LEW-pancreaticoduodenal allografts enjoyed markedly prolonged survival in diabetic BB recipients. In situ reverse transcription-polymerase chain reaction revealed that transferred CTLA4Ig gene was strongly expressed in both endocrine and exocrine tissues on day 3. These results indicate the potential utility of local CD28-B7 costimulatory blockade for prevention of alloimmune and autoimmune destruction of pancreatic grafts in type 1 diabetic hosts. PMID:10078573

  8. [Construction and experimental immunity of recombinant replication-competent canine adenovirus type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus].

    PubMed

    Gao, Yu-Wei; Xia, Xian-Zhu; Wang, Li-Gang; Liu, Dan; Huang, Geng

    2006-04-01

    H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003 (HSN1) HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta EHA and the pPoly2-CAV2 were digested with Nru I /Sal I, respectively. The purified Nru I/Sal I DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA. PMID:16736595

  9. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    NASA Astrophysics Data System (ADS)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  10. human adenoviruses role in ophthalmic pterygium formation

    PubMed Central

    Kelishadi, Mishar; Kelishadi, Mandana; Moradi, Abdolvahab; Javid, Naeme; Bazouri, Masoud; Tabarraei, Alijan

    2015-01-01

    Background: Ophthalmic pterygium is a common benign lesion of unknown origin and the pathogenesis might be vision-threatening. This problem is often associated with exposure to solar light. Recent evidence suggests that potentially oncogenic viruses such as human papillomavirus and Epstein-Barr virus may be involved in the pathogenesis of pterygia. Expression of specific adenovirus genes such as E1A and E1B, which potentially have many functions, may contribute to their oncogenic activity as well as relevance to cellular immortalization. Objectives: For the first time, we aimed to investigate involvement of adenoviruses in pterygium formation. Patients and Methods: Fifty tissue specimens of pterygium from patients undergoing pterygium surgery (as cases), 50 conjunctival swab samples from the same patients and 10 conjunctival biopsy specimens from individuals without pterygium such as patients undergoing cataract surgery (as controls) were analyzed for evidence of adenovirus infection with polymerase chain reaction using specific primers chosen from the moderately conserved region of the hexon gene. Furthermore, β-globin primers were used to access the quality of extracted DNA. Data was analyzed using SPSS (version 16) software. Results: Of 50 patients, 20 were men and 30 women with mean age of 61.1 ± 16.9 years ranged between 22 and 85 years. All samples of pterygia had positive results for adenoviruses DNA with polymerase chain reaction, but none of the negative control groups displayed adenoviruses. The pterygium group and the control groups were β-globin positive. Direct sequencing of PCR products confirmed Adenovirus infection. Conclusions: Adenoviruses might act as a possible cause of pterygium formation and other factors could play a synergistic role in the development. However, further larger studies are required to confirm this hypothesis. PMID:26034543

  11. 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

  12. Comparative Proteomics Study of Streptozotocin-induced Diabetic Nephropathy in Rats Kidneys Transfected with Adenovirus-mediated Fibromodulin Gene

    PubMed Central

    Maleki, Akram; Ramazani, Ali; Foroutan, Maryam; Biglari, Alireza; Ranjzad, Parisa; Mellati, Ali Awsat

    2014-01-01

    Background Transforming Growth Factor-beta (TGF-β) activation appears to be crucial for tissue injury in Diabetic Nephropathy (DN). Fibromodulin, the small leucine-rich proteoglycan, has been proposed to be the potent TGF-β modulator. In this study, the therapeutic effects of fibromodulin in the kidneys of streptozotocin (STZ)-induced diabetic rats were investigated. Methods Diabetic rats received intraperitoneal (IP) injections of recombinant adenovirus expression vectors (RAd5) containing fibromodulin (RAd-FMOD) and were killed after 10 weeks. Proteins were isolated from the rat kidney and separated using two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed using Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Results Ten spots were identified using MALDI-TOF-MS. The identified proteins were primarily responsible for cell metabolism, cytoskeleton formation, and oxidative stress. RAd-FMOD treatment markedly attenuated the albuminuria in diabetic rats. Conclusion Taken together, these results provide a valuable clue in exploring the mechanism underlying the therapeutic effects of fibromodulin in diabetic nephropathy suggesting that it can be a potential agent in the treatment of this disease. PMID:24834312

  13. Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adenovirus vector containing the CD4 gene.

    PubMed Central

    Yasukawa, M; Inoue, Y; Ohminami, H; Sada, E; Miyake, K; Tohyama, T; Shimada, T; Fujita, S

    1997-01-01

    It has been reported recently that CD4 is a major component of the receptor for human herpesvirus 7 (HHV-7), which has been newly identified as a T-lymphotropic virus. To investigate further the role of CD4 in HHV-7 infection, we examined the susceptibility to HHV-7 infection of various CD4-negative or weakly positive cell lines into which the cDNA for CD4 was transferred using an adenovirus vector (Adex1CACD4). Of 13 cell lines transduced with Adex1CACD4, including T-lymphoid, B-lymphoid, monocytoid, and myeloid cell lines, one T-lymphoid cell line, one monocytoid cell line, and two cell lines established from the blast crisis of chronic myelogenous leukemia showed high susceptibility to HHV-7 infection. Taken together with the results of previous studies, these data suggest strongly that CD4 is a major component of the binding receptor for HHV-7. This study also shows that HHV-7 may be able to infect CD4-positive hematopoietic precursor cells as well as T lymphocytes. PMID:8995705

  14. A sight on the current nanoparticle-based gene delivery vectors

    PubMed Central

    2014-01-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/in vivo transformation efficiency. PMID:24936161

  15. A sight on the current nanoparticle-based gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

    2014-05-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

  16. Human serum albumin-polyethylenimine nanoparticles for gene delivery.

    PubMed

    Rhaese, Stephanie; von Briesen, Hagen; Rübsamen-Waigmann, Helga; Kreuter, Jörg; Langer, Klaus

    2003-09-19

    Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration. PMID:14499197

  17. Single-Walled Carbon Nanotube Transporter for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Ke, Pu-Chun

    2005-03-01

    Recent studies have shown great promises in integrating nanomaterials in biomedicine. To explore the feasibility of using single-walled carbon nanotubes (SWNTs) as transporters for gene delivery, we have investigated the binding of SWNTs and RNA polymer poly(rU), and the diffusion and the translocation of the SWNT-poly(rU) complexes. Through single-molecule fluorescence imaging, we have found that the pi- stacking dominates the hydrophobic interactions between the carbon rings on tubes and the nitrogenous bases of RNA. Our diffusion study has further demonstrated the feasibility of tracking the motion of water soluble SWNT-poly(rU) complexes. The uptake of SWNT-poly(rU) by breast cancer cells MCF7 was observed using confocal scanning fluorescence microscopy. It was evident that the complexes could penetrate through cell membrane into cytoplasm and cell nucleus. Our cell culture, MTS assay, and radioisotope labeling showed the negligible cytotoxicity of surface modified SWNTs with RNA polymer and amino acids in cell growth medium. These studies have paved the way for gene transfection using SWNTs as transporters.

  18. Characterisation of the Equine adenovirus 2 genome.

    PubMed

    Giles, Carla; Vanniasinkam, Thiru; Barton, Mary; Mahony, Timothy J

    2015-09-30

    Equine adenovirus 2 (EAdV-2) is one of two serotypes of adenoviruses known to infect equines. Initial studies did not associate EAdV-2 infections with any specific clinical syndromes, although more recent evidence suggests that EAdV-2 may be associated with clinical and subclinical gastrointestinal infections of foals and adults respectively. In contrast, Equine adenovirus 1 is well recognised as a pathogen associated with upper respiratory tract infections of horses. In this study the complete genome sequence of EAdV-2 is reported. As expected, genes common to the adenoviruses were identified. Phylogenetic reconstructions using selected EAdV-2 genes confirmed the classification of this virus within the Mastadenovirus genus, and supported the hypothesis that EAdV-2 and EAdV-1 have evolved from separate lineages within the adenoviruses. One spliced open reading frame was identified that encoded for a polypeptide with high similarity to the pIX and E1b_55K adenovirus homologues and was designated pIX_E1b_55K. In addition to this fused version of E1b_55K, a separate E1b_55K encoding gene was also identified. These polypeptides do not appear to have evolved from a gene duplication event as the fused and unfused E1b_55K were most similar to E1b_55K homologues from the Atadenovirus and Mastadenovirus genera respectively. The results of this study suggest that EAdV-2 has an unusual evolutionary history that warrants further investigation. PMID:26220513

  19. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

    PubMed Central

    Figueiredo, Marxa; Esenaliev, Rinat

    2012-01-01

    This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid) (PLGA) or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound) composed either of polymers (PLGA, polystyrene) or other contrast agent materials (Optison, SonoVue microbubbles). The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a) echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b) PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery. PMID:22506124

  20. In Vivo Gene Delivery with L-Tyrosine Polyphosphate Nanoparticles

    PubMed Central

    Ditto, Andrew J.; Reho, John J.; Shah, Kush N.; Smolen, Justin A.; Holda, James H.; Ramirez, Rolando J.; Yun, Yang H.

    2013-01-01

    The concept of gene therapy is promising; however, the perceived risks and side effects associated with this technology have severely dampened the researchers’ enthusiasm.1–3 Thus, the development of a non-viral gene vector without immunological effects and with high transfection efficiency is necessary. Currently, most non-viral vectors have failed to achieve the in vivo transfection efficiencies of viral vectors due their toxicity,4 rapid clearance,5, 6 and/or inappropriate release rates.7 Although our previous studies have successfully demonstrated the controlled-release of plasmid DNA (pDNA) polyplexes encapsulated into nanoparticles formulated with L-tyrosine polyphosphate (LTP-pDNA nanoparticles),8, 9 the in vivo transfection capabilities and immunogenicity of this delivery system has yet to be examined. Thus, we evaluate LTP-pDNA nanoparticles in an in vivo setting via injection into rodent uterine tissue. Our results demonstrate through X-gal staining and immunohistochemistry of uterine tissue that transfection has successfully occurred after a nine-day incubation. In contrast, the results for the control nanoparticles show similar results to shams. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) from the injected tissues confirms the transfection in vivo. To examine the immunogenicity, the LTP nanoparticles have been evaluated in a mouse model. No significant differences in the activation of the innate immune system are observed. These data provide the first report for the potential use of controlled-release nanoparticles formulated from an amino acid based polymer as an in vivo non-viral vector for gene therapy. PMID:23510151

  1. Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

    PubMed Central

    2010-01-01

    Background Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization. Method Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools. Results CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry. Conclusions Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after

  2. Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product.

    PubMed Central

    Raychaudhuri, P; Bagchi, S; Neill, S D; Nevins, J R

    1990-01-01

    Previous experiments have demonstrated that the DNA-binding activity of the E2F transcription factor is increased upon adenovirus infection and that both the E1A and E4 genes are required for activation. In this study, we demonstrated that this enhanced binding of E2F to the E2 promoter is the result of two events. (i) There is stimulation of the DNA-binding activity of the E2F factor; this stimulation is E1A dependent but independent of E4. (ii) There is also induction of a stabilized interaction between E2F molecules bound to adjacent promoter sites; induction of stable E2F binding requires E4 gene function. This two-step activation process was also demonstrated in vitro. A heat-stable fraction from extracts of adenovirus-infected cells, which contains the 19-kilodalton E4 protein, was capable of stimulating stable E2F binding in an ATP-independent manner and appeared to involve direct interaction of the E4 protein with E2F. An extract from virus-infected cells devoid of the E4 19-kilodalton protein stimulated E2F DNA binding without forming the stable complex. This reaction required ATP. We conclude that activation of E2F during adenovirus infection is a two-step process involving a change in both the DNA-binding activity of the factor and the capacity to stabilize the interaction through protein-protein contacts. Images PMID:2139893

  3. Enhanced Transduction and Replication of RGD-Fiber Modified Adenovirus in Primary T Cells

    PubMed Central

    Sengupta, Sadhak; Ulasov, Ilya V.; Thaci, Bart; Ahmed, Atique U.; Lesniak, Maciej S.

    2011-01-01

    Background Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45% of splenic T cells were transduced by Ad-RGD. Conclusions Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin. PMID:21464908

  4. Stimulation of innate immunity by in vivo cyclic di-GMP synthesis using adenovirus.

    PubMed

    Koestler, Benjamin J; Seregin, Sergey S; Rastall, David P W; Aldhamen, Yasser A; Godbehere, Sarah; Amalfitano, Andrea; Waters, Christopher M

    2014-11-01

    The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination. PMID:25230938

  5. Phylogenetic and pathogenic characterization of novel adenoviruses isolated from long-tailed ducks (Clangula hyemalis).

