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Sample records for hoxd10 gene delivery

  1. 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.

  2. Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

    PubMed Central

    Zardoya, Rafael; Abouheif, Ehab; Meyer, Axel

    1996-01-01

    The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established. PMID:8917540

  3. Decreased HoxD10 expression promotes a proliferative and aggressive phenotype in prostate cancer.

    PubMed

    Mo, R-J; Lu, J-M; Wan, Y-P; Hua, W; Liang, Y-X; Zhuo, Y-J; Kuang, Q-W; Liu, Y-L; He, H-C; Zhong, W-D

    2017-02-19

    HoxD10 gene plays a critical role in cell proliferation in the process of tumor development. However, the protein expression level and the function of HoxD10 in prostate cancer remain unknown. Using tissue microarray, we demonstrate that the protein expression of HoxD10 is commonly decreased in prostate cancer tissues (n = 92) compared to adjacent benign prostate tissues (n = 77). Functionally, knockdown of HoxD10 resulted in significant promotion of prostate cancer cell proliferation. Moreover, knockdown of HoxD10 strikingly stimulated prostate tumor growth in a mouse xenograft model. We also found a significant association between decreased immunohistochemical staining of HoxD10 expression and higher Gleason score (P = 0.031) and advanced clinical pathological stage (P = 0.011). An analysis of the Taylor database revealed that decreased HoxD10 expression predicted worse biochemical recurrence (BCR)-free survival of PCa patients (P = 0.005) and the multivariate analyses further supported that HoxD10 might be an independent predictor for BCR-free survival (P = 0.027). Collectively, our data suggest that the loss of HoxD10 function is common and may thus result in a progressive phenotype in PCa. HoxD10 may function as a biomarker that differentiates patients with BCR disease from the ones that are not after radical prostatectomy, implicating its potential as a therapeutic target.

  4. Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord

    PubMed Central

    Misra, Mala; Shah, Veeral; Carpenter, Ellen; McCaffery, Peter; Lance-Jones, Cynthia

    2009-01-01

    During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement. PMID:19306865

  5. Genetic Variants Contributing to Colistin Cytotoxicity: Identification of TGIF1 and HOXD10 Using a Population Genomics Approach

    PubMed Central

    Eadon, Michael T.; Hause, Ronald J.; Stark, Amy L.; Cheng, Ying-Hua; Wheeler, Heather E.; Burgess, Kimberly S.; Benson, Eric A.; Cunningham, Patrick N.; Bacallao, Robert L.; Dagher, Pierre C.; Skaar, Todd C.; Dolan, M. Eileen

    2017-01-01

    Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity (p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 (p = 6.49 × 10−8), was nominally a cis-expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor β (TGFβ)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10−5). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations. PMID:28335481

  6. Genetic Variants Contributing to Colistin Cytotoxicity: Identification of TGIF1 and HOXD10 Using a Population Genomics Approach.

    PubMed

    Eadon, Michael T; Hause, Ronald J; Stark, Amy L; Cheng, Ying-Hua; Wheeler, Heather E; Burgess, Kimberly S; Benson, Eric A; Cunningham, Patrick N; Bacallao, Robert L; Dagher, Pierre C; Skaar, Todd C; Dolan, M Eileen

    2017-03-18

    Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity (p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 (p = 6.49 × 10(-8)), was nominally a cis-expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor β (TGFβ)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10(-5)). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations.

  7. Interaction between Tbx1 and Hoxd10 and connection with TGFβ-BMP signal pathway during kidney development.

    PubMed

    Fu, Yu; Li, Fei; Zhao, Diana Yue; Zhang, Jing-Shu; Lv, Yuan; Li-Ling, Jesse

    2014-02-15

    Renal malformations are commonly found among patients carrying a 22q11 deletion which renders loss of Tbx1 gene, an important transcriptional factor implicated in a number of developmental processes. Smad1 is known to interact with Tbx1, but the exact mechanism remains unknown. In this study, we have measured the expression of Tbx1 in both murine and human tissues using RT-PCR, and analyzed its protein product and protein-protein interactions with Western blotting and immunoprecipitation assays. Precipitated proteins were verified with mass spectrometry. As discovered, Tbx1 binds with Hoxd10. Tbx1 and Hoxd10 genes also have similar expression profiles during murine kidney development. Based on homology between mouse and human, we hypothesized that such interaction also exists in human. Through a RNA interference experiment using a human embryonic kidney HEK293 cell line, we demonstrated that TBX1 can alter TGF-β/BMP, an important signaling pathway, through interacting with HOXD10. Above findings may shed light on the mechanism of TBX1 mutations leading to renal malformations found in patients carrying a 22q11 deletion.

  8. MicroRNA-376b promotes breast cancer metastasis by targeting Hoxd10 directly

    PubMed Central

    An, Ning; Luo, Xinmei; Zhang, Ming; Yu, Ruilian

    2017-01-01

    Breast cancer is the most common malignant disease in women, and metastasis formed at distant anatomic sites was the major cause of cancer-related mortality. Thus, a novel therapy target and progression biomarker for breast cancer metastasis was necessary. microRNA (miR)-376b has been demonstrated to regulate angiogenesis; however, its role in cancer metastasis remains elusive. In the present study, the expression of miR-376b in normal breast tissue, JC and 4T1 cells was determined by qPCR. Furthermore, in vitro and in vivo experiments were performed to determine the effect of miR-376b on breast cancer metastasis. The direct target of miR-376b was determined by the luciferase assay and western blotting. The results indicated that silencing of miR-376b by the miR-376-mimic significantly inhibited 4T1 cell migration and invasion in vitro. Lung metastasis was also evidently decreased after silencing of miR-376b in 4T1 cells. Moreover, the luciferase assay and western blotting identified that Hoxd10 is the direct target of miR-376b during the regulation of breast cancer metastasis. To the best of our knowledge, the present study was the first to demonstrate the promoting breast cancer metastasis role of miR-376b by directly targeting Hoxd10. Therefore, it would be a novel therapy target and prognostic biomarker for breast cancer. PMID:28123472

  9. Nonviral gene delivery.

    PubMed

    Akita, Hidetaka; Harashima, Hideyoshi

    2008-01-01

    Gene and RNA interference therapies are promising cures for intractable renal failure. However, low delivery efficiency of the therapeutic nucleic acid into the nucleus of the target cell is a significant obstacle in the clinical application of nonviral gene therapy. Various mechanical techniques (hydrodynamic injection, electroporation and ultrasound-microbubble) and topically applied preparations (HVJ liposome and cationic liposome/polymer), which introduce transgenes into specific renal compartments depending on the administration route, have been reported. Additional improvements in renal application of nonviral gene vectors must address the important issue of how to control intracellular trafficking. Therefore, novel vectors based on the 'programmed packaging' concept are desirable in which all functional devices are integrated into a single system so that each function occurs at the appropriate time and correct place. In parallel with development of the carrier, quantitative evaluation of intracellular trafficking is essential to determine the efficacy of the modified devices in the cellular environment. In particular, comparison of the intracellular trafficking of the engineered devices with that of viruses (i.e. adenovirus) is useful in identifying the rate-limiting intracellular processes of the vectors during development.

  10. Electroporation-mediated gene delivery.

    PubMed

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models. Copyright © 2015 Elsevier Inc. All

  11. 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.

  12. 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

  13. Composite Nanoparticles for Gene Delivery

    PubMed Central

    Wang, Yuhua; Huang, Leaf

    2016-01-01

    Nanoparticle-mediated gene and siRNA delivery has been an appealing area to gene therapists when they attempt to treat the diseases by manipulating the genetic information in the target cells. However, the advances in materials science could not keep up with the demand for multifunctional nanomaterials to achieve desired delivery efficiency. Researchers have thus taken an alternative approach to incorporate various materials into single composite nanoparticle using different fabrication methods. This approach allows nanoparticles to possess defined nanostructures as well as multiple functionalities to overcome the critical extracellular and intracellular barriers to successful gene delivery. This chapter will highlight the advances of fabrication methods that have the most potential to translate nanoparticles from bench to bedside. Furthermore, a major class of composite nanoparticle–lipid-based composite nanoparticles will be classified based on the components and reviewed in details. PMID:25409605

  14. Nanoparticle-Mediated Gene Delivery

    NASA Astrophysics Data System (ADS)

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

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

  15. Decationized polyplexes for gene delivery.

    PubMed

    Novo, Luís; Mastrobattista, Enrico; van Nostrum, Cornelus F; Lammers, Twan; Hennink, Wim E

    2015-04-01

    Gene therapy has received much attention in the field of drug delivery. Synthetic, nonviral gene delivery systems have gained increasing attention as vectors for gene therapy mainly due to a favorable immunogenicity profile and ease of manufacturing as compared to viral vectors. The great majority of these formulations are based on polycationic structures, due to their ability to interact with negatively charged nucleic acids to spontaneously form nanoparticles. In recent years, several polycationic systems have demonstrated high transfection in vitro. However, progress toward clinical applications has been slow, mainly because the cationic nature of these systems leads to intolerable toxicity levels, inappropriate biodistribution and unsatisfactory efficiency in vivo, particularly after systemic administration. Decationized polyplexes are a new class of gene delivery systems that have been developed as an alternative for conventional polycation-based systems. The major innovation introduced by decationized polyplexes is that these systems are based on neutral polymers, without any detrimental effect on the physicochemical stability or encapsulation ability, due to the transient presence of cationic charge and disulfide cross-links between the polymer chains by which the nucleic acids are physically entrapped in the particles. This editorial summarizes the most important features of decationized polyplexes and discusses potential implications for the development of new safe and efficient gene delivery systems.

  16. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

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

  17. Gene Delivery to the Airway

    PubMed Central

    Keiser, Nicholas W.; Engelhardt, John F.

    2013-01-01

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

  18. Delivery systems for gene therapy.

    PubMed

    Mali, Shrikant

    2013-01-01

    The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy.

  19. 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

  20. 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.

  1. 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.

  2. Viral and nonviral delivery systems for gene delivery

    PubMed Central

    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

  3. 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

  4. Microneedles: an innovative platform for gene delivery.

    PubMed

    McCaffrey, Joanne; Donnelly, Ryan F; McCarthy, Helen O

    2015-08-01

    The advent of microneedle (MN) technology has provided a revolutionary platform for the delivery of therapeutic agents, particularly in the field of gene therapy. For over 20 years, the area of gene therapy has undergone intense innovation and progression which has seen advancement of the technology from an experimental concept to a widely acknowledged strategy for the treatment and prevention of numerous disease states. However, the true potential of gene therapy has yet to be achieved due to limitations in formulation and delivery technologies beyond parenteral injection of the DNA. Microneedle-mediated delivery provides a unique platform for the delivery of DNA therapeutics clinically. It provides a means to overcome the skin barriers to gene delivery and deposit the DNA directly into the dermal layers, a key site for delivery of therapeutics to treat a wide range of skin and cutaneous diseases. Additionally, the skin is a tissue rich in immune sentinels, an ideal target for the delivery of a DNA vaccine directly to the desired target cell populations. This review details the advancement of MN-mediated DNA delivery from proof-of-concept to the delivery of DNA encoding clinically relevant proteins and antigens and examines the key considerations for the improvement of the technology and progress into a clinically applicable delivery system.

  5. Inhaled gene delivery: a formulation and delivery approach.

    PubMed

    Gomes Dos Reis, Larissa; Svolos, Maree; Hartwig, Benedikt; Windhab, Norbert; Young, Paul M; Traini, Daniela

    2017-03-01

    Gene therapy is a potential alternative to treat a number of diseases. Different hurdles are associated with aerosol gene delivery due to the susceptibility of plasmid DNA (pDNA) structure to be degraded during the aerosolization process. Different strategies have been investigated in order to protect and efficiently deliver pDNA to the lungs using non-viral vectors. To date, no successful therapy involving non-viral vectors has been marketed, highlighting the need for further investigation in this field. Areas covered: This review is focused on the formulation and delivery of DNA to the lungs, using non-viral vectors. Aerosol gene formulations are divided according to the current delivery systems for the lung: nebulizers, dry powder inhalers and pressurized metered dose inhalers; highlighting its benefits, challenges and potential application. Expert opinion: Successful aerosol delivery is achieved when the supercoiled DNA structure is protected during aerosolization. A formulation strategy or compounds that can protect, stabilize and efficiently transfect DNA into the cells is desired in order to produce an effective, low-cost and safe formulation. Nebulizers and dry powder inhalers are the most promising approaches to be used for aerosol delivery, due to the lower shear forces involved. In this context it is also important to highlight the importance of considering the 'pDNA-formulation-device system' as an integral part of the formulation development for a successful nucleic acid delivery.

  6. Current strategies for myocardial gene delivery

    PubMed Central

    Katz, Michael G.; Swain, JaBaris D.; Tomasulo, Catherine E.; Sumaroka, Marina; Fargnoli, Anthony; Bridges, Charles R.

    2013-01-01

    Existing methods of cardiac gene delivery can be classified by the site of injection, interventional approach and type of cardiac circulation at the time of transfer. General criteria to assess the efficacy of a given delivery method include: global versus regional myocardial transduction, technical complexity and the pathophysiological effects associated with its use, delivery-related collateral expression and the delivery-associated inflammatory and immune response. Direct gene delivery (intramyocardial, endocardial, epicardial) may be useful for therapeutic angiogenesis and for focal arrhythmia therapy but with gene expression which is primarily limited to regions in close proximity to the injection site. An often unappreciated limitation of these techniques is that they are frequently associated with substantial systemic vector delivery. Percutaneous infusion of vector into the coronary arteries is minimally invasive and allows for transgene delivery to the whole myocardium. Unfortunately, efficiency of intracoronary delivery is highly variable and the short residence time of vector within the coronary circulation and significant collateral organ expression limit its clinical potential. Surgical techniques, including the incorporation of cardiopulmonary bypass with isolated cardiac recirculation, represent novel delivery strategies that may potentially overcome these limitations; yet, these techniques are complex with inherent morbidity that must be thoroughly evaluated before safe translation into clinical practice. Characteristics of the optimal technique for gene delivery include low morbidity, increased myocardial transcapillary gradient, extended vector residence time in the coronary circulation and exclusion of residual vector from the systemic circulation after delivery to minimize extracardiac expression and to mitigate a cellular immune response. This article is part of a Special Section entitled “Special Section: Cardiovascular Gene Therapy”. PMID

  7. History of Polymeric Gene Delivery Systems.

    PubMed

    Zhang, Peng; Wagner, Ernst

    2017-04-01

    As an option for genetic disease treatment and an alternative for traditional cancer chemotherapy, gene therapy achieves significant attention. Nucleic acid delivery, however, remains a main challenge in human gene therapy. Polymer-based delivery systems offer a safer and promising route for therapeutic gene delivery. Over the past five decades, various cationic polymers have been optimized for increasingly effective nucleic acid transfer. This resulted in a chemical evolution of cationic polymers from the first-generation polycations towards bioinspired multifunctional sequence-defined polymers and nanocomposites. With the increasing of knowledge in molecular biological processes and rapid progress of macromolecular chemistry, further improvement of polymeric nucleic acid delivery systems will provide effective tool for gene-based therapy in the near future.