    PubMed

    Counihan, Katrina L; Skerratt, Lee F; Franson, J Christian; Hollmén, Tuula E

    2015-11-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections. PMID:26342465

  6. Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

    PubMed Central

    Knust, B; Brüggemann, U; Doerfler, W

    1989-01-01

    In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene. Images PMID:2473219

  7. Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression.

    PubMed Central

    Carter, T H; Blanton, R A

    1978-01-01

    Relative abundances of early virus RNA species in the cytoplasm of cells infected with wild-type adenovirus type 5 (WT Ad5) and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization kinetics using separated strands of HindIII restriction endonuclease fragments of Ad5 DNA. 1-beta-D-Arabinofuranosylcytosine (ara-C) was used to limit transcription to early virus genes in cells infected by WT virus. At 40.5 degrees C, a restrictive temperature for ts125, three to seven times as much virus RNA from all four early regions of the genome accumulated in the cytoplasm of cells infected by the mutant as accumulated in cells infected by WT. At 32 degrees C, no such difference in the relative abundances of cytoplasmic virus RNA was observed. The capacity to synthesize a 72,000-dalton (72K) virus polypeptide, presumably the single-stranded DNA-binding protein that is defective in ts125 at restrictive temperatures, was compared in cells infected at 40.5 degrees C in the presence of ara-C with the mutant or WT Ad5. The rate of 72K polypeptide synthesis, measured by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of [35S]methionine-labeled polypeptides and autoradiography, was greater at 15 h after infection in ts125-infected cells than in cells infected by WT. A time course experiment showed that the rate of synthesis of the 72K polypeptide increased continuously in ts125-infected cells during the first 15 h of infection, relative to the rate in WT-infected cells. These data are consistent with the hypothesis that Ad5 early gene expression is modulated by the product of an early gene, the 72K DNA-binding protein. Images PMID:203722

  8. An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

    PubMed Central

    Luo, Jing; Li, Caixia; Chen, Jianlin; Wang, Gang; Gao, Rong; Gu, Zhongwei

    2015-01-01

    Transfection efficiency was the primary goal for in vitro gene delivery mediated by nonviral gene carriers. Here, we report a modified gene transfection method that could greatly increase the efficiency of, and accelerate the process mediated by, 25 kDa branched polyethyleneimine and Lipofectamine™ 2000 in a broad range of cell strains, including tumor, normal, primary, and embryonic stem cells. In this method, the combination of transfection procedure with optimized complexation volume had a determinant effect on gene delivery result. The superiorities of the method were found to be related to the change of cellular endocytosis pathway and decrease of particle size. The efficient and simple method established in this study can be widely used for in vitro gene delivery into cultured cells. We think it may also be applicable for many more nonviral gene delivery materials than polyethyleneimine and liposome. PMID:25767387

  9. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

    Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.; Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J.; Mercier, George T.; Barry, Michael A.

    2015-08-15

    We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

  10. Nonviral gene delivery systems by the combination of bubble liposomes and ultrasound.

    PubMed

    Omata, Daiki; Negishi, Yoichi; Suzuki, Ryo; Oda, Yusuke; Endo-Takahashi, Yoko; Maruyama, Kazuo

    2015-01-01

    The combination of therapeutic ultrasound (US) and nano/microbubbles is an important system for establishing a novel and noninvasive gene delivery system. Genes are delivered more efficiently using this system compared with a conventional nonviral vector system such as the lipofection method, resulting in higher gene expression. This higher efficiency is due to the gene being delivered into the cytosol and bypassing the endocytosis pathway. Many in vivo studies have demonstrated US-mediated gene delivery with nano/microbubbles, and several gene therapy feasibility studies for various diseases have been reported. In addition, nano/microbubbles can deliver genes site specifically by the control of US exposure site. In the present review, we summarize the gene delivery systems by the combination of nano/microbubbles and US, describe their properties, and assess applications and challenges of US theranostics. PMID:25620007

  11. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  12. Potential of bovine herpesvirus 4 as a gene delivery vector.

    PubMed

    Donofrio, Gaetano; Cavirani, Sandro; Simone, Taddei; van Santen, Vicky L

    2002-03-01

    A cloning system was developed for construction of BHV-4 recombinants and recombinant virus BHV-4EGFPDeltaTK containing an enhanced green fluorescent protein (EGFP) gene was constructed. The host range of BHV-4EGFPDeltaTK was characterized in vitro. When cell lines from various species and tissues were infected, most of the non-bovine cell lines exhibited neither cytopathic effect (CPE) nor supported viral replication, but EGFP expression was clearly observed. Next, embryonic stem cells were infected and induced to either non-specific or neural differentiation to determine whether they could survive and differentiate after BHV-4EGFPDeltaTK infection. Embryonic stem cells were infected successfully, as indicated by EGFP expression prior to differentiation, and EGFP expression could be detected in many differentiated cells. No CPE was noted. Therefore, BHV-4EGFPDeltaTK infection caused neither cell death nor interfered with non-specific or neural differentiation of embryonic stem cells. Finally, to assess the capability of BHV-4EGFPDeltaTK to infect post-mitotic neurons, cultures from brains of 2-weeks old mice were infected. No death of neuronal cells due to infection was observed and EGFP expression persisted for at least 15 days. Several biological characteristics of BHV-4 demonstrated previously make it a good candidate for a gene delivery vector. These include: little or no pathogenicity, unlikely oncogenicity, ability to establish persistent infection, and capability of herpesviruses to accommodate large amounts of foreign genetic material. These findings add the ability to infect several cell types coming from different animal species, usually without CPE, lack of interference with differentiation, and ability to maintain transgene expression in both undifferentiated and differentiated cells. PMID:11849683

  13. Oligochitosan polyplexes as carriers for retinal gene delivery.

    PubMed

    Puras, G; Zarate, J; Díaz-Tahoces, A; Avilés-Trigueros, Marcelino; Fernández, E; Pedraz, J L

    2013-01-23

    Non-viral gene therapy represents a promising approach for the treatment of retinal diseases. However, the lack of an efficient carrier hampers the implementation of this therapy. In this study, we evaluated low molecular weight ultrapure oligochitosans for the delivery of the pCMS-EGFP plasmid into the rat retina cells after subretinal and intravitreal administrations. Polyplexes were technologically characterized. Resulting polyplexes based on ultrapure oligochitosans were slightly spherical, protected the plasmid against enzymatic digestion, and their charge and size values ranged from 8 to 14 millivolts and from 150 to 69 nm respectively depending on the N/P ratio. In HEK-293 cultured cells, transfection efficiency significantly increased from 12% to 30% when pH decreased from 7.4 to 7.1 (data normalized to Lipofectamine™ 2000). However, no significant transfection was detected in ARPE-19 cultured cells. Subretinal administrations transfected mainly the pigmented cells of the retinal pigment epithelium and the light sensitive photoreceptor cells, whereas intravitreal injections transfected cells in the ganglion cell layer, blood vessels in the inner layers of the retina and photoreceptors. These results support the potential use of oligochitosans for delivering genetic material into retinal cells in vivo. PMID:23201002

  14. Effect of adenovirus-mediated RNA interference on endogenous microRNAs in a mouse model of multidrug resistance protein 2 gene silencing.

    PubMed

    Narvaiza, Iñigo; Aparicio, Oscar; Vera, María; Razquin, Nerea; Bortolanza, Sergia; Prieto, Jesús; Fortes, Puri

    2006-12-01

    RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery. PMID:17020948

  15. Mapping a new gene that encodes an 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2.

    PubMed Central

    Wold, W S; Cladaras, C; Magie, S C; Yacoub, N

    1984-01-01

    The DNA sequence of the early E3 transcription unit of adenovirus 2 (Ad2) (J. Hérissé et al., Nucleic Acids Res. 8:2173-2192, 1980), indicates that an open reading frame exists between nucleotides 1860 and 2163 that could encode a protein of Mr 11,600 (11.6K). We have determined the DNA sequence of the corresponding region in Ad5 (closely related to Ad2) and have established that this putative gene is conserved in Ad5 (a 10.5K protein). To determine whether this protein is expressed, we prepared an antiserum in rabbits against a synthetic peptide corresponding to amino acids 66 to 74 in the 11.6K protein of Ad2. The peptide antiserum immunoprecipitated a ca. 13K-14K protein doublet, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from [35S]methionine-labeled Ad2- or Ad5-early-infected KB cells. The antiserum also immunoprecipitated a 13K-14K protein doublet translated in vitro from Ad2 or Ad5 early E3-specific mRNA purified by hybridization to Ad2 EcoRI-D (nucleotides -236 to 2437). The synthetic peptide successfully competed with the 13K-14K protein doublet in immunoprecipitation experiments, thereby confirming the specificity of the antiserum. As deduced from the DNA sequence, the 11.6K protein (and the corresponding 10.5K Ad5 protein) has a conserved 22-amino-acid hydrophobic domain, suggesting that the protein may be associated with membranes. We conclude that a gene located at nucleotides 1860 to 2143 in the Ad2 E3 transcription unit (nucleotides 1924 to 2203) in the Ad5 E3 transcription unit) encodes an 11.6K protein (10.5K in Ad5). Images PMID:6492252

  16. 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

  17. Gene Delivery in salivary glands: from the bench to the clinic

    PubMed Central

    Samuni, Yuval; Baum, Bruce J.

    2011-01-01

    In vivo gene delivery has long been seen as providing opportunities for the development of novel treatments for disorders refractory to existing therapies. Over the last two decades, salivary glands have proven to be a useful, if somewhat unconventional, target tissue for studying several potential clinical applications of therapeutic gene delivery. Herein, we follow the progress, address some problems and assess the outlook for clinical applications of salivary gland gene delivery. Our experience with these tissues provides a roadmap for the process of moving an idea from the laboratory bench to patients. PMID:21763423

  18. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  19. Image-guided, Intravascular Hydrodynamic Gene Delivery to Skeletal Muscle in Pigs

    PubMed Central

    Kamimura, Kenya; Zhang, Guisheng; Liu, Dexi

    2009-01-01

    Development of an effective, safe, and convenient method for gene delivery to muscle is a critical step toward gene therapy for muscle-associated diseases. Toward this end, we have explored the possibility of combining the image-guided catheter insertion technique with the principle of hydrodynamic delivery to achieve muscle-specific gene transfer in pigs. We demonstrate that gene transfer efficiency of the procedure is directly related to flow rate, injection pressure, and injection volume. The optimal gene delivery was achieved at a flow rate of 15 ml/second with injection pressure of 300 psi and injection volume equal to 1.5% of body weight. Under such a condition, hydrodynamic injection of saline containing pCMV-Luc (100 µg/ml) resulted in luciferase activity of 106 to 107 relative light units (RLU)/mg of proteins extracted from the targeted muscle 5 days after hydrodynamic gene delivery. Result from immunohistochemical analysis revealed 70–90% transfection efficiency of muscle groups in the hindlimb and persistent reporter gene expression for 2 months in transfected cells. With an exception of transient edema and elevation of creatine phosphokinase, no permanent tissue damage was observed. These results suggest that the image-guided, intravenous hydrodynamic delivery is an effective and safe method for gene delivery to skeletal muscle. PMID:19738603

  20. Group D Adenoviruses Infect Primary Central Nervous System Cells More Efficiently than Those from Group C

    PubMed Central

    Chillon, Miguel; Bosch, Assumpció; Zabner, Joseph; Law, Lane; Armentano, Donna; Welsh, Michael J.; Davidson, Beverly L.

    1999-01-01

    Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector. PMID:9971839

  1. Polyethylenimine-mediated gene delivery to the lung and therapeutic applications

    PubMed Central

    Di Gioia, Sante; Conese, Massimo

    2008-01-01

    Nonviral gene delivery is now considered a promising alternative to viral vectors. Among nonviral gene delivery agents, polyethylenimine (PEI) has emerged as a potent candidate for gene delivery to the lung. PEI has some advantages over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. However, intracellular (mainly the nuclear membrane) and extracellular obstacles still hamper its efficiency in vitro and in vivo, depending on the route of administration and the type of PEI. Nuclear delivery has been increased by adding nuclear localization signals. To overcome nonspecific interactions with biological fluids, extracellular matrix components and nontarget cells, strategies have been developed to protect polyplexes from these interactions and to increase target specificity and gene expression. When gene delivery into airway epithelial cells of the conducting airways is necessary, aerosolization of complexes seems to be better suited to guarantee higher transgene expression in the airway epithelial cells with lower toxicity than observed with either intratracheal or intravenous administration. Aerosolization, indeed, is useful to target the alveolar epithelium and pulmonary endothelium. Proof-of-principle that PEI-mediated gene delivery has therapeutic application to some genetic and acquired lung disease is presented, using as genetic material either plasmidic DNA or small-interfering RNA, although optimization of formulation and delivery protocols and limitation of toxicity need further studies. PMID:19920904

  2. Current Progress in Gene Delivery Technology Based on Chemical Methods and Nano-carriers

    PubMed Central

    Jin, Lian; Zeng, Xin; Liu, Ming; Deng, Yan; He, Nongyue

    2014-01-01

    Gene transfer methods are promising in the field of gene therapy. Current methods for gene transfer include three major groups: viral, physical and chemical methods. This review mainly summarizes development of several types of chemical methods for gene transfer in vitro and in vivo by means of nano-carriers like; calcium phosphates, lipids, and cationic polymers including chitosan, polyethylenimine, polyamidoamine dendrimers, and poly(lactide-co-glycolide). This review also briefly introduces applications of these chemical methods for gene delivery. PMID:24505233

  3. Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers.