  8. Stimuli-responsive polymers in gene delivery.

    PubMed

    Piskin, Erhan

    2005-07-01

    Recent interest in clinical therapy has been directed to deliver nucleic acids (DNA, RNA or short-chain oligonucleotides) that alter gene expression within a specific cell population, thereby manipulating cellular processes and responses, which in turn stimulate immune responses or tissue regeneration, or blocks expression at the level of transcription or translation for treatment of several diseases. Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects. Mainly cationic polymeric systems are used as nonviral vectors, although usually with low levels of transfection. Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.

  9. Diseases originate and terminate by genes: unraveling nonviral gene delivery.

    PubMed

    Swami, Rajan; Singh, Indu; Khan, Wahid; Ramakrishna, Sistla

    2013-12-01

    The world is driving in to the era of transformation of chemical therapeutic molecules to biological genetic material therapeutics, and that is where the biological drugs especially "genes" come into existence. These genes worked as "magical bullets" to specifically silence faulty genes responsible for progression of diseases. Viral gene delivery research is far ahead of nonviral gene delivery technique. However, with more advancement in polymer science, new ways are opening for better and efficient nonviral gene delivery. But efficient delivery method is always considered as a bottleneck for gene delivery as success of which will decide the fate of gene in cells. During the past decade, it became evident that extracellular as well as intracellular barriers compromise the transfection efficiency of nonviral vectors. The challenge for gene therapy research is to pinpoint the rate-limiting steps in this complex process and implement strategies to overcome the biological physiochemical and metabolic barriers encountered during targeting. The synergy between studies that investigate the mechanism of breaking in and breaking out of nonviral gene delivery carrier through various extracellular and intracellular barriers with desired characteristics will enable the rational design of vehicles and revolutionize the treatment of various diseases.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

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

  12. Computer-assisted hydrodynamic gene delivery.

    PubMed

    Suda, Takeshi; Suda, Kieko; Liu, Dexi

    2008-06-01

    The recently developed hydrodynamic delivery method makes it possible to deliver DNA and RNA into parenchyma cells by intravascular injection of nucleic acid-containing solution. While this procedure is effective in rodents, it is difficult to perform in large animals, because manual control while delivering the injection cannot be sufficiently reliable for achieving a just-right hydrodynamic pressure in targeted tissue. In order to overcome this problem, we have developed a computer-controlled injection device that uses real-time intravascular pressure as a regulator. Using the new injection device, and mouse liver as the model organ, we demonstrated continuous injection at a single pressure and different pressures, and also serial (repeated) injections at intervals of 250 ms, by programming the computer according to the need. When assessed by reporter plasmids, the computer-controlled injection device exhibits gene delivery efficiency similar to that of conventional hydrodynamic injection. The device is also effective in gene delivery to kidney and muscle cells in rats, with plasmids or adenoviral vectors as gene carriers. Successful gene delivery to liver and kidney was also demonstrated in pigs, with the computer-controlled injection being combined with image-guided catheterization. These results represent a significant advance in in vivo gene delivery research, with potential for use in gene therapy in humans.

  13. Intracellular trafficking of hybrid gene delivery vectors.

    PubMed

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

    2015-06-10

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

  14. 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

  15. Polysaccharide-based Nanoparticles for Gene Delivery.

    PubMed

    Huh, Myung Sook; Lee, Eun Jung; Koo, Heebeom; Yhee, Ji Young; Oh, Keun Sang; Son, Sohee; Lee, Sojin; Kim, Sun Hwa; Kwon, Ick Chan; Kim, Kwangmeyung

    2017-04-01

    Nanoparticles based on nanotechnology and biotechnology have emerged as efficient carriers for various biopharmaceutical agents including proteins and genes. In particular, polysaccharides have attracted interest of many researchers in the drug delivery field due to their advantages such as biocompatibility, biodegradability, low toxicity, and ease of modification. A number of polysaccharides including chitosan, hyaluronic acid, and dextran, and their derivatives have been widely used as polymeric backbones for the formation of nanoparticles, which can be provided as valuable gene delivery carriers. In this review, we introduce the chemical and physical natures of different polysaccharides particularly used in biomedical applications, and then discuss recent progress in the development of polysaccharide-based nanoparticles for gene delivery.

  16. Gene therapy prospects--intranasal delivery of therapeutic genes.

    PubMed

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  17. Cardiac gene therapy: optimization of gene delivery techniques in vivo.

    PubMed

    Katz, Michael G; Swain, JaBaris D; White, Jennifer D; Low, David; Stedman, Hansell; Bridges, Charles R

    2010-04-01

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

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

    PubMed Central

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

    2010-01-01

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

  19. Bioreducible polymers for gene silencing and delivery.

    PubMed

    Son, Sejin; Namgung, Ran; Kim, Jihoon; Singha, Kaushik; Kim, Won Jong

    2012-07-17

    Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various

  20. 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

  1. Non-viral gene delivery using nanoparticles.

    PubMed

    Ditto, Andrew J; Shah, Parth N; Yun, Yang H

    2009-11-01

    Although the potential benefits of gene therapy for the treatment of acquired and inherited genetic diseases have been demonstrated through preclinical studies, the results of human gene therapy trials have been disappointing. Recombinant viruses are the primary vectors of choice because of their ability to protect genetic materials, cross cellular membranes, escape from endosomes and transport their genetic materials into the nucleus. Unfortunately, viral vectors have been unable to gain widespread clinical application because of their toxicity and immunogenicity. Consequently, the need for safer alternatives has led to the development of liposomes, cationic polyplexes, microparticles and nanoparticles. Although these alternative vectors have shown promise, degradable nanoparticles are the only non-viral vectors that can provide a targeted intracellular delivery with controlled release properties. Furthermore, the potential advantage of degradable nanoparticles over their non-degradable counterparts is the reduced toxicity and the avoidance of accumulation within the target tissue after repeated administration. In this article, current non-viral gene delivery devices are reviewed with a special emphasis on nanoparticle gene delivery systems. Also, the authors highlight their philosophy and efforts on the development of l-tyrosine-based polyphosphate nanoparticle-based non-viral gene delivery systems and assess the potential benefits and shortcomings of their approach.

  2. Therapeutic gene editing: delivery and regulatory perspectives

    PubMed Central

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

    2017-01-01

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

  3. Therapeutic gene editing: delivery and regulatory perspectives.

    PubMed

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

    2017-04-10

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

  4. 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.

  5. The evolution of heart gene delivery vectors.

    PubMed

    Wasala, Nalinda B; Shin, Jin-Hong; Duan, Dongsheng

    2011-10-01

    Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. Copyright © 2011 John Wiley & Sons, Ltd.

  6. Gene delivery technologies for cardiac applications

    PubMed Central

    Katz, MG; Fargnoli, AS; Pritchette, LA; Bridges, CR

    2013-01-01

    Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality in the Western society. Advances in understanding the molecular pathology of these diseases, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed these conditions within the reach of gene-based therapy. One of the cornerstones of limiting the effectiveness of gene therapy is the establishment of clinically relevant methods of genetic transfer. Recently there have been advances in direct and transvascular gene delivery methods with the use of new technologies. Current research efforts in IHD are focused primarily on the stimulation of angiogenesis, modify the coronary vascular environment and improve endothelial function with localized gene-eluting catheters and stents. In contrast to standard IHD treatments, gene therapy in HF primarily targets inhibition of apoptosis, reduction in adverse remodeling and increase in contractility through global cardiomyocyte transduction for maximal efficacy. This article will review a variety of gene-transfer strategies in models of coronary artery disease and HF and discuss the relative success of these strategies in improving the efficiency of vector-mediated cardiac gene delivery. PMID:22418063

  7. Gene delivery using liposome technology.

    PubMed

    Kikuchi, H; Suzuki, N; Ebihara, K; Morita, H; Ishii, Y; Kikuchi, A; Sugaya, S; Serikawa, T; Tanaka, K

    1999-11-01

    Development of more reliable liposomal formulations and preparation methods which can be used for gene therapy instead of commonly used viral vectors is expected. We have already developed the freeze-dried empty (non-drug-containing) liposomes (FDEL) method for mass-production of liposomal products. After these freeze-dried empty liposomes are rehydrated with aqueous drug solutions, many kinds of drugs can be encapsulated highly efficiently, and particle size can be controlled well. This study evaluated the usefulness of this FDEL method for preparation of liposomes containing DNA with a particular attention to the stability of DNA. When the liposomes were prepared by the conventional lipid-film method on a relatively large scale with use of a Potter-homogenizer (a teflon homogenizer), significant degradation and conformational change of DNA was observed during homogenization. Loss of DNA was also significant after extrusion for sizing and sterilization; residual DNA in the final preparation was hardly detected. When the FDEL method was used, on the other hand, no degradation, conformational change or loss of DNA was observed, and particle size was easily controlled. Moreover, there was no significant difference in luciferase activity between the lipid-film method used on a small scale with use of a vortex mixer and the FDEL method after transfection of tumor cells (HRA, HEC-1A and Colo320DM) by the liposomes containing DNA (PGV-C). These findings suggest that the FDEL method is very useful for preparation of liposomes containing DNA.

  8. Future prospects for gene delivery systems.

    PubMed

    Kuşcu, Lale; Sezer, Ali Demir

    2017-10-01

    Gene therapy is the challenging area of biotechnology. Despite its promise for critical diseases, it has serious safety and efficiency issues, particularly with regards to gene transfer systems. Areas covered: We examined the current situation with gene transfer systems and addressed problems this technology. We then searched patent applications about in the area from the Patentscope online system, the international patent database. We analyzed the data obtained to get a general idea about gene delivery systems designed for future use and assessed approaches for more efficient, safer and valid delivery systems. Expert opinion: When quality assurance terms are fulfilled, some of these issues (genetic changes, mutations) could be minimized during the production process. Modification of vectors for improving their efficiency and safety or development of alternative transfer systems could be the solutions for these problems. Gene transfer technologies are important for gene therapy and should demonstrate effective, target-specific and acceptable safety profiles. For this reason, searching for alternatives to current systems is a necessity.

  9. 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

  10. Polymers for gene delivery across length scales

    NASA Astrophysics Data System (ADS)

    Putnam, David

    2006-06-01

    A number of human diseases stem from defective genes. One approach to treating such diseases is to replace, or override, the defective genes with normal genes, an approach called 'gene therapy'. However, the introduction of correctly functioning DNA into cells is a non-trivial matter, and cells must be coaxed to internalize, and then use, the DNA in the desired manner. A number of polymer-based synthetic systems, or 'vectors', have been developed to entice cells to use exogenous DNA. These systems work across the nano, micro and macro length scales, and have been under continuous development for two decades, with varying degrees of success. The design criteria for the construction of more-effective delivery vectors at each length scale are continually evolving. This review focuses on the most recent developments in polymer-based vector design at each length scale.

  11. Nanoparticle motivated gene delivery for ophthalmic application

    PubMed Central

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

    2015-01-01

    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 (siRNA), micro RNA (miRNA), etc., 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 due to exogenous and endogenous degradation modalities. Nanotechnology, a promising and sophisticated cutting edge tool, works as a protective shelter for these therapeutic nucleic acids. They are able to 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 non-viral strategies being used to deliver therapeutic genes in order to treat various devastating ocular diseases. PMID:26109528

  12. 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.

  13. Fetal muscle gene therapy/gene delivery in large animals.

    PubMed

    Abi-Nader, Khalil N; David, Anna L

    2011-01-01

    Gene delivery to the fetal muscles is a potential strategy for the early treatment of muscular dystrophies. In utero muscle gene therapy can also be used to treat other genetic disorders such as hemophilia, where the missing clotting proteins may be secreted from the treated muscle. In the past few years, studies in small animal models have raised the hopes that a phenotypic cure can be obtained after fetal application of gene therapy. Studies of efficacy and safety in large animals are, however, essential before clinical application can be considered in the human fetus. For this reason, the development of clinically applicable strategies for the delivery of gene therapy to the fetal muscles is of prime importance. In this chapter, we describe the protocols for in utero ultrasound-guided gene delivery to the ovine fetal muscle in early gestation. In particular, procedures to inject skeletal muscle groups such as the thigh and thoracic musculature and targeting the diaphragm in the fetus are described in detail.

  14. FUNCTIONAL NANOPARTICLES FOR MOLECULAR IMAGING GUIDED GENE DELIVERY

    PubMed Central

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

    2010-01-01

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

  15. Synthetic virology: engineering viruses for gene delivery.

    PubMed

    Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

  16. 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

  17. 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

  18. [Developments in gene delivery vectors for ocular gene therapy].

    PubMed

    Khabou, Hanen; Dalkara, Deniz

    2015-05-01

    Gene therapy is quickly becoming a reality applicable in the clinic for inherited retinal diseases. Its remarkable success in safety and efficacy, in clinical trials for Leber's congenital amaurosis (LCA) type II generated significant interest and opened up possibilities for a new era of retinal gene therapies. Success in these clinical trials was mainly due to the favorable characteristics of the retina as a target organ. The eye offers several advantages as it is readily accessible and has some degree of immune privilege making it suitable for application of viral vectors. The viral vectors most frequently used for retinal gene delivery are lentivirus, adenovirus and adeno-associated virus (AAV). Here we will discuss the use of these viral vectors in retinal gene delivery with a strong focus on favorable properties of AAV. Thanks to its small size, AAV diffuses well in the inter-neural matrix making it suitable for applications in neural retina. Building on this initial clinical success with LCA II, we have now many opportunities to extend this proof-of-concept to other retinal diseases using AAV as a vector. This article will discuss what are some of the most imminent cellular targets for such therapies and the AAV toolkit that has been built to target these cells successfully. We will also discuss some of the challenges that we face in translating AAV-based gene therapies to the clinic.

  19. Microfluidic methods for non-viral gene delivery.

    PubMed

    Lai, Wing-Fu

    2015-01-01

    Microfluidics is a compelling technology that shows considerable promise in applications ranging from gene expression profiling to cell-based assays. Owing to its capacity to enable generation of single droplets and multiple droplet arrays with precisely controlled composition and a narrow size distribution, recently microfluidics has been exploited for delivery of genes. This article provides an overview of recent advances in microfluidic gene delivery, and speculates the prospects for further research. The objectives of this article are to illustrate the potential roles played by microfluidics in gene delivery research, and to shed new light on strategies to enhance the efficiency of gene therapy.

  20. 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

  1. 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

  2. Gene delivery with viral vectors for cerebrovascular diseases.

    PubMed

    Gan, Yu; Jing, Zheng; Stetler, Ruth Anne; Cao, Guodong

    2013-01-01

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

  3. Gene delivery in tissue engineering and regenerative medicine.

    PubMed

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

    2015-11-01

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

  4. 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.

  5. Investigation of a thiolated polymer in gene delivery

    NASA Astrophysics Data System (ADS)

    Bacalocostantis, Irene

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

  6. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches. © 2013.

  7. Cardiac Gene Delivery in Large Animal Models: Antegrade Techniques.

    PubMed

    Watanabe, Shin; Leonardson, Lauren; Hajjar, Roger J; Ishikawa, Kiyotake

    2017-01-01

    Percutaneous antegrade coronary injection is among the least invasive cardiac selective gene delivery methods. However, transduction efficiency is quite low with a simple bolus antegrade injection. In order to improve the transduction efficiency using antegrade delivery, several additional approaches have been proposed.In this chapter, we briefly discuss important elements associated with intracoronary delivery methods and present protocols for three different catheter-based antegrade delivery techniques in a preclinical large animal model. Despite the lower transduction efficacy relative to more invasive delivery techniques, antegrade techniques have the advantage of being clinically well established and having safer profiles which is important when treating patients with cardiac disease.

  8. State-of-the-art human gene therapy: part I. Gene delivery technologies.

    PubMed

    Wang, Dan; Gao, Guangping

    2014-01-01

    Safe and effective gene delivery is a prerequisite for successful gene therapy. In the early age of human gene therapy, setbacks due to problematic gene delivery vehicles plagued the exciting therapeutic outcome. However, gene delivery technologies rapidly evolved ever since. With the advancement of gene delivery techniques, gene therapy clinical trials surged during the past decade. As the first gene therapy product (Glybera) has obtained regulatory approval and reached clinic, human gene therapy finally realized the promise that genes can be medicines. The diverse gene delivery techniques available today have laid the foundation for gene therapy applications in treating a wide range of human diseases. Some of the most urgent unmet medical needs, such as cancer and pandemic infectious diseases, have been tackled by gene therapy strategies with promising results. Furthermore, combining gene transfer with other breakthroughs in biomedical research and novel biotechnologies opened new avenues for gene therapy. Such innovative therapeutic strategies are unthinkable until now, and are expected to be revolutionary. In part I of this review, we introduced recent development of non-viral and viral gene delivery technology platforms. As cell-based gene therapy blossomed, we also summarized the diverse types of cells and vectors employed in ex vivo gene transfer. Finally, challenges in current gene delivery technologies for human use were discussed.

  9. Overcoming Nonviral Gene Delivery Barriers: Perspective and Future

    PubMed Central

    Jones, Charles H.; Chen, Chih-Kaung; Ravikrishnan, Anitha; Rane, Snehal; Pfeifer, Blaine A.

    2013-01-01

    A key end goal of gene delivery research is to develop clinically-relevant vectors that can be used to combat elusive diseases such as AIDS. Despite promising engineering strategies, efficiency and ultimately gene modulation efficacy of nonviral vectors have been hindered by numerous in vitro and in vivo barriers that have resulted in sub-viral performance. In this perspective, we concentrate on the gene delivery barriers associated with the two most common classes of nonviral vectors, cationic-based lipids and polymers. We present the existing delivery barriers and summarize current vector-specific strategies to overcome said barriers. PMID:24093932

  10. Biodegradable polyphosphoester micelles for gene delivery.

    PubMed

    Wen, Jie; Mao, Hai-Quan; Li, Weiping; Lin, Kevin Y; Leong, Kam W

    2004-08-01

    A new biodegradable polyphosphoester, poly[[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate] (PCEP) was synthesized and investigated for gene delivery. Carrying a positive charge in its backbone and a lipophilic cholesterol structure in the side chain, PCEP self-assembled into micelles in aqueous buffer at room temperature with an average size of 60-100 nm. It could bind and protect plasmid DNA from nuclease digestion. Cell proliferation assay indicated a lower cytotoxicity for PCEP than for poly-L-lysine and Lipofectamine. The IC50 determined by the WST-1 assay was 69.8, 51.6, and 12.1 microg/mL for PCEP, Lipofectamine, and poly-L-lysine, respectively. PCEP efficiently delivered DNA to several cell lines such as HEK293, Caco-2, and HeLa. The highest efficiency was achieved when PCEP/DNA complex was prepared in Opti-MEM with a +/- charge ratio of 1.5-2. The transfection efficiency did not change significantly when the complex was used 3 days after preparation. The addition of chloroquine to the formulation increased transfection efficiency 10- to 50-fold compared to the complex alone. In vivo studies showed a luciferase expression by PCEP/DNA complexes in muscle increasing with time during 3 months, although the expression level was lower than that by direct injection of naked DNA. In addition to biodegradability and lower toxicity, the PCEP micelle carrier offers structural versatility. The backbone charge density and the side chain lipophilicity are two parameters that can be varied through copolymerization and monomer variation to optimize the transfection efficiency.

  11. 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.

  12. 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

  13. Next Generation Delivery System for Proteins and Genes of Therapeutic Purpose: Why and How?

    PubMed Central

    Priya Doss, C. George; Lee, Sang-Soo

    2014-01-01

    Proteins and genes of therapeutic interests in conjunction with different delivery systems are growing towards new heights. “Next generation delivery systems” may provide more efficient platform for delivery of proteins and genes. In the present review, snapshots about the benefits of proteins or gene therapy, general procedures for therapeutic protein or gene delivery system, and different next generation delivery system such as liposome, PEGylation, HESylation, and nanoparticle based delivery have been depicted with their detailed explanation. PMID:25126554

  14. Biodegradable polymer-metal complexes for gene and drug delivery.

    PubMed

    Hosseinkhani, Hossein; Hosseinkhani, Mohsen

    2009-01-01

    The delivery of genes and drugs into cells has increasingly attracted attention for the generation of genetically engineered cells. Successful drug delivery will have enormous academic, clinical, and practical impacts on gene therapy, cell and molecular biology, pharmaceutical and food industries, and bio-production. The major aim of gene therapy is to deliver genetic materials into cells effectively, genetically modifying and repairing cell functions with the possibility of inducing therapeutic healing of disease. The genetic material includes DNA, RNA, antisense, decoy DNA, and ribozymes. The aim is that the appropriate transfection would allow diseased cells to return to a healthy condition. The genetic manipulation is often manifested in the mechanisms of intracellular actions of genes and proteins, and may play an important role in making clear the key genes associated with various diseases. Based on fundamental and scientific knowledge, the delivery technology of genetic material should be applicable to producing various proteins of pharmaceutical value (e.g. cytokines, growth factors, and antibodies) and also to producing seeds resistant to harmful insects and cold weather damage. This implies that the cells might be enhanced to produce valuable pharmaceutical and food products. For each approach, it is important, for successful gene expression, to select an appropriate gene to be delivered as well as to develop the gene delivery technology to enhance transfection efficiency. This review will provide an overview of the enhanced gene expression of plasmid DNA complexed with new non-viral gene delivery vehicles by biodegradable biopolymer-metal complex, introducing our recent research data to emphasize the technical feasibility of biopolymer-metal complexes in gene therapy and biotechnology.

  15. 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.

  16. Characterisation of gene delivery using liposomal bubbles and ultrasound

    NASA Astrophysics Data System (ADS)

    Koshima, Risa; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Nomura, Tetsuya; Negishi, Yoichi; Utoguchi, Naoki; Kudo, Nobuki; Maruyama, Kazuo

    2011-09-01

    The combination of nano/microbubbles and ultrasound is a novel technique for a non-viral gene deliver. We have previously developed novel ultrasound sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumors in vivo. The delivery of genes by bubble liposomes depended on the intensity of the applied ultrasound. The transfection efficiency plateaued at 0.7 W/cm2 ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, bubble liposomes were able to introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells. We conclude that the combination of Bubble liposomes and ultrasound is a good method for gene transfer in vivo.

  17. Bioengineered Silk Protein-Based Gene Delivery Systems

    PubMed Central

    Numata, Keiji; Subramanian, Balajikarthick; Currie, Heather A.; Kaplan, David L.

    2009-01-01

    Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(l-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30-lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm, showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems. PMID:19577803

  18. 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

  19. Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

    PubMed

    Kang, Mijeong; Kim, Bongsoo

    2015-01-01

    Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

  20. Micelles and nanoparticles for ultrasonic drug and gene delivery.

    PubMed

    Husseini, Ghaleb A; Pitt, William G

    2008-06-30

    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 the 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 nanocarriers has tremendous potential because of the wide variety of drugs and genes that could be delivered to targeted tissues by fairly non-invasive means.

  1. 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

  2. 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.

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

    PubMed

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

    2013-05-15

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

  4. Biomaterial Scaffolds for Controlled, Localized Gene Delivery of Regenerative Factors

    PubMed Central

    Gower, Robert Michael; Shea, Lonnie D.

    2013-01-01

    Significance Biomaterials play central roles in tissue regeneration by maintaining a space for tissue growth and facilitating its integration with the host. The regenerative capacity of materials can be enhanced through delivery of factors that promote tissue formation. Gene delivery is a versatile strategy to obtain sustained production of tissue inductive factors. Biomaterial scaffolds capable of gene delivery have been shown to induce transgene expression and tissue growth. Critical Issues The widespread application of biomaterial scaffold systems requires identifying the design principles for the material and vectors that modulate transgene expression temporally and spatially. These technologies and others will ultimately enable spatial and temporal control over expression to recreate the cellular organization and gene expression required for formation of complex tissues. Recent Advances The design parameters for the biomaterials and vectors that modulate the extent and duration of transgene expression and the distribution of transgene-expressing cells within and around the injury are emerging. The cellular interactions with the biomaterial, such as adhesion or migration rate, can influence expression. Furthermore, modulating the interaction between the vector and biomaterial can control vector release while minimizing the exposure to harsh processing conditions. Future Directions Biomaterial scaffolds that deliver genes encoding for regenerative factors may provide a platform for regenerating complex tissues such as skin, blood vessels, and nerves. Biomaterials capable of localized gene delivery can synergistically target multiple cell processes and will have application to the regeneration of many tissues, with great promise for clinical therapies. PMID:24527333

  5. Practical delivery of genes to the marketplace.

    PubMed

    Fischhoff, David A; Cline, Molly N

    2009-01-01

    Although new technologies in genomics are powerful tools for discovering genes and gaining insight into their function, discovery of a gene itself does not ensure its practical application. Commercialization of transgenic crop plants has now taken place for more than a decade. Plant biotechnology, which can be seen as an extension of traditional plant breeding for crop improvement, offers one way to boost food, feed, fiber, and fuel production and has provided significant environmental and economic benefits. Like plant breeding, biotechnology introduces new traits with specific benefits into plants, and does so in a selective, precise, and controlled manner. Several steps are necessary before commercializing a crop with a biotechnology trait, including not only gene discovery and product development but also regulatory clearance, stewardship evaluation, and stakeholder dialogue. Examples will be drawn from the work at Monsanto on the development and commercialization of glyphosate-tolerant soybeans, which is representative of the first wave of agronomic traits.

  6. 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

  7. 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

  8. Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial. PMID:21326169

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-03-01

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

  11. Bioengineered Silk Gene Delivery System for Nuclear Targeting

    PubMed Central

    Yigit, Sezin; Tokareva, Olena; Varone, Antonio; Georgakoudi, Irene

    2015-01-01

    Gene delivery research has gained momentum with the use of lipophilic vectors that mimic viral systems to increase transfection efficiency. However, maintaining cell viability with these systems remains a major challenge. Therefore biocompatible and nontoxic biopolymers that are designed by combining non-immunological viral mimicking components with suitable carriers have been explored to address these limitations. In the present study recombinant DNA technology was used to design a multi-functional gene delivery system for nuclear targeting, while also supporting cell viability. Spider dragline silk recombinant proteins were modified with DNA condensing units and the proton sponge endosomal escape pathway was utilized for enhanced delivery. Short-term transfection efficiency in a COS-7 cell line (adherent kidney cells isolated from African green monkey) was enhanced compared to lipofectamine and polyethyleneimine (PEI), as was cell viability with these recombinant bio-polyplexes. Endosomal escape and consequent nuclear targeting were shown with fluorescence microscopy. PMID:24889658

  12. 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

  13. Delivery of gene-expressing fragments using quantum dot

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Hanada, Sanshiro; Fujioka, Kouki; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2009-02-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced the gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/geneconstruct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of QD-constructs, 3) the surface electronic charges varied according to the structure of QD/gene-constructs, and 4) the particle size of QD/gene complex varied according to the structure and amounts of gene fragments. Using this QD/geneconstruct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes. These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental pollution including human health under certain conditions. Here we propose the further research for the immune and physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale products as well as nanocrystal

  14. Hyperbranched cationic amylopectin derivatives for gene delivery.

    PubMed

    Zhou, Yanfang; Yang, Bin; Ren, Xianyue; Liu, Zhenzhen; Deng, Zheng; Chen, Luming; Deng, Yubin; Zhang, Li-Ming; Yang, Liqun

    2012-06-01

    A series of hyperbranched cationic amylopectin derivatives conjugated with 1,2-ethylenediamine, diethylenetriamine and 3-(dimethylamino)-1-propylamine residues, named as EDA-Amp, DETA-Amp and DMAPA-Amp, were synthesized by the N,N'-carbonyldiimidazole activation method at room temperature. Their structures were characterized by FTIR and (1)H NMR analyses, and their buffering capability was assessed by acid-base titration. The amylopectin derivatives exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI) in the hemolysis and MTT assays. Atomic force microscopy and optical microscopy confirmed that the amylopectin derivatives exhibited lower damage for erythrocytes than bPEI. The amylopectin derivatives could bind and condense plasmid DNA (pDNA) to form the complexes with the size ranging from 100 to 300 nm. The resultant complexes showed higher transfection efficiency in 293T cells than in A549 cells. The DMAPA-Amp derivative-mediated gene transfection for Forkhead box O1 exhibited higher protein expression than that of the EDA-Amp and DETA-Amp derivatives in 293T cells, which was analyzed by western blot, flow cytometry and Hoechst staining assay. On the basis of these data, amylopectin derivatives exhibit potential as nonviral gene vectors. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  15. Specific gene delivery to liver sinusoidal and artery endothelial cells.

    PubMed

    Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

    2013-09-19

    Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

  16. 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

  17. Novel gemini cationic lipids with carbamate groups for gene delivery.

    PubMed

    Zhao, Yi-Nan; Qureshi, Farooq; Zhang, Shu-Biao; Cui, Shao-Hui; Wang, Bing; Chen, Hui-Ying; Lv, Hong-Tao; Zhang, Shu-Fen; Huang, Leaf

    2014-05-21

    To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study.