    PubMed

    McErlean, Emma M; McCrudden, Cian M; McCarthy, Helen O

    2016-09-01

    The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration. PMID:27582234

  4. Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery.

    PubMed

    Kobayashi, Yuji; Kamimura, Kenya; Abe, Hiroyuki; Yokoo, Takeshi; Ogawa, Kohei; Shinagawa-Kobayashi, Yoko; Goto, Ryo; Inoue, Ryosuke; Ohtsuka, Masato; Miura, Hiromi; Kanefuji, Tsutomu; Suda, Takeshi; Tsuchida, Masanori; Aoyagi, Yutaka; Zhang, Guisheng; Liu, Dexi; Terai, Shuji

    2016-01-01

    Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy. PMID:27574785

  5. Complexation of oppositely charged polyelectrolytes in gene delivery and biology

    NASA Astrophysics Data System (ADS)

    Shklovskii, Boris

    2009-03-01

    Charge inversion of a DNA double helix by a positively charged flexible polymer (polyelectrolyte) is widely used to facilitate DNA contact with negative cell membranes for gene delivery. Motivated by this application in the first part of the talk I study the phase diagram a solution of long polyanions (PA) with a shorter polycations (PC) as a function the ratio of total charges of PC and PA in the solution, x, and the concentration of monovalent salt. Each PA attracts many PCs to form a complex. When x= 1, the complexes are neutral and condense in a macroscopic drop. When x is far away from 1, complexes are strongly charged and stable. PA are overcharged by PC at x > 1 and undercharged by PC at x < 1. As x approaches 1, PCs attached to PA disproportionate between complexes. Some complexes become neutral and condensed in a macroscopic drop while others become even stronger charged and stay free. The second part of the talk deals with biological example of PA -PC complexes namely self-assembly of vegetable viruses from long ss-RNA molecule paying role of scaffold and identical capsid proteins with long positive tails. I show that optimization Coulomb energy of the virus leads to the charge of RNA twice larger than the total charge of the capsid, in agreement with the experimental data. Then I discuss kinetics of the Coulomb complexation driven virus self-assembly. Capsid proteins stick to unassembled chain of ss RNA (which we call ``antenna'') and slide on it towards the assembly site. I show that at excess of capsid proteins such one-dimensional diffusion accelerates self-assembly more than ten times. On the other hand at excess of ss-RNA, antenna slows self-assembly down. Several experiments are proposed to verify the role of ss-RNA antenna in self-assembly.

  6. Novel endosomolytic peptides for enhancing gene delivery in nanoparticles.

    PubMed

    Ahmad, Aqeel; Ranjan, Sanjeev; Zhang, Weikai; Zou, Jing; Pyykkö, Ilmari; Kinnunen, Paavo K J

    2015-02-01

    Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection. PMID:25445677

  7. Development of the Liposomes Entrapped Ultrasound Imaging Gas (``Bubble Liposomes'') as Novel Gene Delivery Carriers

    NASA Astrophysics Data System (ADS)

    Suzuki, Ryo; Tanaka, Kumiko; Sawamura, Kaori; Takizawa, Tomoko; Utoguchi, Naoki; Negishi, Yoichi; Hagisawa, Kohsuke; Nishioka, Toshihiko; Maruyama, Kazuo

    2006-05-01

    Recently, microbubbles and ultrasound have been investigated with a view to improving the transfection efficiency of nonviral delivery systems for gene by cavitation. However, microbubbles had some problems in terms of stability and targeting ability. To solve these problems, we paid attention to liposomes that had many advantages such as stable and safe in vivo and easy to modify targeting ligand. Previously, we have represented that liposomes are good drug and gene delivery carriers. In addition, we developed that the liposomes ("Bubble liposomes") were entrapped with perfluoropropane known as ultrasound imaging gas. In this study, we assessed about feasibility of "Bubble liposomes" as gene delivery tool utilized cavitation by ultrasound irradiation. "Bubble liposomes" could effectively deliver plasmid DNA to cells by combination of ultrasound irradiation without cyototoxicity. This result suggested that "Bubble liposomes" might be a new class of tool for gene delivery.

  8. Biosensor-controlled gene therapy/drug delivery with nanoparticles for nanomedicine

    NASA Astrophysics Data System (ADS)

    Prow, Tarl W.; Rose, William A.; Wang, Nan; Reece, Lisa M.; Lvov, Yuri; Leary, James F.

    2005-04-01

    Nanomedicine involves cell-by-cell regenerative medicine, either repairing cells one at a time or triggering apoptotic pathways in cells that are not repairable. Multilayered nanoparticle systems are being constructed for the targeted delivery of gene therapy to single cells. Cleavable shells containing targeting, biosensing, and gene therapeutic molecules are being constructed to direct nanoparticles to desired intracellular targets. Therapeutic gene sequences are controlled by biosensor-activated control switches to provide the proper amount of gene therapy on a single cell basis. The central idea is to set up gene therapy "nanofactories" inside single living cells. Molecular biosensors linked to these genes control their expression. Gene delivery is started in response to a biosensor detected problem; gene delivery is halted when the cell response indicates that more gene therapy is not needed. Cell targeting of nanoparticles, both nanocrystals and nanocapsules, has been tested by a combination of fluorescent tracking dyes, fluorescence microscopy and flow cytometry. Intracellular targeting has been tested by confocal microscopy. Successful gene delivery has been visualized by use of GFP reporter sequences. DNA tethering techniques were used to increase the level of expression of these genes. Integrated nanomedical systems are being designed, constructed, and tested in-vitro, ex-vivo, and in small animals. While still in its infancy, nanomedicine represents a paradigm shift in thinking-from destruction of injured cells by surgery, radiation, chemotherapy to cell-by-cell repair within an organ and destruction of non-repairable cells by natural apoptosis.

  9. 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

  10. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

    PubMed

    Ribault, S; Neuville, P; Méchine-Neuville, A; Augé, F; Parlakian, A; Gabbiani, G; Paulin, D; Calenda, V

    2001-03-16

    Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders. PMID:11249869

  11. [Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

    PubMed

    Ma, Jing; Zhang, Xiao-Guang; Chen, Hong; Li, Kui-Biao; Zhang, Xiao-Mei; Zhang, Ke; Yang, Liang; Xu, Hong; Shu, Yue-Long; Tan, Wen-Jie; Zeng, Yi

    2009-09-01

    The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation. PMID:19954107

  12. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    SciTech Connect

    Freytag, Svend O.; Stricker, Hans; Lu, Mei; Elshaikh, Mohamed; Aref, Ibrahim; Pradhan, Deepak; Levin, Kenneth; Kim, Jae Ho; Peabody, James; Siddiqui, Farzan; Barton, Kenneth; Pegg, Jan; Zhang, Yingshu; Cheng, Jingfang; Oja-Tebbe, Nancy; Bourgeois, Renee; Gupta, Nilesh; Lane, Zhaoli; Rodriguez, Ron; DeWeese, Theodore; and others

    2014-06-01

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

  13. Low-density lipoprotein receptor gene therapy using helper-dependent adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia.

    PubMed

    Nomura, S; Merched, A; Nour, E; Dieker, C; Oka, K; Chan, L

    2004-10-01

    We tested the efficacy of low-density lipoprotein receptor (LDLR) therapy using helper-dependent adenovirus (HD-Ad), comparing it with that of very low-density lipoprotein receptor (VLDLR), an LDLR homolog. We treated high cholesterol diet fed LDLR-/- mice with a single intravenous injection of HD-Ad expressing monkey LDLR (1.5 x 10(13) or 5 x 10(12) VP/kg) or VLDLR. Throughout the 24-week experiment, plasma cholesterol of LDLR-treated mice was lower than that of VLDLR-treated mice, which was in turn lower than that of PBS-treated mice. Anti-LDLR antibodies developed in 2/10 mice treated with high-dose HD-Ad-LDLR but in none (0/14) of the other treatment groups. HD-Ad-treated mice displayed significant retardation of atherosclerotic lesion progression. We next tested the long-term efficacy of low-dose HD-Ad-LDLR injected into 12-week-old LDLR-/- mice. After 60 weeks, atherosclerosis lesions covered approximately 50% of the surface of aortas of control mice, whereas aortas of treated mice were essentially lesion-free. The lipid lowering effect of HD-Ad-LDLR lasted at least 108 weeks (>2 years) when all control mice had died. In addition to retarding lesion progression, treatment caused lesion remodeling from a vulnerable-looking to a more stable-appearing phenotype. In conclusion, HD-Ad-mediated LDLR gene therapy is effective in conferring long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia. PMID:15269711

  14. Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury.

    PubMed

    Wheeler, M D; Katuna, M; Smutney, O M; Froh, M; Dikalova, A; Mason, R P; Samulski, R J; Thurman, R G

    2001-12-10

    The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress. PMID:11779401

  15. Synthesis and inflammatory response of a novel silk fibroin scaffold containing BMP7 adenovirus for bone regeneration.

    PubMed

    Zhang, Yufeng; Wu, Chengtie; Luo, Tao; Li, Shue; Cheng, Xiangrong; Miron, Richard J

    2012-10-01

    Gene therapy has garnished tremendous awareness for the repair of osseous defects. It exhibits high efficiency gene transfer and osteogenic differentiation potential making it well suitable for the sustained delivery of growth factors to local tissues. In the present study a simplified solution-based in situ biomimetic synthesis method is demonstrated for bone morphogenetic protein 7 (BMP7) adenovirus combined with silk fibroin scaffolds. This scaffold not only provides the three dimensional space for bone ingrowth, but also releases the BMP7 adenovirus which targets its secretion by host cells in vivo. Scaffolds were tested both in vitro for their osteogenic potential as well as in vivo in a critical-size calvarial defect in mice. Scaffolds loaded with bone morphogenetic protein 7 adenovirus (adBMP7) were able to sustain release of adBMP7 for up to 21 days and support cell proliferation and differentiation to bone forming osteoblasts. Calvarial defects treated with scaffolds containing adBMP7 significantly induced new bone formation in vivo. To demonstrate immuno-compatibility with host tissues, IL-2, IL-6 and TNF-α were measured up to 4 weeks post-implantation. Although these scaffolds demonstrated an initial pro-inflammatory response, levels of IL-2, IL-6 and TNF-α returned to baseline control values at either 2 or 4 weeks post-implantation demonstrating long term compatibility for growth factor delivery via gene therapy. The results from the present study indicate the promise of gene delivery scaffold systems for robust, low cost, and high quality bone tissue engineering applications. PMID:22796416

  16. Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

    SciTech Connect

    Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E.; Leeuwen, M. van; Boheemen, S. van; Jong, A.A.W. de; Osterhaus, A.D.M.E.; Smits, S.L.; Kuiken, T.

    2013-05-25

    Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. - Highlights: ► Lesions typical for adenovirus infection detected in cloacal bursa of dead gulls. ► Confirmation of adenovirus infection by electron microscopy and deep sequencing. ► Sequence analysis indicates that it is a novel adenovirus in the genus Aviadenovirus. ► The novel (Gull) adenovirus was detected in multiple organs of two species of gulls.

  17. 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

  18. Rapid generation of fowl adenovirus 9 vectors.

    PubMed

    Pei, Yanlong; Griffin, Bryan; de Jong, Jondavid; Krell, Peter J; Nagy, Éva

    2015-10-01

    Fowl adenoviruses (FAdV) have the largest genomes of any fully sequenced adenovirus genome, and are widely considered as excellent platforms for vaccine development and gene therapy. As such, there is a strong need for stream-lined protocols/strategies for the generation of recombinant adenovirus genomes. Current genome engineering strategies rely upon plasmid based homologous recombination in Escherichia coli BJ5183. This process is time-consuming, involves multiple cloning steps, and low efficiency recombination. This report describes a novel system for the more rapid generation of recombinant fowl adenovirus genomes using the lambda Red recombinase system in E. coli DH10B. In this strategy, PCR based amplicons with around 50 nt long homologous arms, a unique SwaI site and a chloramphenicol resistance gene fragment (CAT cassette), are introduced into the FAdV-9 genome in a highly efficient and site-specific manner. To demonstrate the efficacy of this system we generated FAdV-9 ORF2, and FAdV-9 ORF11 deleted, CAT marked and unmarked FAdV-9 infectious clones (FAdmids), and replaced either ORF2 or ORF11, with an EGFP expression cassette or replaced ORF2 with an EGFP coding sequence via the unique SwaI sites, in approximately one month. All recombinant FAdmids expressed EGFP and were fully infectious in CH-SAH cells. PMID:26238923

  19. Protective role of adenovirus vector-mediated interleukin-10 gene therapy on endogenous islet β-cells in recent-onset type 1 diabetes in NOD mice

    PubMed Central

    LI, CHENG; ZHANG, LIJUAN; CHEN, YANYAN; LIN, XIAOJIE; LI, TANG

    2016-01-01

    The aim of the present study was to provide an animal experimental basis for the protective effect of the adenoviral vector-mediated interleukin-10 (Ad-mIL-10) gene on islet β-cells during the early stages of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. A total of 24 female NOD mice at the onset of diabetes were allocated at random into three groups (n=8 per group): Group 1, intraperitoneally injected with 0.1 ml Ad-mIL-10; group 2, intraperitoneally injected with 0.1 ml adenovirus vector; and group 3, was a diabetic control. In addition to groups 1, 2 and 3, 8 age- and gender-matched NOD mice were intraperitoneally injected with 0.1 ml PBS and assigned to group 4 as a normal control. All mice were examined weekly for body weight, urine glucose and blood glucose values prior to onset of diabetes, and at 1, 2 and 3 weeks after that, and all mice were sacrificed 3 weeks after injection. Serum levels of interleukin (IL)-10, interferon (IFN)-γ, IL-4, insulin and C-peptide were evaluated, and in addition the degree of insulitis and the local expression of IL-10 gene in the pancreas were detected. The apoptosis rate of pancreatic β-cells was determined using a TUNEL assay. Compared with groups 2 and 3, IL-10 levels in the serum and pancreas were elevated in group 1. Serum IFN-γ levels were decreased while serum IL-4 levels and IFN-γ/IL-4 ratio were significantly increased in group 1 (P<0.01). C-peptide and insulin levels were higher in group 1 compared with groups 2 and 3, (P<0.01). Furthermore, compared with groups 2 and 3, the degree of insulitis, islet β-cell apoptosis rate and blood glucose values did not change significantly (P>0.05). The administration of the Ad-mIL-10 gene induced limited immune regulatory and protective effects on islet β-cell function in NOD mice with early T1D, while no significant reduction in insulitis, islet β-cell apoptosis rate and blood glucose was observed. PMID:27168782

  20. Functions of and interactions between the A and B blocks in adenovirus type 2-specific VARNA1 gene.

    PubMed

    Cannon, R E; Wu, G J; Railey, J F

    1986-03-01

    The internal transcriptional control region (ITCR) of VARNA1 gene consists of a 33-base-pair (bp) interblock sequence and two 12-bp sequence blocks that are highly conserved in most of the genes transcribed by RNA polymerase III. To define the functions of and study the interactions between the two blocks, we have constructed mutants with altered interblock sequence or spacing for transcription. The results of transcription efficiencies and competing strengths indicated that the interblock sequence was dispensable and the A and B blocks were essential for transcription control. One of the major functions of the interblock sequence was to maintain an optimal spacing for an intimate interaction between the two essential blocks. Shortening or elongating the interblock spacing in the mutants beyond this range drastically decreased the transcription efficiencies and competing strengths of these mutated genes. To further study how the interaction between the two blocks leads to initiation, the start sites and sizes of RNA products of the mutants were determined. When the interblock spacing was less than 105 bp, the wild-type start site was dictated by the A block after an interaction with the B block through proteins. However, when the interblock spacing was longer than 105 bp, several new start sites located closer to the B block were preferentially used. This suggests that new start sites may be dictated by the B block when its interaction with the A block is weakened by longer spacing. The mechanisms of interaction between the bipartite domain in this gene leading to initiation are different from those in tRNAs and Alu-family RNA genes. PMID:3456587

  1. Functions of and interactions between the A and B blocks in adenovirus type 2-specific VARNA1 gene.

    PubMed Central

    Cannon, R E; Wu, G J; Railey, J F

    1986-01-01

    The internal transcriptional control region (ITCR) of VARNA1 gene consists of a 33-base-pair (bp) interblock sequence and two 12-bp sequence blocks that are highly conserved in most of the genes transcribed by RNA polymerase III. To define the functions of and study the interactions between the two blocks, we have constructed mutants with altered interblock sequence or spacing for transcription. The results of transcription efficiencies and competing strengths indicated that the interblock sequence was dispensable and the A and B blocks were essential for transcription control. One of the major functions of the interblock sequence was to maintain an optimal spacing for an intimate interaction between the two essential blocks. Shortening or elongating the interblock spacing in the mutants beyond this range drastically decreased the transcription efficiencies and competing strengths of these mutated genes. To further study how the interaction between the two blocks leads to initiation, the start sites and sizes of RNA products of the mutants were determined. When the interblock spacing was less than 105 bp, the wild-type start site was dictated by the A block after an interaction with the B block through proteins. However, when the interblock spacing was longer than 105 bp, several new start sites located closer to the B block were preferentially used. This suggests that new start sites may be dictated by the B block when its interaction with the A block is weakened by longer spacing. The mechanisms of interaction between the bipartite domain in this gene leading to initiation are different from those in tRNAs and Alu-family RNA genes. Images PMID:3456587

  2. Defining the functional domains in the control region of the adenovirus type 2 specific VARNA1 gene.

    PubMed

    Wu, G J; Railey, J F; Cannon, R E

    1987-04-01

    The outer boundaries of the internal transcriptional control region in the VARNA1 gene have been located from positions +10 to +69. To further define the detailed organization of the functional domains in this region and the function(s) of the 5' flanking sequence, and to obtain a more detailed insight into other transcriptionally important sequences, we have constructed 77 mutants with deletion endpoints at almost every one to five base-pairs in the entire region from -30 to +160 for transcriptional studies. Using our highly active crude extract under our assay conditions, and quantitatively measuring the transcriptional efficiency and competing strength of each mutant, we have revealed new features of important transcriptional control sequences and defined the transcriptional functions of several functional domains in this gene. The essential domain is from +59/+63 to +66/+68, which corresponds to the B block sequence. This is smaller than that defined previously. The second most important domain is the region from +12/14 to +40, which includes the A block sequence that dictates the wild-type major start site and amplifies the events started by the B block region, mediated through factors and RNA polymerase III. Furthermore, the domain from -5 to +11 affects the use of certain start site(s). Moreover, the 5' flanking region from -30 to +1 contributes 80 to 90% of the overall transcriptional efficiency of the gene. Finally, our transcriptional studies of mutants deleted of the A block sequence and all of the upstream sequence indicated that an intimate interaction between the two blocks is essential for initiation of transcription. Furthermore, the B block sequence is more important than the A block sequence in the transcription reaction. The mechanism and control of transcriptional initiation in the VARNA1 gene is similar to that in some tRNA genes, but differs from that in others. PMID:3625769

  3. A Photo-Degradable Gene Delivery System for Enhanced Nuclear Gene Transcription

    PubMed Central

    Hoyoung, Lee; Yeji, Kim; Patrick G., Schweickert; Stephen F., Konieczny; You-Yeon, Won

    2013-01-01

    There currently exists a significant gap in our understanding of how the detailed chemical characteristics of polycation gene carriers influence their delivery performances in overcoming an important cellular-level transport barrier, i.e., intranuclear gene transcription. In this study, a UV-degradable gene carrier material (ENE4-1) was synthesized by crosslinking low molecular weight branched polyethylenimine (bPEI-2k) molecules using UV-cleavable o-nitrobenzyl urethane (NBU) as the linker molecule. NBU degrades upon exposure to mild UV irradiation. Therefore, this UV-degradable carrier allows us to control the chemical characteristics of the polymer/DNA complex (polyplex) particles at desired locations within the intracellular environment. By using this photolytic DNA carrier, we found that the exact timing of the UV degradation significantly influences the gene transfection efficiencies of ENE4-1/DNA(pGL2) polyplexes in HeLa cells. Interestingly, even if the polyplexes were UV-degraded at different intracellular locations/times, their nuclear entry efficiency was not influenced by the location/timing of UV degradation. The UV treatment did not influence the size or binding strength of the polyplexes. However, we confirmed that the degradation of the carrier molecules impacts the chemical characteristics of the polyplexes (it produces carbamic acid and nitrosobenzyl aldehyde groups on ENE4-1). We believe that these anionic acid groups enhance the interaction of the polyplexes with nuclear transcription proteins and thus the final gene expression levels; this effect was found to occur, even though UV irradiation itself has a general effect of reducing transfection efficiencies. Excess (uncomplexed) ENE4-1 polymers appear to not play any role in the UV-enhanced gene transcription phenomenon. PMID:24172855

  4. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment.

    PubMed

    Kang, Lin; Gao, Zhonggao; Huang, Wei; Jin, Mingji; Wang, Qiming

    2015-05-01

    The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems. PMID:26579443

  5. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment

    PubMed Central

    Kang, Lin; Gao, Zhonggao; Huang, Wei; Jin, Mingji; Wang, Qiming

    2015-01-01

    The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems. PMID:26579443

  6. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin; Kannan, Rangaramanujam M.

    2015-02-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform.Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE

  7. Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF

    PubMed Central

    Im, S-A; Kim, J-S; Gomez-Manzano, C; Fueyo, J; Liu, T-J; Cho, M-S; Seong, C-M; Lee, S N; Hong, Y-K; Yung, W K A

    2001-01-01

    Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-αVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-αVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-αVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF 165 cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-αVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336478

  8. 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.

  9. Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-07-28

    Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'. PMID:27298431

  10. Up-Scaled Synthesis and Characterization of Nonviral Gene Delivery Particles for Transient In Vitro and In Vivo Transgene Expression.

    PubMed

    Taschauer, Alexander; Geyer, Antonia; Gehrig, Sebastian; Maier, Julia; Sami, Haider; Ogris, Manfred

    2016-06-01

    Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery. Nanoparticle tracking analysis of the upscaled formulations involved single nanoparticle tracking, thereby generating highly resolved biophysical characterization. Gene delivery performance was investigated by luciferase gene expression in cells and three-dimensional bioluminescence imaging in mice. PMID:27169568

  11. Structure of human adenovirus

    SciTech Connect

    Nemerow, Glen R.; Stewart, Phoebe L.; Reddy, Vijay S.

    2012-07-11

    A detailed structural analysis of the entire human adenovirus capsid has been stymied by the complexity and size of this 150 MDa macromolecular complex. Over the past 10 years, the steady improvements in viral genome manipulation concomitant with advances in crystallographic techniques and data processing software has allowed structure determination of this virus by X-ray diffraction at 3.5 {angstrom} resolution. The virus structure revealed the location, folds, and interactions of major and minor (cement proteins) on the inner and outer capsid surface. This new structural information sheds further light on the process of adenovirus capsid assembly and virus-host cell interactions.

  12. 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

  13. Microbubbles in Ultrasound-Triggered Drug and Gene Delivery

    PubMed Central

    Hernot, Sophie; Klibanov, Alexander L.

    2008-01-01

    Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection is observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials. PMID:18486268

  14. Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

    NASA Astrophysics Data System (ADS)

    Au, Giang H. T.; Y Shih, Wan; Shih, Wei-Heng

    2015-01-01

    Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control.