  18. Novel gemini cationic lipids with carbamate groups for gene delivery

    PubMed Central

    Zhao, Yi-Nan; Qureshi, Farooq; Zhang, Shu-Biao; Cui, Shao-Hui; Wang, Bing; Chen, Hui-Ying; Lv, Hong-Tao; Zhang, Shu-Fen; Huang, Leaf

    2014-01-01

    To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study. PMID:25045521

  19. 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

  20. Functional lipids and lipoplexes for improved gene delivery

    PubMed Central

    Zhang, Xiao-Xiang; McIntosh, Thomas J.; Grinstaff, Mark W.

    2013-01-01

    Cationic lipids are the most common non-viral vectors used in gene delivery with a few currently being investigated in clinical trials. However, like most other synthetic vectors, these vectors suffer from low transfection efficiencies. Among the various approaches to address this challenge, functional lipids (i.e., lipids responding to a stimuli) offer a myriad of opportunities for basic studies of nucleic acid–lipid interactions and for in vitro and in vivo delivery of nucleic acid for a specific biological/medical application. This manuscript reviews recent advances in pH, redox, and charge-reversal sensitive lipids. PMID:21621581

  1. Chitosan nanoparticles for oral drug and gene delivery

    PubMed Central

    Bowman, Katherine; Leong, Kam W

    2006-01-01

    Chitosan is a widely available, mucoadhesive polymer that is able to increase cellular permeability and improve the bioavailability of orally administered protein drugs. It can also be readily formed into nanoparticles able to entrap drugs or condense plasmid DNA. Studies on the formulation and oral delivery of such chitosan nanoparticles have demonstrated their efficacy in enhancing drug uptake and promoting gene expression. This review summarizes some of these findings and highlights the potential of chitosan as a component of oral delivery systems. PMID:17722528

  2. Gene delivery by dendrimers operates via a cholesterol dependent pathway.

    PubMed

    Manunta, Maria; Tan, Peng Hong; Sagoo, Pervinder; Kashefi, Kirk; George, Andrew J T

    2004-01-01

    Understanding the cellular uptake and intracellular trafficking of dendrimer-DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer-DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.

  3. Reducible, Dibromomaleimide-linked Polymers for Gene Delivery

    PubMed Central

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

    2014-01-01

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

  4. Reducible, dibromomaleimide-linked polymers for gene delivery.

    PubMed

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

    2015-01-01

    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 azide-alkyne 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 azide-fluorophore 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.

  5. Gene delivery to periodontal tissue using Bubble liposomes and ultrasound.

    PubMed

    Sugano, M; Negishi, Y; Endo-Takahashi, Y; Hamano, N; Usui, M; Suzuki, R; Maruyama, K; Aramaki, Y; Yamamoto, M

    2014-06-01

    Periodontitis is the most common inflammatory disease caused by oral biofilm infection. For efficient periodontal treatment, it is important to enhance the outcome of existing regenerative therapies. The physical action of an ultrasound may be able to deliver a therapeutic gene or drugs into the local area of the periodontium being treated for periodontal regeneration. Previously, we developed "Bubble liposomes" as a useful carrier for gene or drug delivery, and reported that delivery efficiency was increased with high-frequency ultrasound in vitro and in vivo. Hence, the aim of the present study was to examine the possibility of delivering genes into gingival tissues using Bubble liposomes and ultrasound. We attempted to deliver naked plasmid DNA encoding luciferase or enhanced green fluorescent protein (EGFP) into the lower labial gingiva of Wistar rats using Bubble liposomes, with or without ultrasound exposure. Ultrasound parameters were optimized for intensity (0-4.0 W/cm(2) ) and exposure time (0-120 s) to establish the most efficient conditions for exposure. The efficacy and duration of gene expression in the gingiva were investigated using a luciferase assay and fluorescence microscopy. The strongest relative luciferase activity was observed when rats were treated under the following ultrasound conditions: 2.0 W/cm(2) intensity and 30 s of exposure time. Relative luciferase activity, 1 d after gene delivery, was significantly higher in gingiva treated using Bubble liposomes and ultrasound than in gingiva of the other treatment groups. Histological analysis also showed that distinct EGFP-expressing cells were observed in transfected gingiva when rats were treated under optimized conditions. From these results, the combination of Bubble liposomes and ultrasound provides an efficient technique for delivering plasmid DNA into the gingiva. This technique can be applied for the delivery of a variety of therapeutic molecules into target tissue, and may serve as a

  6. Layered double hydroxide nanoparticles in gene and drug delivery.

    PubMed

    Ladewig, Katharina; Xu, Zhi Ping; Lu, Gao Qing Max

    2009-09-01

    Layered double hydroxides (LDHs) have been known for many decades as catalyst and ceramic precursors, traps for anionic pollutants, catalysts and additives for polymers, but their successful synthesis on the nanometer scale a few years ago opened up a whole new field for their application in nanomedicine. The delivery of drugs and other therapeutic/bioactive molecules (e.g., peptides, proteins, nucleic acids) to mammalian cells is an area of research that is of tremendous importance to medicine and provides manifold applications for any new developments in the area of nanotechnology. Among the many different nanoparticles that have been shown to facilitate gene and/or drug delivery, LDH nanoparticles have attracted particular attention owing to their many desirable properties. This review aims to report recent progress in gene and drug delivery using LDH nanoparticles. It summarizes the advantages and disadvantages of using LDH nanoparticles as carriers for nucleic acids and drugs against the general background of bottlenecks that are encountered by cellular delivery systems. It describes further the models that have been proposed for the internalization of LDH nanoparticles into cells so far and discusses the intracellular fate of the particles and their cargo. The authors offer some remarks on how this field of research will progress in the near future and which challenges need to be overcome before LDH nanoparticles can be used in a clinical setting.

  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. Novel cholesterol-based cationic lipids for gene delivery.

    PubMed

    Medvedeva, Darya A; Maslov, Mikhail A; Serikov, Roman N; Morozova, Nina G; Serebrenikova, Galina A; Sheglov, Dmitry V; Latyshev, Alexander V; Vlassov, Valentin V; Zenkova, Marina A

    2009-11-12

    Gene therapy based on gene delivery is a promising strategy for the treatment of human disease. Here we present data on structure/biological activity of new biodegradable cholesterol-based cationic lipids with various heterocyclic cationic head groups and linker types. Enhanced accumulation of nucleic acids in the cells mediated by the lipids was demonstrated by fluorescent microscopy and flow cytometry. Light scattering and atomic force microscopy were used to find structure/transfection activity correlations for the lipids. We found that the ability of the lipids to stimulate intracellular accumulation of the oligodeoxyribonucleotides and plasmid DNA correlates well with their ability to form in solution lipid/NA complexes of sizes that do not exceed 100 nm. Screening of the lipids revealed the most promising transfection agents both in terms of low toxicity and efficient delivery: cholesterol-based lipids with positively charged pyridine and methyl imidazole head groups and either the ester or carbamate linker.

  9. 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

  10. Characterization of novel cationic amphiphiles for gene delivery

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoxiang

    Gene therapy has drawn vast interest for treating, preventing, or controlling a myriad of diseases. The two most common methods for gene delivery use either synthetic or viral vectors. Viral vectors (infection) are by far the most effective and efficient means of DNA delivery, but their use is tempered by safety and immunogenicity concerns. Consequently, there has been a significant effort to develop and evaluate non- viral vectors, which include cationic amphiphiles and polymers, and more recently anionic amphiphiles. Non-viral vectors have the advantages of ease of production, better stability and low immunogenicity. At the same time, they also have a number of limitations, including low in vitro and in vivo transfection efficiencies, and cytotoxicity in many instances. My research project has been focused on design, development and characterizations of novel amphiphilic lipids for gene delivery. Through rational design and characterization of the amphiphile structures, it not only yielded vectors showing high transfection activities, but also provided information of the structure-activity relationship. These results provide us with a better understanding on the transfection process and future directions to further optimize the amphiphile structures. More specifically, my dissertation research included the following three parts: (i) characterization of novel lipopeptides possessing di- or tri- peptide head groups; (ii) determination of the effect of spacer (between the cationic domain and the hydrophobic domain of charge-reversal amphiphiles) length, rigidity and hydrophilicity on gene delivery; (iii) identification of the cellular uptake pathway and the transfection mechanism of a known enzyme-sensitive charge-reversal amphiphile.

  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. Copyright © 2014 Elsevier B.V. All rights reserved.

  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. Smart Micro/Nano-robotic Systems for Gene Delivery.

    PubMed

    Pedram, Alireza; Pishkenari, Hossein Nejat

    2017-01-01

    Small scale robotics have attracted growing attention for the prospect of targeting and accessing cell-sized sites, necessary for high precision biomedical applications and drug/gene delivery. The loss of controlled gene therapy, inducing systemic side effects and reduced therapeutic efficiency, can be settled utilizing these intelligent carriers. Newly proposed solutions for the main challenges of control, power supplying, gene release and final carrier extraction/degradation have shifted these smart miniature robots to the point of being employed for practical applications of transferring oligonucleotides (pDNA, siRNA, mRNA, etc.) in near future. In this paper, different scenarios and their endeavors to address the vital working demands and steps, in particular, carrier attachment and release, cell internalization, manipulation concerns as well as actuation systems are discussed.This review highlights some promising experimental results showing controlled gene release of robotic systems in comparison with current non-specific gene delivery methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Recent advances in nonviral vectors for gene delivery.

    PubMed

    Guo, Xia; Huang, Leaf

    2012-07-17

    Gene therapy has long been regarded a promising treatment for many diseases, whether acquired (such as AIDS or cancer) or inherited through a genetic disorder. A drug based on a nucleic acid, however, must be delivered to the interior of the target cell while surviving an array of biological defenses honed by evolution. Successful gene therapy is thus dependent on the development of an efficient delivery vector. Researchers have pursued two major vehicles for gene delivery: viral and nonviral (synthetic) vectors. Although viral vectors currently offer greater efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, nonviral vectors can readily transfect cells in culture, but efficient nanomedicines remain far removed from the clinic. Overcoming the obstacles associated with nonviral vectors to improve the delivery efficiency and therapeutic effect of nucleic acids is thus an active area of current research. The difficulties are manifold, including the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, and managing the targeting ability of the carriers with respect to the cells of interest. Modifying the surface with poly(ethylene glycol), that is, PEGylation, is the predominant method used to reduce the binding of plasma proteins to nonviral vectors and minimize clearance by the RES after intravenous administration. Nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors because of the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral and anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal models. Other approaches include (i) designing and synthesizing novel

  15. Hyaluronic acid enhances gene delivery into the cochlea.

    PubMed

    Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

    2012-03-01

    Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

  16. 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.

  17. Multivalent dendrimer vectors with DNA intercalation motifs for gene delivery.

    PubMed

    Wong, Pamela T; Tang, Kenny; Coulter, Alexa; Tang, Shengzhuang; Baker, James R; Choi, Seok Ki

    2014-11-10

    Poly(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and with 3,8-diamino-6-phenylphenanthridinium ("DAPP")-derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon coincubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: KD = 0.22 nM for G5(FA)(DAPP) versus 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line and showed that RF-conjugated dendrimers were 1 to 2 orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery.

  18. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  19. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    PubMed Central

    Fillat, Cristina; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano

    2011-01-01

    The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed. PMID:24212620

  20. 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

  1. Intratympanic Gene Delivery of Antimicrobial Molecules in Otitis Media.

    PubMed

    Moon, Sung K; Lim, David J

    2015-04-01

    Otitis media (OM) in children is clinically important because of its detrimental effects on the development of language and motor coordination and is the most common reason for prescription of antibiotics. A recent bacteriological change in OM pathogens such as emergence of antibiotic resistance and vaccination-mediated pathogenic shift urges us to develop a new non-antibiotic strategy. The middle ear epithelium abundantly secretes a variety of antimicrobial molecules suppressing the viability of the common OM pathogens. Recently, we have demonstrated that the adenoviral vector is able to deliver the β-defensin 2 gene to the middle ear epithelial cells in vitro and in vivo, and adenovirus-mediated overexpression of β-defensin 2 is protective for experimental OM. There are many hurdles limiting successful clinical application of gene delivery to the respiratory epithelium of the tubotympanum; however, intratympanic gene therapy with β-defensin 2 is a promising alternative or adjuvant strategy for the management of OM.

  2. Gene delivery: intelligent but just at the beginning.

    PubMed

    Pişkin, E; Dinçer, S; Türk, M

    2004-01-01

    Gene therapy is used to treat genetic disorders, which may be achieved both ex vivo and in vivo. Gene-delivery systems usually include a carrier system which both protects the gene expression plasmid and allows its extracellular and intracellular trafficking. Viruses are used in most of the clinical trials today; however, they do have important drawbacks. Non-viral vectors based on lipids, water-soluble polycations, other non-condensing polymers and nano- or microparticles/capsules have been proposed. Cationic polymers, especially carrying novel targeting ligands. are receiving increasing attention. Intelligent polymers with temperature, pH, and light sensitivities for a controllable and effective non-viral transfection have recently been introduced but are just at the beginning. Our preliminary studies showed that block copolymers of N-isopropylacrylamide-acrylic acid with poly(ethylene imine) could be one example of these novel non-viral vectors.

  3. Current Status of Gene Delivery and Gene Therapy in Lacrimal Gland using Viral Vectors

    PubMed Central

    Selvam, Shivaram; Thomas, Padmaja B.; Hamm-Alvarez, Sarah F.; Schechter, Joel E.; Stevenson, Douglas; Mircheff, Austin K.; Trousdale*, Melvin D.

    2006-01-01

    Gene delivery is one of the biggest challenges in the field of gene therapy. It involves the efficient transfer of transgenes into somatic cells for therapeutic purposes. A few major drawbacks in gene delivery include inefficient gene transfer and lack of sustained transgene expression. However, the classical method of using viral vectors for gene transfer has circumvented some of these issues. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. The transfer of genetic material into lacrimal epithelial cells and tissues, both in vitro and in vivo, has been critical for the study of tear secretory mechanisms and autoimmunity of the lacrimal gland. These studies will help in the development of therapeutic interventions for autoimmune disorders such as Sjögren’s syndrome and dry eye syndromes which are associated with lacrimal dysfunction. These studies are also critical for future endeavors which utilize the lacrimal gland as a reservoir for the production of therapeutic factors which can be released in tears, providing treatment for diseases of the cornea and posterior segment. This review will discuss the developments related to gene delivery and gene therapy in the lacrimal gland using several viral vector systems. PMID:17056149

  4. 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.

  5. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    PubMed

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.

  6. 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.