  15. Image-guided, Lobe-specific Hydrodynamic Gene Delivery to Swine Liver

    PubMed Central

    Kamimura, Kenya; Suda, Takeshi; Xu, Wei; Zhang, Guisheng; Liu, Dexi

    2009-01-01

    Image-guided, lobe-specific hydrodynamic gene delivery to liver was assessed in pigs. The procedure involved image-guided insertion of a balloon catheter to the hepatic vein of the selected lobe from the jugular vein and hydrodynamic injection of plasmid DNA using a newly developed computer-controlled injection device. We demonstrated that the impact of the procedure was regional with minimal effects on neighboring lobes. Level of gene expression resulted from the procedure was 107 relative light units (RLU)/mg in the targeted lobes and 102–105 RLU/mg in the nontargeted lobes 4 hours after hydrodynamic injection of pCMV-Luc plasmids. Occlusion of blood flow in the inferior vena cava (IVC) or IVC plus portal vein (PV) was effective in elevating hydrodynamic pressure in the targeted vasculature but did not enhance gene delivery efficiency. Physiological examination on pigs with IVC occlusion revealed transient decreases of blood pressure and respiration rate. Removal of occlusion from IVC resulted in a rapid and transient increase in heart rate. Occlusion of the PV and hepatic vein showed no effect on physiological and cardiac activities. No major changes in serum composition were observed. These results suggest that (i) image-guided, lobe-specific hydrodynamic procedure is effective for regional gene delivery to liver, (ii) blockade in IVC should be avoided for hydrodynamic gene delivery to the liver, and (iii) clinical application of hydrodynamic gene delivery to liver is feasible. PMID:19156134

  16. Gene delivery to the neurulating embryo during culture

    EPA Science Inventory

    Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of g...

  17. 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

  18. Development of a Targeted Gene-Delivery System Using Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chang, Chih-Hsiang; Chao, Yun-Peng; Kao, Ming-Ching

    2016-01-01

    A gene-delivery system based on microbes is useful for development of targeted gene therapy of non-phagocytic cancer cells. Here, the feasibility of the delivery system is illustrated by targeted delivery of a transgene (i.e., eukaryotic GFP) by Escherichia coli to HER2/neu-positive cancer cells. An E. coli strain was engineered with surface display of the anti-HER2/neu affibody. To release the gene cargo, a programmed lysis system based on phage ϕX174 gene E was introduced into the E. coli strain. As a result, 3 % of HER2/neu-positive cells that were infected with engineered E. coli were able to express the GFP. PMID:26846805

  19. Role of nanotechnology and gene delivery systems in TRAIL-based therapies

    PubMed Central

    Naoum, George E; Tawadros, Fady; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Tabassum, Sobia; Buchsbaum, Donald J; Arafat, Waleed

    2016-01-01

    Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles’ implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery. PMID:27594905

  20. Role of nanotechnology and gene delivery systems in TRAIL-based therapies.

    PubMed

    Naoum, George E; Tawadros, Fady; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Tabassum, Sobia; Buchsbaum, Donald J; Arafat, Waleed

    2016-01-01

    Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles' implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery. PMID:27594905

  1. Placental expression of imprinted genes varies with sampling site and mode of delivery

    PubMed Central

    Janssen, A.B.; Tunster, S.J.; Savory, N.; Holmes, A.; Beasley, J.; Parveen, S.A.R.; Penketh, R.J.A.; John, R.M.

    2015-01-01

    Imprinted genes, which are monoallelically expressed by virtue of an epigenetic process initiated in the germline, are known to play key roles in regulating fetal growth and placental development. Numerous studies are investigating the expression of these imprinted genes in the human placenta in relation to common complications of pregnancy such as fetal growth restriction and preeclampsia. This study aimed to determine whether placental sampling protocols or other factors such as fetal sex, gestational age and mode of delivery may influence the expression of imprinted genes predicted to regulate placental signalling. Methods Term placentas were collected from Caucasian women delivering at University Hospital of Wales or Royal Gwent Hospital within two hours of delivery. Expression of the imprinted genes PHLDA2, CDKN1C, PEG3 and PEG10 was assayed by quantitative real time PCR. Intraplacental gene expression was analysed (N = 5). Placental gene expression was compared between male (N = 11) and female (N = 11) infants, early term (N = 8) and late term (N = 10) deliveries and between labouring (N = 13) and non-labouring (N = 21) participants. Results The paternally expressed imprinted genes PEG3 and PEG10 were resilient to differences in sampling site, fetal sex, term gestational age and mode of delivery. The maternally expressed imprinted gene CDKN1C was elevated over 2-fold (p < 0.001) in placenta from labouring deliveries compared with elective caesarean sections. In addition, the maternally expressed imprinted gene PHLDA2 was elevated by 1.8 fold (p = 0.01) in samples taken at the distal edge of the placenta compared to the cord insertion site. Conclusion These findings support the reinterpretation of existing data sets on these genes in relation to complications of pregnancy and further reinforce the importance of optimising and unifying placental collection protocols for future studies. PMID:26162698

  2. Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases.

    PubMed

    David, A L; Peebles, D M; Gregory, L; Waddington, S N; Themis, M; Weisz, B; Ruthe, A; Lawrence, L; Cook, T; Rodeck, C H; Coutelle, C

    2006-07-01

    Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the beta-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase immunohistochemistry of fetal tissues. Expression of beta-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia. PMID:16839275

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

    SciTech Connect

    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 lysosomes 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.

  4. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

  5. A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

    NASA Astrophysics Data System (ADS)

    Li, Peng; Liu, Donghua; Sun, Xiaoli; Liu, Chunxi; Liu, Yongjun; Zhang, Na

    2011-06-01

    The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.

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

    NASA Astrophysics Data System (ADS)

    Zhang, Bingqi

    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 lysosomes 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.

  7. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells†

    PubMed Central

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin

    2016-01-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform. PMID:25213606

  8. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology

    NASA Astrophysics Data System (ADS)

    Chen, Shuyuan; Ding, Jia-Huan; Bekeredjian, Raffi; Yang, Bing-Zhi; Shohet, Ralph V.; Johnston, Stephen A.; Hohmeier, Hans E.; Newgard, Christopher B.; Grayburn, Paul A.

    2006-05-01

    This study describes a method of gene delivery to pancreatic islets of adult, living animals by ultrasound targeted microbubble destruction (UTMD). The technique involves incorporation of plasmids into the phospholipid shell of gas-filled microbubbles, which are then infused into rats and destroyed within the pancreatic microcirculation with ultrasound. Specific delivery of genes to islet beta cells by UTMD was achieved by using a plasmid containing a rat insulin 1 promoter (RIP), and reporter gene expression was regulated appropriately by glucose in animals that received a RIP-luciferase plasmid. To demonstrate biological efficacy, we used UTMD to deliver RIP-human insulin and RIP-hexokinase I plasmids to islets of adult rats. Delivery of the former plasmid resulted in clear increases in circulating human C-peptide and decreased blood glucose levels, whereas delivery of the latter plasmid resulted in a clear increase in hexokinase I protein expression in islets, increased insulin levels in blood, and decreased circulating glucose levels. We conclude that UTMD allows relatively noninvasive delivery of genes to pancreatic islets with an efficiency sufficient to modulate beta cell function in adult animals. diabetes | gene therapy | ultrasound

  9. In Vitro and In Vivo Effective Gene Delivery with Novel Liposomal Bubbles

    NASA Astrophysics Data System (ADS)

    Nishiie, Norihito; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Taira, Yuichiro; Utoguchi, Naoki; Negishi, Yoichi; Maruyama, Kazuo

    2010-03-01

    Microbubbles, which were ultrasound contrast agents, could improve the transfection efficiency by cavitation with ultrasound exposure. However, conventional microbubbles had some problems regarding size and targeting ability. To solve these problems, we paid attention to liposomes that had many advantages as drug, antigen and gene delivery carriers. Because they can easily be controlled their size and added a targeting function. And we succeeded to prepare novel liposomal bubbles (Bubble liposomes) entrapping perfluoropropane which was utilized for contrast enhancement in ultrasonography. In this study, we assessed the feasibility of Bubble liposomes as gene delivery tools utilized cavitation by ultrasound exposure. In vitro gene delivery, Bubble liposomes could deliver plasmid DNA to many cell types such as tumor cells, T cell line and endothelial cells without cytotoxicity. In vivo gene delivery, Bubble liposomes could effectively deliver plasmid DNA into mouse femoral artery. This method was more effectively than conventional lipofection. We conclude that Bubble liposomes are unique and efficient gene delivery tools in vitro and in vivo.

  10. DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery.

    PubMed

    Pierrat, Philippe; Casset, Anne; Kereselidze, Dimitri; Lux, Marie; Pons, Françoise; Lebeau, Luc

    2016-07-01

    Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments. PMID:26990218

  11. Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

    PubMed

    Saito, Y; Swanson, X; Mhashilkar, A M; Oida, Y; Schrock, R; Branch, C D; Chada, S; Zumstein, L; Ramesh, R

    2003-11-01

    The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers. PMID:14528320

  12. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

    PubMed

    Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

    2016-03-01

    Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed. PMID:26725041

  13. 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

  14. 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. PMID:24008081

  15. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    PubMed Central

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  16. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

    PubMed

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  17. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2.

    PubMed

    Zhang, Zheng; Wang, Guoxian; Li, Chen; Liu, Danping

    2013-08-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

  18. Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo

    PubMed Central

    Tyler, MA; Ulasov, IV; Sonabend, AM; Nandi, S; Han, Y; Marler, S; Roth, J; Lesniak, MS

    2008-01-01

    Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone. PMID:19078993

  19. Engineering biodegradable and multifunctional peptide-based polymers for gene delivery

    PubMed Central

    2013-01-01

    The complex nature of in vivo gene transfer establishes the need for multifunctional delivery vectors capable of meeting these challenges. An additional consideration for clinical translation of synthetic delivery formulations is reproducibility and scale-up of materials. In this review, we summarize our work over the last five years in developing a modular approach for synthesizing peptide-based polymers. In these materials, bioactive peptides that address various barriers to gene delivery are copolymerized with a hydrophilic backbone of N-(2-hydroxypropyl)methacrylamide (HPMA) using reversible-addition fragmentation chain-transfer (RAFT) polymerization. We demonstrate that this synthetic approach results in well-defined, narrowly-disperse polymers with controllable composition and molecular weight. To date, we have investigated the effectiveness of various bioactive peptides for DNA condensation, endosomal escape, cell targeting, and degradability on gene transfer, as well as the impact of multivalency and polymer architecture on peptide bioactivity. PMID:24156736

  20. Gene Delivery from Supercharged Coiled-coil Protein and Cationic Lipid Hybrid Complex

    PubMed Central

    More, Haresh T.; Frezzo, Joseph A.; Dai, Jisen; Yamano, Seiichi; Montclare, Jin K.

    2014-01-01

    A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP•DNA(8:1)•FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat•FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP•DNA(8:1)•FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery. PMID:24875765

  1. First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

    PubMed

    Lima, Francisco Esmaile de Sales; Cibulski, Samuel Paulo; Elesbao, Felipe; Carnieli Junior, Pedro; Batista, Helena Beatriz de Carvalho Ruthner; Roehe, Paulo Michel; Franco, Ana Cláudia

    2013-10-01

    This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses. PMID:23828618

  2. Gene Delivery to the Retina: From Mouse to Man

    PubMed Central

    Bennett, Jean; Chung, Daniel C.; Maguire, Albert

    2013-01-01

    With the recent progress in identifying disease-causing genes in humans and in animal models, there are more and more opportunities for using retinal gene transfer to learn more about retinal physiology and also to develop therapies for blinding disorders. Success in preclinical studies for one form of inherited blindness have led to testing in human clinical trials. This paves the way to consider a number of other retinal diseases as ultimate gene therapy targets in human studies. The information presented here is designed to assist scientists and clinicians to use gene transfer to probe the biology of the retina and/or to move appropriate gene-based treatment studies from the bench to the clinic. PMID:22365778

  3. 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

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

    PubMed

    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

  5. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology

    PubMed Central

    Chen, Shuyuan; Ding, Jia-huan; Bekeredjian, Raffi; Yang, Bing-zhi; Shohet, Ralph V.; Johnston, Stephen A.; Hohmeier, Hans E.; Newgard, Christopher B.; Grayburn, Paul A.

    2006-01-01

    This study describes a method of gene delivery to pancreatic islets of adult, living animals by ultrasound targeted microbubble destruction (UTMD). The technique involves incorporation of plasmids into the phospholipid shell of gas-filled microbubbles, which are then infused into rats and destroyed within the pancreatic microcirculation with ultrasound. Specific delivery of genes to islet beta cells by UTMD was achieved by using a plasmid containing a rat insulin 1 promoter (RIP), and reporter gene expression was regulated appropriately by glucose in animals that received a RIP–luciferase plasmid. To demonstrate biological efficacy, we used UTMD to deliver RIP–human insulin and RIP–hexokinase I plasmids to islets of adult rats. Delivery of the former plasmid resulted in clear increases in circulating human C-peptide and decreased blood glucose levels, whereas delivery of the latter plasmid resulted in a clear increase in hexokinase I protein expression in islets, increased insulin levels in blood, and decreased circulating glucose levels. We conclude that UTMD allows relatively noninvasive delivery of genes to pancreatic islets with an efficiency sufficient to modulate beta cell function in adult animals. PMID:16709667

  6. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  7. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  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. Sui generis: gene therapy and delivery systems for the treatment of glioblastoma

    PubMed Central

    Kane, J. Robert; Miska, Jason; Young, Jacob S.; Kanojia, Deepak; Kim, Julius W.; Lesniak, Maciej S.