  7. Nanoparticles facilitate gene delivery to microorganisms via an electrospray process.

    PubMed

    Lee, Yi-Hsuan; Wu, Bing; Zhuang, Wei-Qin; Chen, Da-Ren; Tang, Yinjie J

    2011-02-01

    In this study, we developed a technique for delivering genes to microorganisms via electrospray of gold nanoparticles. During the electrospray process, charged monodisperse nano-droplets (a mixture of pET30a-GFP plasmid and nano-sized gold particles) were accelerated and deposited on a thin layer of non-competent Escherichia coli cells. Via antibiotic selection, transformed cells containing green fluorescent protein appeared on the agar plates. PCR amplification and restriction enzyme analysis further confirmed that pET30a-GFP plasmid had successfully been delivered into the non-competent E. coli cells. The transformation efficiencies were optimized under different electrospray conditions. Among several electrospray buffer solutions, CaCl(2) (0.01M) was found to be the best for gene delivery. Furthermore, gold nanoparticles (NPs, 50 nm diameter) significantly improved plasmid transformation efficiency by 5-7 fold (up to 2×10(6) CFU/μg plasmid) compared with that obtained using naked plasmid. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmids remained unchanged during the electrospray process, but cellular membrane integrity was reduced after being electrosprayed with gold NPs and CaCl(2) buffer solutions. This gene delivery method has the potential to work for many other microorganisms. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Sperm as a noninvasive gene delivery system for preimplantation embryos.

    PubMed

    Chan, P J; Kalugdan, T; Su, B C; Whitney, E A; Perrott, W; Tredway, D R; King, A

    1995-05-01

    To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. Clinical and academic research environment. Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

  9. 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

  10. Arginoplexes: an arginine-anchored nanoliposomal carrier for gene delivery

    NASA Astrophysics Data System (ADS)

    Patel, Ketan; Tyagi, Monica; Monpara, Jasmin; Vora, Lalit; Gupta, Sanjay; Vavia, Pradeep

    2014-04-01

    There is a need of an efficient and safe non-viral gene delivery carrier due to promising future of nucleic acid-based therapeutics in the treatment of intractable diseases. Cytotoxicity and cost are the major concerns with current quaternary ammonium-based cationic liposomes. The major aim of current research work was development and in vitro evaluation of arginine-anchored nanoliposomes for gene delivery. l-Arginine-fatty acid conjugate was synthesized and characterized using IR, NMR, and mass spectroscopy. Synthesized conjugate—lauroyl arginine ethyl ester (LAE) was successfully incorporated into liposomes. Effect of nanocarrier composition on DNA binding was evaluated by preparing solid lipid nanoparticle (SLN) and self nanoemulsifying system (SNES) using same LAE concentration. Effect of cationic head on DNA binding was also evaluated. Arginine-anchored nanoliposomes—arginoplexes (APX) showed superior DNA-binding affinity. Surface PEG was expected to cause hindrance in DNA binding in SLNs and SNES. Guanidino group was found to be a better cationic head for DNA binding compared to primary amine or quaternary amine. Gel retardation assay was performed to optimize the ratio of DNA to LAE in nanocarrier. Serum stability, haemolysis, cytotoxicity, and transfection studies were carried out to evaluate APX. Binding of DNA to APX was found to be stable in the presence of serum, and no degradation of DNA was observed. APX containing 2 mg/ml LAE which exhibited particle size of 72 nm with zeta potential of +57.5 mV, showed lower cytotoxicity and better transfection. APX can be a promising carrier for gene delivery.

  11. Click assembly of magnetic nanovectors for gene delivery

    PubMed Central

    Biswas, Souvik; Gordon, Laura E.; Clark, Geoffrey J.; Nantz, Michael H.

    2011-01-01

    Functionalization of iron oxide nanoparticles with quaternary ammonium ion-based aminooxy and oxime ether substrates provides a flexible route for generating magnetic gene delivery vectors. Using the MCF-7 breast cancer cell line, our findings show that pDNA magnetoplexes derived from the lipid-coated nanoparticle formulation dMLP transfect in the presence of 10% serum with or without magnetic assistance at significantly higher levels than a commonly used cationic liposome formulation, based on luciferase assay. The present ionpairing, click chemistry approach furnishes Fe3O4 nanoparticles with lipid layers. The resultant magnetic nanovectors serve as transfection enhancers for otherwise transfection-inactive materials. PMID:21255832

  12. Design of serum compatible tetrary complexes for gene delivery.

    PubMed

    Bahadur K C, Remant; Thapa, Bindu; Xu, Peisheng

    2012-05-01

    A novel gene delivery system, called PoSC, consisting of PEI, PSP, and HA is described. In contrast to the DNA/PEI/HA ternary system whose transfection efficiency decreases significantly with increasing serum concentration, PoSC exhibits a high transfection efficiency of about 51 and 87% for NIH3T3 and HCT116 cells, respectively, at 50% serum concentration. Furthermore, PoSC shows no cytotoxic effect at its working concentration. The overall results suggest that HA adsorption on cationic complexes enhances the transfection efficiency, while PSP is essential for high transfection efficiency at higher serum concentration.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  14. 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

  15. AAV2-mediated gene delivery to monkey putamen: Evaluation of an infusion device and delivery parameters

    PubMed Central

    Sanftner, Laura M.; Sommer, Jurg M.; Suzuki, Brian M.; Smith, Peter H.; Vijay, Sharmila; Vargas, Joseph A.; Forsayeth, John R.; Cunningham, Janet; Bankiewicz, Krys S.; Kao, Haihwa; Bernal, Jan; Pierce, Glenn F.; Johnson, Kirk W.

    2013-01-01

    In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell® clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic L-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson&apos ;s disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 μL/min to 1μL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 μL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated. PMID:16022872

  16. 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

  17. 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.

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

    PubMed

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

    2015-03-02

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

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

    PubMed

    Frenkel, Victor

    2008-06-30

    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.

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

    PubMed

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

    2014-06-01

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

  1. Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

    PubMed Central

    Fein, David E.; Bucki, Robert; Byfield, Fitzroy; Leszczynska, Katarzyna; Janmey, Paul A.

    2010-01-01

    Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D2S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D2S. D2S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at ∼5 μM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D2S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D2S can improve transfection activity over common lipofection reagents, and D2S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation. PMID:20573781

  2. Gene Delivery Nanoparticles Fabricated by Supercritical Fluid Extraction of Emulsions

    PubMed Central

    Mayo, Aaron S.; Ambati, Balamurali K.; Kompella, Uday B.

    2010-01-01

    Non-viral polymeric gene delivery systems offer increased protection from nuclease degradation, enhanced plasmid DNA (pDNA) uptake, and controlled dosing to sustain the duration of pDNA administration. Such gene delivery systems can be formulated from biocompatible and biodegradable polymers such as poly (d,l-lactic-co-glycolic) acid (PLGA). Experimental loading of hydrophilic macromolecules such as pDNA is low in polymeric particles. The study purpose was to develop a supercritical fluid extraction of emulsions (SFEE) process based on CO2 for preparing pEGFP-PLGA nanoparticles with high plasmid loading and loading efficiency. Another objective was to determine the efficacy of pFlt23k, an anti-angiogenic pDNA capable of inhibiting vascular endothelial growth factor (VEGF) secretion, following nanoparticle formation using the SFEE process. Results indicated that the SFEE process allows high actual loading of pDNA (19.7% w/w), high loading efficiency (> 98%), and low residual solvents (< 50 ppm), due to rapid particle formation from efficient solvent removal provided by the SFEE process. pFlt23K-PLGA nanoparticles were capable of in vitro transfection, significantly reducing secreted VEGF from human lung alveolar epithelial cells (A549) under normoxic and hypoxic conditions. pFlt23K-PLGA nanoparticles did not exhibit cytotoxicity and are of potential value in treating neovascular disorders wherein VEGF levels are elevated. PMID:20025945

  3. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy

    DTIC Science & Technology

    2016-09-01

    To test the safety and efficacy of somatostatin gene delivery as a potential therapeutic approach to epilepsy, an established rodent model is used in...reliably. Animals tested during the reporting period establish that somatostatin gene delivery after development of maximal seizure susceptibility can...observed when gene delivery preceded kindling, but comparable to extant antiepileptic medication. Responder and non-responder cohorts cannot be

  4. Characterization of reducible peptide oligomers as carriers for gene delivery.

    PubMed

    Kiselev, Anton; Egorova, Anna; Laukkanen, Antti; Baranov, Vladislav; Urtti, Arto

    2013-01-30

    The stability of DNA-polyplexes and intracellular DNA release are important features of gene delivery systems. To study these features, we have evaluated reducible cysteine-flanked linear lysine and arginine-rich peptides, modified with histidine residues. The reducible disulfide bonds in cysteine flanked peptides and histidine residues should augment DNA release from the peptide-DNA complexes upon disintegration of the reducible bonds. Template polymerization and oxidative polycondensation were applied to obtain peptide oligomers used for DNA-polyplex preparation. The peptides and DNA-peptide complexes were investigated with physical, chemical and transfection measurements. Physicochemical and transfection properties of DNA-polyplexes depended on the amino acid sequence of the peptidic polymers and type of the polymerization. MALDI-TOF analysis of oxidatively polycondensed products revealed several forms of peptide oligomers corresponding to 5-8 amino acid monomers. DNA-peptide particles based on template-polymerized complexes were more resistant to relaxation by negatively charged heparan sulfate than polyplexes formed with oxidatively condensed peptides. Complexes of DNA with the polycations prepared by oxidative polycondensation exhibited a 100-1000-fold higher level of gene expression compared to DNA/template-polymerized peptide complexes. The most efficient transgene expression was shown with arginine-rich polyplexes. Transfection efficacy of the arginine-rich polyplexes was even 10-fold better than that of DNA/PEI complexes. On average, polyplexes based on cysteine-flanked peptide oligomers showed lower cytotoxicity than non-reducible high molecular weight polylysine/DNA particles. We conclude that reducible peptide oligomers provide efficient DNA transfection and have the potential as vehicles for gene delivery.

  5. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

    PubMed

    Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

    2014-01-01

    Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

  6. 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

  7. Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery.

    PubMed

    Jang, J-H; Koerber, J T; Gujraty, K; Bethi, S R; Kane, R S; Schaffer, D V

    2010-11-01

    Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

  8. Strategies for retargeted gene delivery using vectors derived from lentiviruses.

    PubMed

    Bartosch, Birke; Cosset, Francois-Loic

    2004-12-01

    With the development of the first viral vector systems 20 years ago [Mann et al., 1983; Watanabe and Temin, 1983] gene therapy strategies have come to the forefront of novel therapeutics [Cavazzana-Calvo et al., 2000]. A deeper understanding of vector biology and the molecular mechanisms of disease alongside tremendous advances in vector technology have significantly advanced the field of human gene therapy. Over the last few years several challenges needed to be overcome in order to bring gene therapy strategies closer to the clinic. These hurdles include the preparation of large amounts of stable, high titre vectors, minimising vector-related immunology and last but not least targeting infection and transgene expression to tissue or cells, which in many cases are not or only slowly dividing. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Moreover, lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle thus opening up the possibility to target non-dividing target cells and tissues. Human immunodeficiency virus (HIV) based vectors have been used in vitro and in vivo in a number of situations, however, safety concerns still exist, and therefore the development of vector systems based on primate as well as non-primate lentiviruses is ongoing. Concomitantly with lentiviral vector design, much has been learned about the incorporation of heterologous env proteins on lentiviral cores in order to combine specific targeting properties of envelope glycoproteins with the biological properties of lentiviral vectors. In this review article we will give an overview over advantages lentiviral vector systems offer. We will then discuss the current state of our understanding of the structure and function of viral

  9. Cyclen-based lipidic oligomers as potential gene delivery vehicles.

    PubMed

    Yi, Wen-Jing; Zhang, Qin-Fang; Zhang, Ji; Liu, Qiang; Ren, Laifeng; Chen, Qian-Ming; Guo, Liandi; Yu, Xiao-Qi

    2014-03-01

    A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency.

  10. Magnetofection: A Reproducible Method for Gene Delivery to Melanoma Cells

    PubMed Central

    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

  11. 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

  12. A novel gene delivery system for mammalian cells.

    PubMed

    Gibson, Brian; Duffy, Angela M; Gould Fogerite, Susan; Krause-Elsmore, Sara; Lu, Ruying; Shang, Gaofeng; Chen, Zi-Wei; Mannino, Raphael J; Bouchier-Hayes, David J; Harmey, Judith H

    2004-01-01

    Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

  13. 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.

  14. Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

    PubMed

    Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

    2017-07-01

    Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

  15. Identification of a developmental gene expression signature, including HOX genes, for the normal human colonic crypt stem cell niche: overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis.

    PubMed

    Bhatlekar, Seema; Addya, Sankar; Salunek, Moreh; Orr, Christopher R; Surrey, Saul; McKenzie, Steven; Fields, Jeremy Z; Boman, Bruce M

    2014-01-15

    Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

  16. Biological delivery approaches for gene therapy: strategies to potentiate efficacy and enhance specificity.

    PubMed

    Mohit, Elham; Rafati, Sima

    2013-12-01

    Nowadays many therapeutic agents such as suicide genes, anti-angiogenesis agents, cytokines, chemokines and other therapeutic genes were delivered to cancer cells. Various biological delivery systems have been applied for directing therapeutic gene to target cells. Some of these successful preclinical studies, steps forward to clinical trials and a few are examined in phase III clinical trials. In this review, the biological gene delivery systems were categorized into microorganism and cell based delivery systems. Viral, bacterial, yeast and parasite are among microorganism based delivery systems which are expanded in this review. In cell based approach, different strategies such as tumor cells, stem cells, dendritic cells and sertoli cells will be discussed. Different drawbacks are associated with each delivery system; therefore, many strategies have been improved and potentiated their direction toward specific target cells. Herein, further to the principle of each delivery system, the progresses of these approaches for development of newer generation are discussed.

  17. 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.

  18. 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

  19. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    PubMed

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  20. In vitro non-viral gene delivery with nanofibrous scaffolds

    PubMed Central

    Liang, Dehai; Luu, Yen K.; Kim, Kwangsok; Hsiao, Benjamin S.; Hadjiargyrou, Michael; Chu, Benjamin

    2005-01-01

    Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (β-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1× TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro. PMID:16269820

  1. Lentivirus-Based Stable Gene Delivery into Intestinal Organoids.

    PubMed

    Maru, Yoshiaki; Orihashi, Kaoru; Hippo, Yoshitaka

    2016-01-01

    Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.

  2. Nonionic polymeric micelles for oral gene delivery in vivo.

    PubMed

    Chang, Shwu-Fen; Chang, Han-Yi; Tong, Yaw-Chong; Chen, Sy-Hann; Hsaio, Fei-Chin; Lu, Shao-Chun; Liaw, Jiahorng

    2004-05-01

    The main aim of this study was to investigate the feasibility of using nonionic polymeric micelles of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. The size and appearance of DNA/PEO-PPO-PEO polymeric micelles were examined, respectively, by dynamic light scattering and atomic force microscopy, and their zeta potential was measured. Expression of the delivered lacZ gene in various tissues of nude mice was assessed qualitatively by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of sections and quantitatively by measuring enzyme activity in tissue extracts, using the substrate of beta-galactosidase, chlorophenol red-beta-D-galactopyranoside. In addition, the types of cells expressing the lacZ gene in the duodenum were identified by histological analysis. DNA/PEO-PPO-PEO polymeric micelles are a single population of rounded micelles with a mean diameter of 170 nm and a zeta potential of -4.3 mV. Duodenal penetration of DNA/PEO-PPO-PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 +/- 0.37) x 10(-5) cm/sec at low DNA concentrations (0.026-0.26 microg/microl), but a decrease to (2.89 +/- 0.37) x 10(-5) cm/sec at a concentration of 1.3 microg/microl. Furthermore, when 10 mM RGD peptide or 10 mM EDTA was administered before and concurrent with the administration of DNA/PEO-PPO-PEO polymeric micelles, transport was inhibited ([0.95 +/- 0.57] x 10(-5) cm/sec) by blocking endocytosis or enhanced ([29.8 +/- 5.7] x 10(-5) cm/sec) by opening tight junctions, respectively. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was

  3. 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.

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

    PubMed

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

    2016-11-28

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

  5. Analysis of novel nonviral gene transfer systems for gene delivery to cells of the musculoskeletal system.

    PubMed

    Orth, Patrick; Weimer, Anja; Kaul, Gunter; Kohn, Dieter; Cucchiarini, Magali; Madry, Henning

    2008-02-01

    The aim of the present study was to evaluate the efficacy of novel nonviral gene delivery systems in cells of musculoskeletal origin. Primary cultures of lapine skeletal muscle cells, lapine articular chondrocytes, human cells from fibrous dysplasia and cell lines established from human osteosarcoma (SAOS-2), chondrosarcoma (CS-1), murine skeletal myoblasts (L8) and fibroblasts (NIH 3T3) were transfected with the P. pyralis luc or the E. coli lacZ genes using Nanofectin 1 and 2, Superfect, JetPEI, GeneJammer, Effectene, TransPass D2, FuGENE 6, Lipofectamine 2000, Dreamfect, Metafectene, Escort III, and calcium phosphate. Maximal transfection efficiency in lapine skeletal muscle cells was of 60.8 +/- 21.2% using Dreamfect, 38.9 +/- 5.0% in articular chondrocytes using Gene Jammer, 5.2 +/- 8.0% in human cells from fibrous dysplasia using Lipofectamine 2000, 12.7 +/- 16.2% in SAOS-2 cells using FuGENE 6, 29.9 +/- 3.5% in CS-1 cells using Lipofectamine 2000, 70.7 +/- 8.6% in L8 cells using FuGENE 6, and 48.9 +/- 13.0% in NIH 3T3 cells using Metafectene. When the cells were transfected with a human IGF-I gene, significant amounts of the IGF-I protein were secreted. These results indicate that relatively high levels of transfection can be achieved using novel nonviral gene transfer methods.

  6. Comparison of two kinds of nanomedicine for targeted gene therapy: premodified or postmodified gene delivery systems

    PubMed Central

    Jiang, Zhaoshun; Sun, Cong; Yin, Zhaohui; Zhou, Fang; Ge, Linfu; Liu, Ximin; Kong, Fansheng

    2012-01-01

    Background The applications of ligand-polyethylene glycol (PEG)-modified nanocarriers have now emerged, as well as recognized strategies to provide the vectors with active targeting properties. In this research, premodification and postmodification were compared using the same ligand, ie, a novel conjugated mannan-containing PEG and L-α-phosphatidylethanolamine (PE). Methods Premodified and postmodified solid lipid nanoparticles were prepared and the characteristics of the two kinds of vehicles were evaluated. The modified vectors were then administered intravenously to rats and the in vivo targeting behavior of the complexes was investigated in liver macrophages. Results By carefully formulating the carriers with an optimal ratio of mannan-containing PEG-PE, postmodified vehicles displayed more efficient gene expression in rat Kupffer cells both in vitro and in vivo. Conclusion Postmodified gene carriers are superior to premodified gene vectors, although the latter is also promising for targeted gene delivery. This discovery could guide our future research. PMID:22619539

  7. 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

  8. Molecular Recognition Enables Nanosubstrate-Mediated Delivery of Gene-Encapsulated Nanoparticles with High Efficiency

    PubMed Central

    2015-01-01

    Substrate-mediated gene delivery is a promising method due to its unique ability to preconcentrate exogenous genes onto designated substrates. However, many challenges remain to enable continuous and multiround delivery of the gene using the same substrates without depositing payloads and immobilizing cells in each round of delivery. Herein we introduce a gene delivery system, nanosubstrate-mediated delivery (NSMD) platform, based on two functional components with nanoscale features, including (1) DNA⊂SNPs, supramolecular nanoparticle (SNP) vectors for gene encapsulation, and (2) Ad-SiNWS, adamantane (Ad)-grafted silicon nanowire substrates. The multivalent molecular recognition between the Ad motifs on Ad-SiNWS and the β-cyclodextrin (CD) motifs on DNA⊂SNPs leads to dynamic assembly and local enrichment of DNA⊂SNPs from the surrounding medium onto Ad-SiNWS. Subsequently, once cells settled on the substrate, DNA⊂SNPs enriched on Ad-SiNWS were introduced through the cell membranes by intimate contact with individual nanowires on Ad-SiNWS, resulting in a highly efficient delivery of exogenous genes. Most importantly, sequential delivery of multiple batches of exogenous genes on the same batch cells settled on Ad-SiNWS was realized by sequential additions of the corresponding DNA⊂SNPs with equivalent efficiency. Moreover, using the NSMD platform in vivo, cells recruited on subcutaneously transplanted Ad-SiNWS were also efficiently transfected with exogenous genes loaded into SNPs, validating the in vivo feasibility of this system. We believe that this nanosubstrate-mediated delivery platform will provide a superior system for in vitro and in vivo gene delivery and can be further used for the encapsulation and delivery of other biomolecules. PMID:24708312

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

    PubMed Central

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

    2016-01-01

    Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review. PMID:28228799

  10. Functional magnetic nanoparticles for non-viral gene delivery and MR imaging.

    PubMed

    Xing, Ruijun; Liu, Gang; Zhu, Jinghan; Hou, Yanglong; Chen, Xiaoyuan

    2014-06-01

    Gene therapy is becoming a promising strategy to treat various kinds of genetic and acquired diseases. However, the development of safe, efficient, and targetable gene delivery systems remains a major challenge in gene therapy. The unique material characteristics of magnetic nanoparticles (MNPs), including high surface area, facile surface modification, controllable size, and excellent magnetic properties, make them promising candidates for gene delivery. The engineered MNPs with modifiable functional surfaces and bioactive cores can result in several advantageous diagnostic and therapeutic properties including enhanced magnetic resonance imaging (MRI) signal intensity, long permeation and retention in the circulatory system, specific delivery of therapeutic genes to target sites. In this review, the updated research on the preparation and surface modification of MNPs for gene delivery is summarized.

  11. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?

    PubMed Central

    Mendes, Rita; Fernandes, Alexandra R.; Baptista, Pedro V.

    2017-01-01

    Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA) sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs) have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics. PMID:28257109

  12. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer-The Case for Combined Delivery?

    PubMed

    Mendes, Rita; Fernandes, Alexandra R; Baptista, Pedro V

    2017-03-02

    Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA) sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs) have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

  13. 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.

  14. Ultrasound-targeted HSVtk and Timp3 gene delivery for synergistically enhanced antitumor effects in hepatoma.

    PubMed

    Yu, B-F; Wu, J; Zhang, Y; Sung, H-W; Xie, J; Li, R-K

    2013-05-01

    Cancer gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited by non-targeted and insufficient gene transfer. We evaluated gene therapy targeting hepatocellular carcinoma (HCC) using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system and the tissue inhibitor of metalloproteinase 3 (Timp3) gene. Ultrasound-targeted microbubble destruction (UTMD) targeted gene delivery to the tumor tissue, and the α-fetoprotein promoter targeted HSVtk expression to the HCC cells. Human HepG2 cells transfected with the HSVtk or Timp3 gene demonstrated a reduction in cell viability by >40% compared with the vector control. Cell viability was further inhibited by over 50% with co-transfection of the genes. HepG2 cells were inoculated subcutaneously into athymic mice to induce tumors. UTMD-mediated delivery of HSVtk or Timp3 suppressed tumor growth by >45% and increased survival of tumor-bearing animals (P<0.01 vs vector control). Co-delivery of the genes resulted in a further 30% improvement in tumor suppression and significant extension of animal survival (P<0.01 vs vector control). Targeted gene delivery increased the number of apoptotic cells and decreased the vascular density of the tumors. Targeted co-delivery of the genes synergistically improved the antitumor effects and may provide an effective therapy for HCC.

  15. Temporally controlled multiple-gene delivery in scaffolds: A promising strategy to enhance bone regeneration.

    PubMed

    Liu, Jinsong; Xu, Lihua; Li, Yiming; Ma, Jianfeng

    2011-02-01

    Bone defects sometimes require more effective repair regimens than conventional clinical therapies can provide. On account of this, tissue-engineered scaffolds have emerged as a promising alternative. Scaffolds that release genes encoding growth factors (GFs) offer additional benefits for bone regeneration in comparison with scaffolds providing protein delivery. The present gene delivery systems focus on unitary or dual genes delivery without controlled release. In the meantime, evidences indicate that bone formation is a complex cascade of events, in which time-dependent expression of multiple growth factors is involved. In our hypothesis, a temporally controlled, multi-gene delivery system embedded in a scaffold matrix can be fabricated; such a system is capable of mimicking the expression of growth factor profile in osteogenesis. Consequently, bone regeneration can be promoted by sequential gene expression of multiple growth factors.

  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. Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions

    PubMed Central

    Kamimura, Kenya; Yokoo, Takeshi; Abe, Hiroyuki; Kobayashi, Yuji; Ogawa, Kohei; Shinagawa, Yoko; Inoue, Ryosuke; Terai, Shuji

    2015-01-01

    Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model for experimental research. Recent progress in the development of image-guided procedure for hydrodynamics-based gene delivery in large animals directly supports the clinical applicability of this technique. This review summarizes the current status and recent progress in the development of hydrodynamics-based gene delivery and discusses the future directions for its clinical application. PMID:26308044

  18. Baculovirus as a gene delivery vector for cartilage and bone tissue engineering.

    PubMed

    Lin, Chin-Yu; Lu, Chia-Hsin; Luo, Wen-Yi; Chang, Yu-Han; Sung, Li-Yu; Chiu, Hsin-Yi; Hu, Yu-Chen

    2010-06-01

    Baculovirus is an effective vector for gene delivery into various mammalian cells, including chondrocytes and mesenchymal stem cells, and has been employed for diverse applications. By gene delivery and expression of the growth factor, recombinant baculovirus has been shown to modulate the differentiation state of the cells and stimulates the production of extracellular matrix and tissue formation, hence repairing the damaged cartilage and bone in vivo. This article reviews the studies pertaining to the applications of baculovirus-mediated gene delivery in cartilage and bone tissue engineering and discusses recent progress, future applications and potential hurdles.

  19. 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

  20. Nonviral gene delivery to mesenchymal stem cells using cationic liposomes for gene and cell therapy.

    PubMed

    Madeira, C; Mendes, R D; Ribeiro, S C; Boura, J S; Aires-Barros, M R; da Silva, C L; Cabral, J M S

    2010-01-01

    Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from approximately 2% to approximately 35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and approximately 20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

  1. 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

  2. In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges.

    PubMed

    Mout, Rubul; Ray, Moumita; Lee, Yi-Wei; Scaletti, Federica; Rotello, Vincent M

    2017-03-17

    The successful use of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based gene editing for therapeutics requires efficient in vivo delivery of the CRISPR components. There are, however, major challenges on the delivery front. In this Topical Review, we will highlight recent developments in CRISPR delivery, and we will present hurdles that still need to be overcome to achieve effective in vivo editing.

  3. 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

  4. Factors affecting drug and gene delivery: effects of interaction with blood components.

    PubMed

    Opanasopit, Praneet; Nishikawa, Makiya; Hashida, Mitsuru

    2002-01-01

    Targeted drug delivery systems have been used extensively to improve the pharmacological and therapeutic activities of a wide variety of drugs and genes. In this article, we summarize the factors determining the tissue disposition of delivery systems: the physicochemical and biological characteristics of the delivery system and the anatomic and physiological characteristics of the tissues. There are several modes of drug and gene targeting, ranging from passive to active targeting, and each of these can be achieved by optimizing the design of the delivery system to suit a specific aim. After entering the systemic circulation, either by an intravascular injection or through absorption from an administration site, however, a delivery system encounters a variety of blood components, including blood cells and a range of serum proteins. These components are by no means inert as far as interaction with the delivery system is concerned, and they can sometimes markedly effect its tissue disposition. The interaction with blood components is known to occur with particulate delivery systems, such as liposomes, or with cationic charge-mediated delivery systems for genes. In addition to these rather nonspecific ones, interactions via the targeting ligand of the delivery system can occur. We recently found that mannosylated carriers interact with serum mannan binding protein, greatly altering their tissue disposition in a number of ways that depend on the properties of the carriers involved.

  5. Poly(beta-amino esters): procedures for synthesis and gene delivery.

    PubMed

    Green, Jordan J; Zugates, Gregory T; Langer, Robert; Anderson, Daniel G

    2009-01-01

    Non-viral gene delivery systems are promising as they avoid many problems of viral gene therapy by having increased design flexibility, high safety, large DNA cargo capacity, and ease of manufacture. Here, we describe the use of polymeric vectors, in particular biodegradable poly(beta-amino esters) (PBAEs), for non-viral gene delivery. These polymers are able to self-assemble with DNA and form positively charged gene delivery nanoparticles. Methods for synthesis of these polymers, particle self-assembly, and transfection using these particles are delineated. A standard protocol is presented as well as a high-throughput screening technique that can be used to more quickly optimize transfection parameters for efficient delivery.

  6. 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

  7. 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.

  8. Myocardial gene transfer by selective pressure-regulated retroinfusion of coronary veins: comparison with surgical and percutaneous intramyocardial gene delivery.