    2015-01-01

    Gene therapy offers a multidimensional set of approaches intended to treat and cure glioblastoma (GBM), in combination with the existing standard-of-care treatment (surgery and chemoradiotherapy), by capitalizing on the ability to deliver genes directly to the site of neoplasia to yield antitumoral effects. Four types of gene therapy are currently being investigated for their potential use in treating GBM: (i) suicide gene therapy, which induces the localized generation of cytotoxic compounds; (ii) immunomodulatory gene therapy, which induces or augments an enhanced antitumoral immune response; (iii) tumor-suppressor gene therapy, which induces apoptosis in cancer cells; and (iv) oncolytic virotherapy, which causes the lysis of tumor cells. The delivery of genes to the tumor site is made possible by means of viral and nonviral vectors for direct delivery of therapeutic gene(s), tumor-tropic cell carriers expressing therapeutic gene(s), and “intelligent” carriers designed to increase delivery, specificity, and tumoral toxicity against GBM. These vehicles are used to carry genetic material to the site of pathology, with the expectation that they can provide specific tropism to the desired site while limiting interaction with noncancerous tissue. Encouraging preclinical results using gene therapies for GBM have led to a series of human clinical trials. Although there is limited evidence of a therapeutic benefit to date, a number of clinical trials have convincingly established that different types of gene therapies delivered by various methods appear to be safe. Due to the flexibility of specialized carriers and genetic material, the technology for generating new and more effective therapies already exists. PMID:25746089

  10. Ionic α-Helical Polypeptides towards Non-Viral Gene Delivery

    PubMed Central

    Zhang, Rujing; Song, Ziyuan; Yin, Lichen; Zheng, Nan; Tang, Haoyu; Lu, Hua; Gabrielson, Nathan P.; Lin, Yao; Kim, Kyung; Cheng, Jianjun

    2014-01-01

    The advent of polymeric materials has significantly promoted the development and rapid growth of various technologies in biomedical applications, such as tissue engineering and controlled drug and gene delivery. Water-soluble polypeptides bearing functional side chains and adopting stable secondary structures are a new class of functional polymeric materials of potentially broad applications in medicine and biotechnology. In this article, we summarize our recent effort on the design and synthesis of the water-soluble α-helical ionic polypeptides originally developed in our laboratory and highlight their applications in cell membrane penetration and non-viral gene/siRNA delivery. PMID:25377262

  11. Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.

    PubMed

    Pfeifer, Corinna; Hasenpusch, Guenther; Uezguen, Senta; Aneja, Manish Kumar; Reinhardt, Dietrich; Kirch, Julian; Schneider, Marc; Claus, Sarah; Friess, Wolfgang; Rudolph, Carsten

    2011-08-25

    Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery. PMID:21600251

  12. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene (AdTW68.H5ck). Commercial layer-type chicken groups were singly vaccinated ocularly at 5 days of age, or singly v...

  13. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding a H7 hemagglutinin gene from a low pathogenic North American isolate (AdChNY94.H7). Chickens vaccinate...

  14. 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. PMID:24858708

  15. Gastrointestinal gene delivery by cyclodextrins--in vitro quantification of extracellular barriers.

    PubMed

    O'Neill, Martin J; O'Mahony, Aoife M; Byrne, Colin; Darcy, Raphael; O'Driscoll, Caitriona M

    2013-11-18

    Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers. Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2). Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase. In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers. PMID:24016741

  16. Efficient In Vitro Gene Delivery by Hybrid Biopolymer/Virus Nanobiovectors

    PubMed Central

    Keswani, Rahul; Su, Kai; Pack, Daniel W.

    2014-01-01

    Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific delivery via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLP) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6–9 µg chitosan/109 M-VLP at 10 × 109 M-VLP/ml to 40 µg chitosan /109 M-VLP at 2.5 × 109 M-VLP/ml, χ/M-VLP were ~300–350 nm diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms. PMID:25009978

  17. Local delivery of gene-modifying triplex-forming molecules to epidermis

    PubMed Central

    Rogers, Faye A.; Hu, Rong-Hua; Milstone, Leonard M.

    2012-01-01

    Epidermal keratinocytes are particularly suitable candidates for in situ gene correction. Intraperitoneal administration of a triplex-forming oligonucleotide (TFO) was shown previously to introduce DNA base changes in a reporter gene in skin, without identifying which cells had been targeted. We extend those previous experiments using two triplex-forming molecules (TFMs), a peptide nucleic acid (PNA-Antp) and a TFO (AG30), and two lines of transgenic mice that have the chromosomally integrated λsupFG1 shuttle-reporter transgene. Successful in vivo genomic modification occurs in epidermis and dermis in CD1 transgenic mice following either intraperitoneal or intradermal delivery of the PNA-Antennapedia conjugate. FITC-PNA-Antp accumulates in nuclei of keratinocytes and, after intradermal delivery of the PNA-Antp, chromosomally modified, keratin 5 positive basal keratinocytes persist for at least 10 days. In hairless (SKH1) mice with the λsupFG1 transgene, intradermal delivery of the TFO, AG30, introduces gene modifications in both tail and back skin and those chromosomal modifications persist in basal keratinocytes for 10 days. Hairless mice should facilitate comparison of various targeting agents and methods of delivery. Gene targeting by repeated local administration of oligonucleotides may prove clinically useful for judiciously selected disease-causing genes in the epidermis. PMID:23014335

  18. Prodrug converting enzyme gene delivery by L. monocytogenes

    PubMed Central

    Stritzker, Jochen; Pilgrim, Sabine; Szalay, Aladar A; Goebel, Werner

    2008-01-01

    Background Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Methods Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. Results The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. Conclusion These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors. PMID:18402662

  19. Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery

    PubMed Central

    Shen, ZP; Brayman, AA; Chen, L; Miao, CH

    2013-01-01

    Current ultrasound (US)-mediated gene delivery methods are inefficient due, in part, to a lack of US optimization. We systematically explored the use of microbubbles (MBs), US parameters and plasmid delivery routes to improve gene transfer into the mouse liver. Co-presentation of plasmid DNA (pDNA), 10% Optison MBs and pulsed 1-MHz US at a peak negative pressure of 4.3 MPa significantly increased luciferase gene expression with pDNA delivered by intrahepatic injection to the left liver lobe. Intraportal injection delivered pDNA and MBs to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. Gene expression was also dependent on acoustic pressure over the range of 0–4.3 MPa, with a peak effect at 3 MPa. An average of 85-fold enhancement in gene delivery was achieved. No enhancement was observed below 0.25 MPa. Increasing pulse length while decreasing pulse repetition frequency and exposure time to maintain a constant total energy during exposure did not further improve transfection efficiency, nor did extend the US exposure pre- or postinjection of pDNA. The results indicate that coupled with MBs, US can more efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation. PMID:18385766

  20. PEG shielded MMP sensitive CPPs for efficient and tumor specific gene delivery in vivo.

    PubMed

    Veiman, Kadi-Liis; Künnapuu, Kadri; Lehto, Tõnis; Kiisholts, Kristina; Pärn, Kalle; Langel, Ülo; Kurrikoff, Kaido

    2015-07-10

    Gene therapy has great potential to treat a range of different diseases, such as cancer. For that therapeutic gene can be inserted into a plasmid vector and delivered specifically to tumor cells. The most frequently used applications utilize lipoplex and polyplex approaches where DNA is non-covalently condensed into nanoparticles. However, lack of in vivo efficacy is the major concern that hinders translation of such gene therapeutic applications into clinics. In this work we introduce a novel method for in vivo delivery of plasmid DNA (pDNA) and efficient tumor-specific gene induction using intravenous (i.v) administration route. To achieve this, we utilize a cell penetrating peptide (CPP), PepFect14 (PF14), double functionalized with polyethylene glycol (PEG) and a matrix metalloprotease (MMP) substrate. We show that this delivery vector effectively forms nanoparticles, where the condensed CPP and pDNA are shielded by the PEG, in an MMP-reversible manner. Administration of the complexes results in efficient induction of gene expression specifically in tumors, avoiding normal tissues. This strategy is a potent gene delivery platform that can be used for tumor-specific induction of a therapeutic gene. PMID:25935707

  1. Biodegradable Poly(aminoester)-Mediated p53 Gene Delivery for Cancer Therapy.

    PubMed

    Shen, He; Liu, Min; Zhang, Zhijun

    2016-03-01

    Gene therapy is a promising strategy in cancer treatment. However, efficient gene translation still remains challenging. In the previous work, a hydrolytically degradable poly(aminoester) with good biocompatibility was synthesized. Herein, the poly(aminoester) was explored as a vector for gene delivery and cancer therapy. The experiments revealed that the poly(aminoester) condensed plasmid DNA into nanosized particles via electrostatic interaction. The pEGFP-N1 and pGL-3 were first used as two reporter genes to study intracellular transfection. The poly(aminoester) showed higher GFP expression (33%) than PEI 25 kDa (21%). Intracellular trafficking of Cy3-labelled pGL-3 also indicated that the poly(aminoester) showed superior DNA delivery ability to nucleus compared to PEI 25 kDa. Furthermore, the therapeutic gene (p53) was translated into the breast cancer cell line (MCF-7), and then induced cell apoptosis. These results suggested that the degradable poly(aminoester) is a promising and efficient gene delivery vector for gene therapeutic applications. PMID:27455620

  2. Ethanol enhanced in vivo gene delivery with non-ionic polymeric micelles inhalation.

    PubMed

    Chao, Yen-Chin; Chang, Shwu-Fen; Lu, Shao-Chun; Hwang, Tzyh-Chang; Hsieh, Wei-Hsien; Liaw, Jiahorng

    2007-03-12

    Modifications of both carriers and host barriers have been investigated for efficient inhalation gene delivery to lung. Here we used a biocompatible, non-ionic poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) polymeric micelles (PM) as a carrier and combined it with ethanol to enhance membrane penetration of delivered DNA. The inhalation delivery with six 100 microg doses of pCMV-Lac Z with PM co-formulated with 10%-40% ethanol to nude mice in 2 days at 8 h interval was performed. The beta-galatosidase (beta-Gal) activity was assessed using chlorophenol red-beta-d galactopyranoside (CPRG) and X-gal staining for quantitative and qualitative analysis in tissues. The results showed that beta-Gal activity was significantly increased by 38% in lung around bronchioles when inhalation with PM and 10% ethanol was given. The 10% ethanol also increased the intracellular apparent permeability by 42% in stomach and by 141% in intestine at 48 h after the first dosage of delivery. Also delivery of DNA encoding a functional human cystic fibrosis transmembrane protein (CFTR) using the same inhalation delivery method co-formulated with 10% ethanol, an increased expression of CFTR in lung was detected by immunostaining. We concluded that 10% ethanol co-formulated with the PM system could enhance inhaled gene delivery to airway and gastrointestinal (GI) tract. PMID:17258837

  3. Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems.

    PubMed

    Rezaee, Mehdi; Oskuee, Reza Kazemi; Nassirli, Hooriyeh; Malaekeh-Nikouei, Bizhan

    2016-08-28

    Efficient gene therapy is mainly dependent on the gene transfer capability of gene delivery vectors. Non-viral vectors have become the research interest of many researchers because these vectors are safer than viral vectors. Acquiring the advantages of both polyplexes and lipoplexes, the lipopolyplex (LPP) is a ternary nanocomplex composed of cationic liposome, polycation, and nucleic acid. Considering the polycationic component, ternary complexes (LPPs) are divided into cationic polymer-based LPPs and cationic peptide-based LPPs. Considering the capability of rational design, LPP is an interesting field of research to design a more potent nucleic acid carrier. With the promising transfection activity and safety observed in the LPPs, many researchers have formulated various types of lipids and polycations to achieve an efficient and safe carrier for gene therapy. Here we provide a review on the designed LPPs for efficient delivery of different nucleic acids such as plasmid DNA, siRNA, shRNA, and DNA vaccines. PMID:27317365

  4. Lipopolyplex for Therapeutic Gene Delivery and Its Application for the Treatment of Parkinson’s Disease

    PubMed Central

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

    2016-01-01

    Lipopolyplex is a core-shell structure composed of nucleic acid, polycation and lipid. As a non-viral gene delivery vector, lipopolyplex combining the advantages of polyplex and lipoplex has shown superior colloidal stability, reduced cytotoxicity, extremely high gene transfection efficiency. Following intravenous administration, there are many strategies based on lipopolyplex to overcome the complex biological barriers in systemic gene delivery including condensation of nucleic acids into nanoparticles, long circulation, cell targeting, endosomal escape, release to cytoplasm and entry into cell nucleus. Parkinson’s disease (PD) is the second most common neurodegenerative disorder and severely influences the patients’ life quality. Current gene therapy clinical trials for PD employing viral vectors didn’t achieve satisfactory efficacy. However, lipopolyplex may become a promising alternative approach owing to its stability in blood, ability to cross the blood-brain barrier (BBB) and specific targeting to diseased brain cells. PMID:27092073

  5. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation

    PubMed Central

    Rincon, Melvin Y.; VandenDriessche, Thierry; Chuah, Marinee K.