    PubMed

    Raake, Philip; von Degenfeld, Georges; Hinkel, Rabea; Vachenauer, Robert; Sandner, Torleif; Beller, Sabrina; Andrees, Martin; Kupatt, Christian; Schuler, Gerhard; Boekstegers, Peter

    2004-09-01

    We sought to study adenoviral gene delivery using percutaneous selective pressure-regulated retroinfusion and to compare it directly with surgical and percutaneous intramyocardial delivery (PIMD) for the first time. Intramyocardial delivery (IMD) has been recommended to be the preferred gene delivery strategy so far. However, surgical and percutaneous intramyocardial injection lead to incomplete retention of the injected viral vectors and to limited spatial myocardial distribution. Percutaneous selective pressure-regulated retroinfusion of the coronary veins was developed recently to provide an effective and more homogenous regional myocardial gene transfer. In 15 pigs, adenoviral vectors (Ad2-CMV beta-galactosidase [beta-gal] 5 x 10(9) pfu) were applied via surgical IMD (n = 5), PIMD (n = 5), and selective pressure-regulated retroinfusion (n = 5). Seven days after gene transfer, myocardial beta-gal expression was measured by ELISA. Selective retroinfusion into the anterior cardiac vein substantially increased reporter gene expression (1,039 +/- 79 pg beta-gal/mg protein) in the targeted left anterior descending coronary artery territory when compared with surgical (448 +/- 127, p < 0.05) and PIMD (842 +/- 145, p < 0.05). Both IMD approaches showed an inhomogenous beta-gal expression, particularly along the injection sites, while retroinfusion resulted in a more homogenous transmural gene expression. Percutaneous selective pressure-regulated retroinfusion compares favorably with surgical and percutaneous intramyocardial injection techniques by providing a more homogenous and even more efficient adenoviral gene delivery.

  9. A visible, targeted high-efficiency gene delivery and transfection strategy

    PubMed Central

    2011-01-01

    Background To enhance myocardial angiogenic gene expression, a novel gene delivery strategy was tested. Direct intramyocardial injection of an angiogenic gene with microbubbles and insonation were applied in a dog animal model. Dogs received one of the four different treatments in conjunction with either the enhanced green fluorescence protein (EGFP) gene or the hepatocyte growth factor (HGF) gene: gene with microbubbles (MB) and ultrasound (US); gene with US; gene with MB; or the gene alone. Results Distribution of MB and the gene in the myocardium was visualized during the experiment. Compared with the EGFP gene group, an average 14.7-fold enhancement in gene expression was achieved in the EGFP+MB/US group (P < 0.01). Compared with the HGF gene group, an average 10.7-fold enhancement in gene expression was achieved in the HGF+MB/US group (P < 0.01). In addition, capillary density increased from 20.8 ± 3.4/mm2 in the HGF gene group to 146.7 ± 31.4/mm2 in HGF+MB/US group (P < 0.01). Conclusions Thus, direct intramyocardial injection of an angiogenic gene in conjunction with microbubbles plus insonation synergistically enhances angiogenesis. This method offers an observable gene delivery procedure with enhanced expression efficiency of the delivered gene. PMID:21600027

  10. 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

  11. Electroporation-mediated delivery of genes in rodent models of lung contusion.

    PubMed

    Machado-Aranda, David; Raghavendran, Krishnan

    2014-01-01

    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.

  12. 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

  13. A promising gene delivery system developed from PEGylated MoS2 nanosheets for gene therapy

    PubMed Central

    2014-01-01

    A new class of two-dimensional (2D) nanomaterial, transition metal dichalcogenides (TMDCs) such as MoS2, MoSe2, WS2, and WSe2 which have fantastic physical and chemical properties, has drawn tremendous attention in different fields recently. Herein, we for the first time take advantage of the great potential of MoS2 with well-engineered surface as a novel type of 2D nanocarriers for gene delivery and therapy of cancer. In our system, positively charged MoS2-PEG-PEI is synthesized with lipoic acid-modified polyethylene glycol (LA-PEG) and branched polyethylenimine (PEI). The amino end of positively charged nanomaterials can bind to the negatively charged small interfering RNA (siRNA). After detection of physical and chemical characteristics of the nanomaterial, cell toxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polo-like kinase 1 (PLK1) was investigated as a well-known oncogene, which was a critical regulator of cell cycle transmission at multiple levels. Through knockdown of PLK1 with siRNA carried by novel nanovector, qPCR and Western blot were used to measure the interfering efficiency; apoptosis assay was used to detect the transfection effect of PLK1. All results showed that the novel nanocarrier revealed good biocompatibility, reduced cytotoxicity, as well as high gene-carrying ability without serum interference, thus would have great potential for gene delivery and therapy. PMID:25386104

  14. Development of Liposomal Bubbles with Perfluoropropane Gas as Gene Delivery Carriers

    NASA Astrophysics Data System (ADS)

    Maruyama, Kazuo; Suzuki, Ryo; Sawamura, Kaori; Takizawa, Tomoko; Utoguchi, Naoki; Negishi, Yoichi

    2007-05-01

    Liposomes have some advantages as drug, antigen and gene delivery carriers. Their size can be easily controlled and they can be modified to add a targeting function. Based on liposome technology, we developed novel liposomal bubbles (Bubble liposomes) containing the ultrasound imaging gas, perfluoropropane. We assessed the feasibility of Bubble liposomes as carriers for gene delivery after cavitation induced by ultrasound. At first, we investigated their ability to deliver genes with Bubble liposomes and ultrasound to various types of cells such as mouse sarcoma cells, mouse melanoma cells, human T cell line and human umbilical vein endothelial cells. The results showed that the Bubble liposomes could deliver plasmid DNA to many cell types without cytotoxicity. In addition, we found that Bubble liposomes could effectively deliver plasmid DNA into mouse femoral artery in vivo. The gene transduction with Bubble liposomes was more effectively than conventional lipofection. We conclude that Bubble liposomes are unique and efficient gene delivery carriers in vitro and in vivo.

  15. 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.

  16. 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

  17. Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery.

    PubMed

    Joshi, Chaitanya R; Labhasetwar, Vinod; Ghorpade, Anuja

    2017-03-01

    Neurological diseases and disorders (NDDs) present a significant societal burden and currently available drug- and biological-based therapeutic strategies have proven inadequate to alleviate it. Gene therapy is a suitable alternative to treat NDDs compared to conventional systems since it can be tailored to specifically alter select gene expression, reverse disease phenotype and restore normal function. The scope of gene therapy has broadened over the years with the advent of RNA interference and genome editing technologies. Consequently, encouraging results from central nervous system (CNS)-targeted gene delivery studies have led to their transition from preclinical to clinical trials. As we shift to an exciting gene therapy era, a retrospective of available literature on CNS-associated gene delivery is in order. This review is timely in this regard, since it analyzes key challenges and major findings from the last two decades and evaluates future prospects of brain gene delivery. We emphasize major areas consisting of physiological and pharmacological challenges in gene therapy, function-based selection of a ideal cellular target(s), available therapy modalities, and diversity of viral vectors and nanoparticles as vehicle systems. Further, we present plausible answers to key questions such as strategies to circumvent low blood-brain barrier permeability and most suitable CNS cell types for targeting. We compare and contrast pros and cons of the tested viral vectors in the context of delivery systems used in past and current clinical trials. Gene vector design challenges are also evaluated in the context of cell-specific promoters. Key challenges and findings reported for recent gene therapy clinical trials, assessing viral vectors and nanoparticles are discussed from the perspective of bench to bedside gene therapy translation. We conclude this review by tying together gene delivery challenges, available vehicle systems and comprehensive analyses of

  18. Portal vein delivery of viral vectors for gene therapy for hemophilia.

    PubMed

    Sherman, Alexandra; Schlachterman, Alexander; Cooper, Mario; Merricks, Elizabeth P; Raymer, Robin A; Bellinger, Dwight A; Herzog, Roland W; Nichols, Timothy C

    2014-01-01

    The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B. Although there are multiple delivery routes for both viral and non-viral vectors in animals, portal vein administration delivers vectors directly and efficiently into the liver. Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy.

  19. 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

  20. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  1. 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

  2. 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.

  3. Cationic liposome–nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing

    PubMed Central

    Majzoub, Ramsey N.

    2016-01-01

    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

  4. 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...

  5. Transplantation of novel vascular endothelial growth factor gene delivery system manipulated skeletal myoblasts promote myocardial repair.

    PubMed

    Zhu, Kai; Guo, Changfa; Xia, Yu; Lai, Hao; Yang, Wuli; Wang, Yang; Song, Dongli; Wang, Chunsheng

    2013-10-03

    Skeletal myoblast (SkM) transplantation combined with vascular endothelial growth factor (VEGF) gene delivery has been proposed as a promising therapy for cardiac repair. Nevertheless, the defective gene vectors and unregulable VEGF expression in vivo hinder its application. Therefore, the search for an economical, effective, controllable gene delivery system is quite necessary. In our study, hyperbranched polyamidoamine (h-PAMAM) dendrimer was synthesized as a novel gene delivery vector using a modified method. And hypoxia-regulated human VEGF-165 plasmids (pHRE-hVEGF165) were constructed for controllable VEGF gene expression. The efficiency and feasibility of h-PAMAM-HRE-hVEGF165 gene delivery system manipulated SkM transplantation for cardiac repair were investigated in myocardial infarction models. The h-PAMAM encapsulated pHRE-hVEGF165 could resist nuclease digestion for over 120 min. In primary SkMs, h-PAMAM-pHRE-hVEGF165 gene delivery system showed high transfection efficiency (43.47 ± 2.22%) and minor cytotoxicity (cell viability = 91.38 ± 0.48%). And the transfected SkMs could express hVEGF165 for 18 days under hypoxia in vitro. For myocardial infarction models, intramyocardial transplantation of the transfected SkMs could result in reduction of apoptotic myocardiocytes, improvement of grafted cell survival, decrease of infarct size and interstitial fibrosis, and increase of blood vessel density, which inhibited left ventricle remodeling and improved heart function at the late phase following infarction. These results indicate that h-PAMAM based pHRE-hVEGF165 gene delivery into SkMs is feasible and effective, and may serve as a novel and promising gene therapy strategy in ischemic heart disease. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. 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

  7. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2008-12-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  8. 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.

  9. 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

  10. Mucus Penetrating Nanoparticles: Biophysical Tool and Method of Drug and Gene Delivery

    PubMed Central

    Ensign, Laura M.; Schneider, Craig; Suk, Jung Soo; Cone, Richard; Hanes, Justin

    2013-01-01

    A method that could provide more uniform and longer-lasting drug and gene delivery to mucosal surfaces holds the potential to greatly improve the effectiveness of prophylactic and therapeutic approaches for numerous diseases and conditions, including sexually transmitted infections, cystic fibrosis, chronic rhinosinusitis, inflammatory bowel disease, and glaucoma to name a few. However, the body's natural defenses, including adhesive, rapidly cleared mucus linings coating nearly all entry points to the body not covered by skin, has limited the effectiveness of drug and gene delivery by nanoscale delivery systems. This article discusses the recent development of the “mucus-penetrating particle” or “MPP” nanotechnology, and how it has been used to both enhance understanding of the nanoscale barrier properties of human mucus secretions, and to achieve more uniform and longer-lasting drug delivery to mucosal tissues following topical administration. Drug loaded MPPs possess non-adhesive coatings that allow them to rapidly penetrate mucus layers through openings in the mucus mesh at rates nearly as fast as they would penetrate pure water. Critically, MPPs allow enhanced drug and gene delivery to mucosal tissues without diminishing the protective function of mucus. Recent progress in the development of MPPs as a biophysical tool to probe the length-scale dependent rheological properties of mucosal secretions and as a method for drug and gene delivery is highlighted. PMID:22988559

  11. 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

  12. 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

  13. Ultrasound for drug and gene delivery to the brain.

    PubMed

    Hynynen, Kullervo

    2008-06-30

    Noninvasive, transient, and local image-guided blood-brain barrier disruption (BBBD) has been demonstrated with focused ultrasound exposure in animal models. Most studies have combined low pressure amplitude and low time average acoustic power burst sonications with intravascular injection of pre-formed micro-bubbles to produce BBBD without damage to the neurons. The BBB has been shown to be healed within a few hours after the exposure. The combination of focused ultrasound beams with MR image guidance allows precise anatomical targeting as demonstrated by the delivery of several marker molecules in different animal models. This method may in the future have a significant impact on the diagnosis and treatment of central nervous system (CNS) disorders. Most notably, the delivery of the chemotherapy agents (liposomal Doxorubicin and Herceptin) has been shown in a rat model.

  14. 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.

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

    PubMed

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

    2014-04-01

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

  16. Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals.

    PubMed

    Dean, David A

    2013-10-01

    The use of electroporation to facilitate gene transfer is an extremely powerful and useful method for both in vitro and in vivo applications. One of its great strengths is that it induces functional destabilization and permeabilization of cell membranes throughout a tissue leading to widespread gene transfer to multiple cells and cell types within the electric field. While this is a strength, it can also be a limitation in terms of cell-specific gene delivery. The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. At present, there are relatively few ways to obtain cell-specific targeting of nonviral vectors, molecular probes, small molecules, and imaging agents. We have developed a novel means of restricting gene delivery to desired cell types based on the ability to control the transport of plasmids into the nuclei of desired cell types. In this article, we discuss the mechanisms of this approach and several applications in living animals to demonstrate the benefits of the combination of electroporation and selective nuclear import of plasmids for cell-specific gene delivery.

  17. 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.

  18. 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.

  19. 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

  20. Development of a tailorable and tunable mechanism for cell-responsive substrate-mediated gene delivery

    NASA Astrophysics Data System (ADS)

    Blocker, Kory M.

    Due to the spatial and temporal control as well as the cell-type specificity necessary to extend gene delivery to therapeutic applications, there exists a need to create systems capable of gene transfer that are well-understood and easily manipulated. Furthermore, the creation of such materials will enable further exploration of the correlation between biochemical cues and the resulting cellular responses. In response to this as yet unmet need, a method to promote cell-responsive substrate-mediated gene delivery was developed for this dissertation. Through the use of non-viral gene delivery, flexibility of the vehicle design was incorporated into the system. Using PNA technology, pDNA was able to be specifically tethered to a self-assembled monolayer via an enzymatically-labile peptide tether. This construct was shown to promote cell-responsive delivery while retaining flexibility over the chemical and physical properties of the vehicle and substrate. By alteration of some design parameters including tether number, pDNA surface coverage, and complexation agent, temporal control over the release profile was demonstrated. Furthermore, the ability to extend the applicability of the system was detailed by transitioning to a poly-D-lysine coated substrate upon which the pDNA is immobilized. This dissertation details proof-of-principle work in the formation of a controlled release gene delivery mechanism that may be used to promote understanding of cellular responses to biochemical signaling as well as be extended to use in tissue engineering applications.

  1. Gene transfer to hemophilia A mice via oral delivery of FVIII-chitosan nanoparticles.

    PubMed

    Bowman, Katherine; Sarkar, Rita; Raut, Sanj; Leong, Kam W

    2008-12-18

    Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.