    2015-01-01

    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca2+-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality. PMID:26239654

  6. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

    PubMed

    Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K

    2015-10-01

    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality. PMID:26239654

  7. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

    Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

  8. Ferritin-mediated siRNA delivery and gene silencing in human tumor and primary cells.

    PubMed

    Li, Le; Muñoz-Culla, Maider; Carmona, Unai; Lopez, Maria Paz; Yang, Fan; Trigueros, Cesar; Otaegui, David; Zhang, Lianbing; Knez, Mato

    2016-08-01

    We demonstrate a straightforward method to encapsulate siRNA into naturally available and unmodified human apoferritin. The encapsulation into apoferritin is independent of the sequence of the siRNA and provides superior protection for those sensitive molecules. High efficiency in transfection can be achieved in human tumorigenic cells, human primary mesenchymal stem cells (hMSC) and peripheral blood mononuclear cells (PBMCs). In contrast to Lipofectamine, highly effective gene silencing can be achieved with ferritin as the delivery agent in both tumor cells and PBMCs at low siRNA concentrations (10 nM). As an endogenous delivery agent, apoferritin does not induce immune activation of T- and B-cells in human PBMCs. Apoferritin shows intrinsic anti-inflammatory effects and apoferritin-mediated delivery shows a preference for immune-activated T- and B-cells, a natural selectivity which may turn useful for drug delivery in case of infections or inflammatory diseases. PMID:27187278

  9. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers

    SciTech Connect

    Igor I. Slowing; Juan L. Viveo-Escoto; Chia-Wen Wu; Victor S. Y. Lin

    2008-04-10

    In this review, we highlight the recent research developments of a series of surface-functionalized mesoporous silica nanoparticle (MSN) materials as efficient drug delivery carriers. The synthesis of this type of MSN materials is described along with the current methods for controlling the structural properties and chemical functionalization for biotechnological and biomedical applications. We summarized the advantages of using MSN for several drug delivery applications. The recent investigations of the biocompatibility of MSN in vitro are discussed. We also describe the exciting progress on using MSN to penetrate various cell membranes in animal and plant cells. The novel concept of gatekeeping is introduced and applied to the design of a variety of stimuli-responsive nanodevices. We envision that these MSN-based systems have a great potential for a variety of drug delivery applications, such as the site-specific delivery and intracellular controlled release of drugs, genes, and other therapeutic agents.

  10. Mechanical oscillations enhance gene delivery into suspended cells

    PubMed Central

    Zhou, Z. L.; Sun, X. X.; Ma, J.; Man, C. H.; Wong, A. S. T.; Leung, A. Y.; Ngan, A. H. W.

    2016-01-01

    Suspended cells are difficult to be transfected by common biochemical methods which require cell attachment to a substrate. Mechanical oscillations of suspended cells at certain frequencies are found to result in significant increase in membrane permeability and potency for delivery of nano-particles and genetic materials into the cells. Nanomaterials including siRNAs are found to penetrate into suspended cells after subjecting to short-time mechanical oscillations, which would otherwise not affect the viability of the cells. Theoretical analysis indicates significant deformation of the actin-filament network in the cytoskeleton cortex during mechanical oscillations at the experimental frequency, which is likely to rupture the soft phospholipid bilayer leading to increased membrane permeability. The results here indicate a new method for enhancing cell transfection. PMID:26956215

  11. SV40 in vitro packaging: a pseudovirion gene delivery system.

    PubMed

    Kimchi-Sarfaty, Chava; Gottesman, Michael M

    2012-09-01

    Nuclear extracts of Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encode the simian virus 40 (SV40) major coat protein (VP1). As described in this protocol, these extracts are able to package supercoiled plasmid DNA or RNA interference (RNAi) sequences in the presence of MgCl(2), CaCl(2), and ATP, thus forming SV40 pseudovirions in vitro. Such packaging has numerous advantages over other viral and nonviral delivery systems. Specifically, it provides a wide host range and high transduction efficiency. The only major disadvantage of this system is low expression per transduced cell observed in vitro, likely a result of DNA trapped in the cytoplasmic compartment of the cell that does not enter the cell nucleus. PMID:22949719

  12. Lignin nanotubes as vehicles for gene delivery into human cells.

    PubMed

    Ten, Elena; Ling, Chen; Wang, Yuan; Srivastava, Arun; Dempere, Luisa Amelia; Vermerris, Wilfred

    2014-01-13

    Lignin nanotubes (LNTs) synthesized from the aromatic plant cell wall polymer lignin in a sacrificial alumina membrane template have as useful features their flexibility, ease of functionalization due to the availability of many functional groups, label-free detection by autofluorescence, and customizable optical properties. In this report we show that the physicochemical properties of LNTs can be varied over a wide range to match requirements for specific applications by using lignin with different subunit composition, a function of plant species and genotype, and by choosing the lignin isolation method (thioglycolic acid, phosphoric acid, sulfuric acid (Klason), sodium hydroxide lignin), which influences the size and reactivity of the lignin fragments. Cytotoxicity studies with human HeLa cells showed that concentrations of up to 90 mg/mL are tolerated, which is a 10-fold higher concentration than observed for single- or multiwalled carbon nanotubes (CNTs). Confocal microscopy imaging revealed that all LNT formulations enter HeLa cells without auxiliary agents and that LNTs made from NaOH-lignin penetrate the cell nucleus. We further show that DNA can adsorb to LNTs. Consequently, exposure of HeLa cells to LNTs coated with DNA encoding the green fluorescent protein (GFP) leads to transfection and expression of GFP. The highest transfection efficiency was obtained with LNTs made from NaOH-lignin due to a combination of high DNA binding capacity and DNA delivery directly into the nucleus. These combined features of LNTs make LNTs attractive as smart delivery vehicles of DNA without the cytotoxicity associated with CNTs or the immunogenicity of viral vectors. PMID:24308459

  13. Ultrasound-mediated oncolytic virus delivery and uptake for increased therapeutic efficacy: state of art

    PubMed Central

    Nande, Rounak; Howard, Candace M; Claudio, Pier Paolo

    2015-01-01

    The field of ultrasound (US) has changed significantly from medical imaging and diagnosis to treatment strategies. US contrast agents or microbubbles (MB) are currently being used as potential carriers for chemodrugs, small molecules, nucleic acids, small interfering ribonucleic acid, proteins, adenoviruses, and oncolytic viruses. Oncolytic viruses can selectively replicate within and destroy a cancer cell, thus making them a powerful therapeutic in treating late-stage or metastatic cancer. These viruses have been shown to have robust activity in clinical trials when injected directly into tumor nodules. However limitations in oncolytic virus’ effectiveness and its delivery approach have warranted exploration of ultrasound-mediated delivery. Gene therapy bearing adenoviruses or oncolytic viruses can be coupled with MBs and injected intravenously. Following application of US energy to the target region, the MBs cavitate, and the resulting shock wave enhances drug, gene, or adenovirus uptake. Though the underlying mechanism is yet to be fully understood, there is evidence to suggest that mechanical pore formation of cellular membranes allows for the temporary uptake of drugs. This delivery method circumvents the limitations due to stimulation of the immune system that prevented intravenous administration of viruses. This review provides insight into this intriguing new frontier on the delivery of oncolytic viruses to tumor sites. PMID:27512682

  14. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses.

    PubMed

    Wellehan, James F X; Johnson, April J; Harrach, Balázs; Benkö, Mária; Pessier, Allan P; Johnson, Calvin M; Garner, Michael M; Childress, April; Jacobson, Elliott R

    2004-12-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses. PMID:15542689

  15. Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

    PubMed Central

    Wellehan, James F. X.; Johnson, April J.; Harrach, Balázs; Benkö, Mária; Pessier, Allan P.; Johnson, Calvin M.; Garner, Michael M.; Childress, April; Jacobson, Elliott R.

    2004-01-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses. PMID:15542689

  16. 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

  17. Fabrication of PLGA polymer microspheres for U. S. mediated gene delivery

    NASA Astrophysics Data System (ADS)

    Williamson, Rene G.; Saltzman, William M.; Brandsma, Janet L.

    2001-05-01

    The promises of gene therapy remain unfulfilled because of the lack of a safe and efficient method for transfecting DNA into cells. PLGA has been used as a vehicle for protein, drug, and gene delivery applications because of its biocompatibility and sustained release properties. PLGA polymer microspheres offer advantages of safety and the possibility of sustained intracytoplasmic delivery. The PLGA also protects the plasmid from degradation. Using the double-emulsion microsphere fabrication technique, a new DNA delivery vehicle, comprising of plasmid DNA and octafluoropropane gas encapsulated in PLGA polymer and PVA stabilizer (Sonospheres) was made. The encapsulated gas offers acoustic activity to the microspheres, which enables them to undergo cavitation in an acoustic field. The goal is to lead to increased DNA transfection when these Sonospheres are subjected to an acoustic field in the MHz frequency range. A summary of the fabrication methods and some initial in vitro studies will be presented.

  18. Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

    PubMed

    Zhang, Nian-Hua; Peng, Rui-Qing; Ding, Ya; Zhang, Xiao-Shi

    2016-08-01

    The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity. PMID:27373420

  19. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy. PMID:25674795

  20. A simplified system for generating recombinant E3-deleted canine adenovirus-2.

    PubMed

    Yu, Zuo; Jiang, Qian; Liu, Jiasen; Guo, Dongchun; Quan, Chuansong; Li, Botao; Qu, Liandong

    2015-01-01

    Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector. PMID:25450764

  1. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver

    PubMed Central

    Song, S; Shen, Z; Chen, L; Brayman, AA; Miao, CH

    2015-01-01

    Ultrasound (US) combined with microbubbles (MBs) is a promising technology for non-viral gene delivery. Significant enhancements of gene expression have been obtained in our previous studies. To optimize and prepare for application to larger animal models, the luciferase reporter gene transfer efficacy of lipid-based Definity MBs of various concentrations, pressure amplitudes and a novel unfocused high-intensity therapeutic US (HITU) system were explored. Luciferase expression exhibited a dependence on MB dose over the range of 0–25 vol%, and a strong dependence on acoustic peak negative pressure at over the range of 0–3.2 MPa. Gene expression reached an apparent plateau at MB concentration ≥2.5 vol% or at negative pressures >1.8 MPa. Maximum gene expression in treated animals was 700-fold greater than in negative controls. Pulse train US exposure protocols produced an upward trend of gene expression with increasing quiescent time. The hyperbolic correlation of gene expression and transaminase levels suggested that an optimum gene delivery effect can be achieved by maximizing acoustic cavitation-induced enhancement of DNA uptake and minimizing unproductive tissue damage. This study validated the new HITU system equipped with an unfocused transducer with a larger footprint capable of scanning large tissue areas to effectively enhance gene transfer efficiencies. PMID:21451579

  2. A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells.

    PubMed

    Veiseh, Omid; Kievit, Forrest M; Gunn, Jonathan W; Ratner, Buddy D; Zhang, Miqin

    2009-02-01

    As conventional cancer therapies struggle with toxicity issues and irregular remedial efficacy, the preparation of novel gene therapy vectors could offer clinicians the tools for addressing the genetic errors of diseased tissue. The transfer of gene therapy to the clinic has proven difficult due to safety, target specificity, and transfection efficiency concerns. Polyethylenimine (PEI) nanoparticles have been identified as promising gene carriers that induce gene transfection with high efficiency. However, the inherent toxicity of the material and non-selective delivery are the major concerns in applying these particles clinically. Here, a non-viral nanovector has been developed by PEGylation of DNA-complexing PEI in nanoparticles functionalized with an Alexa Fluor 647 near infrared fluorophore, and the chlorotoxin (CTX) peptide which binds specifically to many forms of cancer. With this nanovector, the potential toxicity to healthy cells is minimized by both the reduction of the toxicity of PEI with the biocompatible copolymer and the targeted delivery of the nanovector to cancer cells, as evaluated by viability studies. The nanovector demonstrated high levels of targeting specificity and gene transfection efficiency with both C6 glioma and DAOY medulloblastoma tumor cells. Significantly, with the CTX as the targeting ligand, the nanovector may serve as a widely applicable gene delivery system for a broad array of cancer types. PMID:18990439

  3. Cationic Surface Modification of PLG Nanoparticles Offers Sustained Gene Delivery to Pulmonary Epithelial Cells

    PubMed Central

    BAOUM, ABDULGADER; DHILLON, NAVNEET; BUCH, SHILPA; BERKLAND, CORY

    2010-01-01

    Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by 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 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression 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. PMID:19911425

  4. Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double-stranded RNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. To determine if such an a...

  5. Dual-responsive aggregation-induced emission-active supramolecular nanoparticles for gene delivery and bioimaging.

    PubMed

    Dong, Ruijiao; Ravinathan, Screenath P; Xue, Lizhe; Li, Nan; Zhang, Yingjian; Zhou, Linzhu; Cao, Chengxi; Zhu, Xinyuan

    2016-06-28

    Dual-responsive aggregation-induced emission-active supramolecular fluorescent nanoparticles are reported, which have the ability to undergo a unique morphological transition combining with a cooperative optical variation in response to pH and light stimuli. The dynamic supramolecular nanoparticles show excellent biocompatibility and effective plasmid DNA condensation capability, further achieving efficient in vitro gene delivery and bioimaging. PMID:27251637

  6. 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

  7. Non-Condensing Polymeric Nanoparticles for Targeted Gene and siRNA Delivery

    PubMed Central

    Xu, Jing; Ganesh, Shanthi; Amiji, Mansoor

    2011-01-01

    Gene therapy has shown a tremendous potential to benefit patients in a variety of disease conditions. However, finding a safe and effective systemic delivery system is the major obstacle in this area. Although viral vectors showed promise for high transfection rate, the immunogenicity associated with these systems has hindered further development. As an alternative to viral gene delivery, this review focuses on application of novel safe and effective non-condensing polymeric systems that have shown high transgene expression when administered systemically or by the oral route. Type B gelatin-based engineered nanocarriers were evaluated for passive and active tumor-targeted delivery and transfection using both reporter and therapeutic plasmid DNA. Additionally, we have shown that nanoparticles-in-microsphere oral system (NiMOS) can efficiently deliver reporter and therapeutic gene constructs in the gastrointestinal tract. Additionally, there has been a significant recent interest in the use small interfering RNA (siRNA) as a therapeutic system for gene silencing. Both gelatin nanoparticles and NiMOS have shown activity in systemic and oral delivery of siRNA, respectively. PMID:21621597

  8. Development of Therapeutic Microbubbles for Enhancing Ultrasound-Mediated Gene Delivery

    PubMed Central

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

    2014-01-01

    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. PMID:24650644

  9. Statistical modelling of the formulation variables in non-viral gene delivery systems.

    PubMed

    Birchall, J C; Waterworth, C A; Luscombe, C; Parkins, D A; Gumbleton, M

    2001-06-01

    Traditionally, optimisation of a gene delivery formulation utilises a study design that involves altering only one formulation variable at any one time whilst keeping the other variables constant. As gene delivery formulations become more complex, e.g. to include multiple cellular and sub-cellular targeting elements, there will be an increasing requirement to generate and analyse data more efficiently and allow examination of the interaction between variables. This study aims to demonstrate the utility of multifactorial design, specifically a Central Composite Design, in modelling the responses size, zeta potential and in vitro transfection efficiency of some prototypic non-viral gene delivery vectors. i.e. cationic liposome-pDNA complexes, and extending the application of the design strategy to more complex vectors, i.e. tri-component lipid:polycation:DNA (LPD). The modelled predictions of how the above responses change as a function of formulation show consistency with an extensive literature base of data obtained using more traditional approaches, and highlight the robustness and utility of the Central Composite Design in examining key formulation variables in non-viral gene delivery systems. The approach should be further developed to maximise the predictive impact of data across the full range of pharmaceutical sciences. PMID:11697203

  10. Controlling mesenchymal stem cell gene expression using polymer-mediated delivery of siRNA

    PubMed Central

    Benoit, Danielle S.W.; Boutin, Molly E.

    2012-01-01

    siRNA treatment has great promise to specifically control gene expression and select cell behaviors but have delivery challenges limiting their use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effective delivery siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA, which is vastly superior to a commercially-available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), is cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT:siRNA treatment results in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There is no statistically significant differences on cell surface markers of diblock copolymer:siRNA or DharmaFECT:siRNA treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there is no discernable differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitate uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC

  11. Synthesis of Electroneutralized Amphiphilic Copolymers with Peptide Dendrons for Intramuscular Gene Delivery.

    PubMed

    Pu, Linyu; Wang, Jiali; Li, Na; Chai, Qiuxia; Irache, Juan M; Wang, Gang; Tang, James Zhenggui; Gu, Zhongwei

    2016-06-01

    Intramuscular gene delivery materials are of great importance in plasmid-based gene therapy system, but there is limited information so far on how to design and synthesize them. A previous study showed that the peptide dendron-based triblock copolymer with its components arranged in a reversed biomembrane architecture could significantly increase intramuscular gene delivery and expression. Herein, we wonder whether copolymers with biomembrane-mimicking arrangement may have similar function on intramuscular gene delivery. Meanwhile, it is of great significance to uncover the influence of electric charge and molecular structure on the function of the copolymers. To address the issues, amphiphilic triblock copolymers arranged in hydrophilic-hydrophobic-hydrophilic structure were constructed despite the paradoxical characteristics and difficulties in synthesizing such hydrophilic but electroneutral molecules. The as-prepared two copolymers, dendronG2(l-lysine-OH)-poly propylene glycol2k(PPG2k)-dendronG2(l-lysine-OH) (rL2PL2) and dendronG3(l-lysine-OH)-PPG2k-dendronG3(l-lysine-OH) (rL3PL3), were in similar structure but had different hydrophilic components and surface charges, thus leading to different capabilities in gene delivery and expression in skeletal muscle. rL2PL2 was more efficient than Pluronic L64 and rL3PL3 when mediating luciferase, β-galactosidase, and fluorescent protein expressions. Furthermore, rL2PL2-mediated growth-hormone-releasing hormone expression could significantly induce mouse body weight increase in the first 21 days after injection. In addition, both rL2PL2 and rL3PL3 showed good in vivo biosafety in local and systemic administration. Altogether, rL2PL2-mediated gene expression in skeletal muscle exhibited applicable potential for gene therapy. The study revealed that the molecular structure and electric charge were critical factors governing the function of the copolymers for intramuscular gene delivery. It can be concluded that, combined

  12. Multifunctional disulfide-based cationic dextran conjugates for intravenous gene delivery targeting ovarian cancer cells.

    PubMed

    Song, Yanyan; Lou, Bo; Zhao, Peng; Lin, Chao

    2014-07-01

    A folate-decorated, disulfide-based cationic dextran conjugate having dextran as the main chain and disulfide-linked 1,4-bis(3-aminopropyl)piperazine (BAP) residues as the grafts was designed and successfully prepared as a multifunctional gene delivery vector for targeted gene delivery to ovarian cancer SKOV-3 cells in vitro and in vivo. Initially, a new bioreducible cationic polyamide (denoted as pSSBAP) was prepared by polycondensation reaction of bis(p-nitrophenyl)-3,3'-dithiodipropanoate, a disulfide-containing monomer, and BAP. It was found that the pSSBAP was highly efficient for in vitro gene delivery against MCF-7 and SKOV-3 cell lines. Subsequently, two cationic dextran conjugates with different amounts of BAP residues (denoted as Dex-SSBAP6 and Dex-SSBAP30, respectively) were synthesized by coupling BAP to disulfide-linked carboxylated dextran or coupling pSSBAP-oligomer to p-nitrophenyl carbonated dextran. Both two conjugates were able to bind DNA to form nanosized polyplexes with an improved colloidal stability in physiological conditions. The polyplexes, however, were rapidly dissociated to liberate DNA in a reducing environment. In vitro transfection experiments revealed that the polyplexes of Dex-SSBAP30 efficiently transfected SKOV-3 cells, yielding transfection efficiency that is comparable to that of linear polyethylenimine or lipofectamine 2000. AlamarBlue assay showed that the conjugates had low cytotoxicity in vitro at a high concentration of 100 mg/L. Further, Dex-SSBAP30 has primary amine side groups and thus allows for folate (FA) conjugation, yielding FA-coupled Dex-SSBAP30 (Dex-SSBAP30-FA). It was found that Dex-SSBAP30-FA was efficient for targeted gene delivery to SKOV-3 tumor xenografted in a nude mouse model by intravenous injection, inducing a higher level of gene expression in the tumor as compared to Dex-SSBAP30 lacking FA and comparable gene expression to linear polyethylenimine as one of the most efficient polymeric vectors for

  13. Phylogenetic Analyses of Novel Squamate Adenovirus Sequences in Wild-Caught Anolis Lizards

    PubMed Central

    Ascher, Jill M.; Geneva, Anthony J.; Ng, Julienne; Wyatt, Jeffrey D.; Glor, Richard E.

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity. PMID:23593364

  14. Viral vectors and delivery strategies for CNS gene therapy

    PubMed Central

    Gray, Steven J; Woodard, Kenton T; Samulski, R Jude

    2015-01-01

    This review aims to provide a broad overview of the targets, challenges and potential for gene therapy in the CNS, citing specific examples. There are a broad range of therapeutic targets, with very different requirements for a suitable viral vector. By utilizing different vector tropisms, novel routes of administration and engineered promoter control, transgenes can be targeted to specific therapeutic applications. Viral vectors have proven efficacious in preclinical models for several disease applications, spurring several clinical trials. While the field has pushed the limits of existing adeno-associated virus-based vectors, a next generation of vectors based on rational engineering of viral capsids should expand the application of gene therapy to be more effective in specific therapeutic applications. PMID:22833965

  15. Cardiovascular gene delivery: The good road is awaiting☆

    PubMed Central

    Brewster, L.P.; Brey, E.M.; Greisler, H.P.

    2012-01-01

    Atherosclerotic cardiovascular disease is a leading cause of death worldwide. Despite recent improvements in medical, operative, and endovascular treatments, the number of interventions performed annually continues to increase. Unfortunately, the durability of these interventions is limited acutely by thrombotic complications and later by myointimal hyperplasia followed by progression of atherosclerotic disease over time. Despite improving medical management of patients with atherosclerotic disease, these complications appear to be persisting. Cardiovascular gene therapy has the potential to make significant clinical inroads to limit these complications. This article will review the technical aspects of cardiovascular gene therapy; its application for promoting a functional endothelium, smooth muscle cell growth inhibition, therapeutic angiogenesis, tissue engineered vascular conduits, and discuss the current status of various applicable clinical trials. PMID:16769148

  16. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

    PubMed

    Tolmachov, Oleg E

    2014-08-01

    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  17. Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation.

    PubMed

    Zhao, Xiaoli; Li, Zhaoyang; Pan, Haobo; Liu, Wenguang; Lv, Minmin; Leung, Frankie; Lu, William W

    2013-05-01

    Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid-base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease. PMID:23395816

  18. 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. PMID:27021743

  19. Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

    PubMed Central

    Kamau, Sarah W.; Hassa, Paul O.; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hofmann-Amtenbrink, Margarethe; von Rechenberg, Brigitte; Hottiger, Michael O.

    2006-01-01

    New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs. PMID:16540591

  20. A Magnetic Nanoparticle-Based Multiple-Gene Delivery System for Transfection of Porcine Kidney Cells

    PubMed Central

    Wang, Yan; Cui, Haixin; Li, Kui; Sun, Changjiao; Du, Wei; Cui, Jinhui; Zhao, Xiang; Chen, Wenjie

    2014-01-01

    Superparamagnetic nanoparticles are promising candidates for gene delivery into mammalian somatic cells and may be useful for reproductive cloning using the somatic cell nuclear transfer technique. However, limited investigations of their potential applications in animal genetics and breeding, particularly multiple-gene delivery by magnetofection, have been performed. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using magnetic Fe3O4 nanoparticles as gene carriers. After surface modification by polyethylenimine, the spherical magnetic Fe3O4 nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNAGFP) or red (DNADsRed) fluorescent protein. At weight ratios of DNAGFP or DNADsRed to magnetic nanoparticles lower than or equal to 10∶1 or 5∶1, respectively, the DNA molecules were completely bound by the magnetic nanoparticles. Atomic force microscopy analyses confirmed binding of the spherical magneti