  2. 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

  3. Effect of strontium ions substitution on gene delivery related properties of calcium phosphate nanoparticles.

    PubMed

    Hanifi, A; Fathi, M H; Mir Mohammad Sadeghi, H

    2010-09-01

    Gene therapy has been considered a strategy for delivery of therapeutic nucleic acids to a specific site. Calcium phosphates are one gene delivery vector group of interest. However, low transfection efficiency has limited the use of calcium phosphate in gene delivery applications. Present work aims at studying the fabrication of strontium substituted calcium phosphate nanoparticles with improved gene delivery related properties. Strontium substituted calcium phosphate was prepared using a simple sol gel method. X-ray diffraction analysis, Fourier transform infrared spectroscopy, transmission electron microscopy, specific surface area analysis, zeta potential measurement and ion release evaluation were used to characterize the samples. This characterization showed strontium and carbonate co-substituted calcium phosphate which resulted in nano size particles with low crystallinity, high specific surface area, positive surface charge, and a high dissolution rate. These improved properties could increase the DNA concentration on the vector as well as the endosomal escape of the complex that leads to higher transfection efficiency of this novel gene delivery vector.

  4. Intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models.

    PubMed

    O'Neill, Martin J; Bourre, Ludovic; Melgar, Silvia; O'Driscoll, Caitriona M

    2011-03-01

    The future of nucleic acid-based therapeutics is dependent on achieving successful delivery. Recently, there has been an increasing interest in delivery via the gastrointestinal tract. Gene therapy via this route has many advantages, including non-invasive access and the versatility to treat local diseases, such as inflammatory bowel disease, as well as systemic diseases, such as haemophilia. However, the intestine presents several distinct barriers and, therefore, the design of robust non-viral delivery systems is key to future success. Several non-viral delivery strategies have provided evidence of activity in vivo. To facilitate the design of more efficient and safe gene medicines, more physiologically relevant models, at both the in vitro and in vivo levels, are essential.

  5. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

    PubMed

    Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

    2016-03-02

    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.

  6. The effects of a multifunctional oligomer and its incorporation strategies on the gene delivery efficiency of poly(L-lysine).

    PubMed

    Zhou, Dezhong; Li, Congxin; Hu, Yuling; Zhou, Hao; Chen, Jiatong; Zhang, Zhengpu; Guo, Tianying

    2012-05-14

    A novel multifunctional oligomer is synthesized and incorporated to enhance the gene delivery efficiency of PLL via non-electrostatic assembly and covalent grafting strategies. The improvement of the gene delivery efficiency is dependent on the gene carrying complex properties, and the properties are dependent on the oligomer incorporation strategy.

  7. 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

  8. Cerebellomedullary Cistern Delivery for AAV-Based Gene Therapy: A Technical Note for Nonhuman Primates

    PubMed Central

    Samaranch, Lluis; Bringas, John; Pivirotto, Philip; Sebastian, Waldy San; Forsayeth, John; Bankiewicz, Krystof

    2016-01-01

    Accessing cerebrospinal fluid (CSF) from the craniocervical junction through the posterior atlanto-occipital membrane via cerebellomedullary injection (also known as cisternal puncture or cisterna magna injection) has become a standard procedure in preclinical studies. Such delivery provides broader coverage to the central and peripheral nervous system unlike local parenchymal delivery alone. As a clinical application, this approach offers a more reliable method for neurological gene replacement delivery in infants, where skull-mounted devices are not indicated. Here we describe a consistent, precise, and safe method for CSF injection with minimal equipment and technical skills. PMID:26757202

  9. Cerebellomedullary Cistern Delivery for AAV-Based Gene Therapy: A Technical Note for Nonhuman Primates.

    PubMed

    Samaranch, Lluis; Bringas, John; Pivirotto, Philip; Sebastian, Waldy San; Forsayeth, John; Bankiewicz, Krystof

    2016-02-01

    Accessing cerebrospinal fluid (CSF) from the craniocervical junction through the posterior atlanto-occipital membrane via cerebellomedullary injection (also known as cisternal puncture or cisterna magna injection) has become a standard procedure in preclinical studies. Such delivery provides broader coverage to the central and peripheral nervous system unlike local parenchymal delivery alone. As a clinical application, this approach offers a more reliable method for neurological gene replacement delivery in infants, where skull-mounted devices are not indicated. Here we describe a consistent, precise, and safe method for CSF injection with minimal equipment and technical skills.

  10. 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

  11. 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

  12. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    PubMed

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.

  13. 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

  14. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  15. Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

    PubMed

    Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

    2014-06-01

    Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

  16. In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

    PubMed Central

    Tani, Hideki; Limn, Chang Kwang; Yap, Chan Choo; Onishi, Masayoshi; Nozaki, Masami; Nishimune, Yoshitake; Okahashi, Nobuo; Kitagawa, Yoshinori; Watanabe, Rie; Mochizuki, Rika; Moriishi, Kohji; Matsuura, Yoshiharu

    2003-01-01

    Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy. PMID:12941888

  17. Synthesis and Characterization of Biodegradable HPMA-Oligolysine Copolymers for Improved Gene Delivery

    PubMed Central

    Burke, Rob S.; Pun, Suzie H.

    2009-01-01

    Bioactive peptides, including DNA-binding, endosomal release and cell targeting peptides, have been integrated into synthetic gene carriers to improve delivery efficiencies by enabling the vectors to overcome barriers to gene delivery. Our overall goal is to develop multifunctional, peptide-based polymers that incorporate motifs to condense DNA and facilitate sequential trafficking steps. One approach is to polymerize vinyl-terminated peptides by radical polymerization. In this work, cationic oligolysine peptides were designed to contain vinyl termini with internal reducible linkers. These peptides were copolymerized with HPMA to form biodegradable, DNA-condensing copolymers for gene delivery. The polymerization conditions were optimized by varying the initiator to monomer ratios, macromonomer to comonomer ratios, and reactant concentrations. The synthesized copolymers were shown to possess several important properties required for in vivo gene delivery applications, including (i) efficient DNA binding and condensation, (ii) the ability to stabilize particles against salt-induced aggregation, (iii) the ability to resist extracellular polyplex unpackaging, (iv) biocompatibility and the potential to be degraded into nontoxic components after cellular uptake, and (v) efficient delivery of plasmid to cultured cells. PMID:19968270

  18. Structure-function investigations of DNA condensing agents with application to gene delivery

    NASA Astrophysics Data System (ADS)

    Evans, Heather Marie

    Lipid-based systems are notoriously poor for gene delivery, and their use has been primarily empirical. In order to improve these systems, it is imperative to obtain a greater understanding of molecular interactions between DNA and positively charged molecules. A variety of cationic molecules have been studied with DNA, in an attempt to correlate structural properties of these assemblies (using x-ray diffraction) with their efficiency as DNA carriers for gene delivery (using a luciferase assay). Several systems have been studied, some of which use the same charged amine moieties presented in three distinct morphologies: the multivalent salts spermine and spermidine, dendrimers, and dendrimeric lipids. The dendrimers somewhat approximate the properties of histories, cylindrical proteins that condense intracellular DNA. Structural studies of histone and DNA have also been conducted in order to better understand these interactions and their possible relevance to the gene delivery pathway. In addition, empirical evidence suggests that for successful in vivo gene delivery, cholesterol should be used as a helper lipid. The delivery efficiency and structural behavior of cholesterol and other sterol molecules have been studied in ternary lipid mixtures.

  19. Inverted Quasi-Spherical Droplets on Polydopamine-TiO2 Substrates for Enhancing Gene Delivery.

    PubMed

    Kim, Seung-Hyun; Lee, Mihyun; Cho, Mira; Kim, Il-Sun; Park, Kook In; Lee, Haeshin; Jang, Jae-Hyung

    2017-08-15

    Devising efficient gene delivery systems is crucial to enhancing the therapeutic efficacy of gene-cell therapy approaches. Herein, inverted quasi-spherical (iQS) droplet systems, which enhance gene delivery efficiencies by reducing the path lengths of gene vectors, mediating motions of vectors at early stages, and raising the contact frequencies of vectors with cells, are developed by adopting the principle of 3D hanging-drop cell culture. Micrometer-sized polydopamine (pDA) holes are created on superhydrophobic titanium isopropoxide (TiO2 )-coated substrates by physical scraping; droplets are loaded on the pDA holes, and inversion of the substrate generates iQS droplets with large contact angles. Both human neural stem cells (hNSCs) and adeno-associated viral vectors are simultaneously incorporated into the iQS droplets to assess gene delivery efficiencies. The steep angles of iQS droplets and enhanced cell/vector contact frequencies facilitate the viral association with hNSCs and enhancing cell-cell interactions, thereby significantly promoting gene delivery efficiencies. Even with reduced viral quantities/exposure times and cell numbers, the iQS droplet systems elicit sufficient gene expression (i.e., interleukin-10). The ability of the iQS droplet systems to maximize beneficial gene delivery effects with minimal materials (e.g., medium, cells, and vectors) should enable their extensive use as a platform for preparing genetically stimulated cellular therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. 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

  1. Sonoporation increases therapeutic efficacy of inducible and constitutive BMP2/7 in vivo gene delivery.

    PubMed

    Feichtinger, Georg A; Hofmann, Anna T; Slezak, Paul; Schuetzenberger, Sebastian; Kaipel, Martin; Schwartz, Ernst; Neef, Anne; Nomikou, Nikolitsa; Nau, Thomas; van Griensven, Martijn; McHale, Anthony P; Redl, Heinz

    2014-02-01

    An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery.

  2. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

  3. 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

  4. Structure-activity relationship of dendrimers engineered with twenty common amino acids in gene delivery.

    PubMed

    Wang, Fei; Hu, Ke; Cheng, Yiyun

    2016-01-01

    Systematic explorations on the structure-activity relationship of surface-engineered dendrimers are essential to design high efficient and safe gene vectors. The chemical diversity of residues in naturally occurring amino acids allows us to generate a library of dendrimers with various surface properties. Here, we synthesized a total number of 40 dendrimers engineered with the twenty common amino acids and investigated their performances in gene delivery. The results show that gene transfection efficacy of the synthesized materials depends on both the type of amino acid and the conjugation ratio. Dendrimers engineered with cationic and hydrophobic amino acids possess relatively higher transfection efficacies. Engineering dendrimers with cationic amino acids such as arginine and lysine facilitates polyplex formation and cellular uptake, with histidine improves endosomal escape of the polyplexes, and with hydrophobic amino acids such as tyrosine and phenylalanine modulates the balance between hydrophobicity and hydrophilicity on dendrimer surface, which is beneficial for efficient cellular internalization. Dendrimers engineered with anionic or hydrophilic amino acids show limited transfection efficacy due to poor DNA binding capacity and/or limited cellular uptake. In the aspect of cytotoxicity, dendrimers engineered with arginine, lysine, tyrosine, phenylalanine and tryptophan show much higher cytotoxicity than other engineered dendrimers. These results are helpful for us to tailor the surface chemistry of dendrimers for efficient gene delivery. Cationic polymers such as dendrimers were widely used as gene vectors but are limited by relatively low delivery efficacy and high toxicity. To achieve efficient and low toxic gene delivery, the polymers were modified with various ligands. However, these ligand-modified polymers in gene delivery are reported by independent researchers using different polymer scaffolds and cell lines. It is hard to provide structure

  5. Sui generis: gene therapy and delivery systems for the treatment of glioblastoma.

    PubMed

    Kane, J Robert; Miska, Jason; Young, Jacob S; Kanojia, Deepak; Kim, Julius W; Lesniak, Maciej S

    2015-03-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Pezzoli, Daniele; Candiani, Gabriele

    2013-03-01

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

  7. Receptor-mediated, tumor-targeted gene delivery using folate-terminated polyrotaxanes.

    PubMed

    Zhou, Yi; Wang, He; Wang, Chengxi; Li, Yueshan; Lu, Wenfeng; Chen, Shuifang; Luo, Jiandong; Jiang, Yongnan; Chen, Jianhai

    2012-05-07

    Safe and effective gene delivery is essential to the success of gene therapy. We synthesized and characterized a novel nonviral gene delivery system in which folate (FA) molecules were functioned as blockers on cationic polyrotaxanes (PR) composed of poly(ethylenimine) (PEI)(600)-grafted α-cyclodextrin rings linearized on polyethylene glycol to form FA-terminated PR-PEI(600) (FPP). The FA terminal caps of FPP target cell surfaces abundant in FA receptor (FR), a common feature of tumor cells. The structure of FPP was characterized by using (1)H nuclear magnetic resonance ((1)H NMR). The delivery particle was composed of chemically bonded PEG (4000), α-cyclodextrins (CD), and PEI (600 Da) at a molar ratio of 1:17:86.7, and the particle size and zeta potential of FPP/pDNA polyplexes were measured using dynamic light scattering. FPP/pDNA exhibited a lower cytotoxicity, strong specificity to FR, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. Furthermore, the FPP/DNA complex showed an enhanced antitumor effect in the nude mice compared with other delivery systems, such as PEI-25K. Together, these results suggest that FPP may be useful for gene therapy.

  8. 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.

  9. 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.

  10. Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

    PubMed

    Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Sáenz-García, José; Probst, Christian Macagnan; DaRocha, Wanderson Duarte

    2017-06-01

    New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Gene delivery strategies for the treatment of mucopolysaccharidoses.

    PubMed

    Baldo, Guilherme; Giugliani, Roberto; Matte, Ursula

    2014-03-01

    Mucopolysaccharidosis (MPS) disorders are genetic diseases caused by deficiencies in the lysosomal enzymes responsible for the degradation of glycosaminoglycans. Current treatments are not able to correct all disease symptoms and are not available for all MPS types, which makes gene therapy especially relevant. Multiple gene therapy approaches have been tested for different types of MPS, and our aim in this study is to critically analyze each of them. In this review, we have included the major studies that describe the use of adeno-associated retroviral and lentiviral vectors, as well as relevant non-viral approaches for MPS disorders. Some protocols such as the use of adeno-associated vectors and lentiviral vectors are approaching the clinic for these disorders and, along with combined approaches, seem to be the future of gene therapy for MPS.

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

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2014-10-01

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

  13. 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.

  14. Poly(ethylene glycol)-block-cationic polylactide nanocomplexes of differing charge density for gene delivery.

    PubMed

    Chen, Chih-Kuang; Jones, Charles H; Mistriotis, Panagiotis; Yu, Yun; Ma, Xiaoni; Ravikrishnan, Anitha; Jiang, Ming; Andreadis, Stelios T; Pfeifer, Blaine A; Cheng, Chong

    2013-12-01

    Representing a new type of biodegradable cationic block copolymer, well-defined poly(ethylene glycol)-block-cationic polylactides (PEG-b-CPLAs) with tertiary amine-based cationic groups were synthesized by thiol-ene functionalization of an allyl-functionalized diblock precursor. Subsequently the application of PEG-b-CPLAs as biodegradable vectors for the delivery of plasmid DNAs (pDNAs) was investigated. Via the formation of PEG-b-CPLA:pDNA nanocomplexes by spontaneous electrostatic interaction, pDNAs encoding luciferase or enhanced green fluorescent protein were successfully delivered to four physiologically distinct cell lines (including macrophage, fibroblast, epithelial, and stem cell). Formulated nanocomplexes demonstrated high levels of transfection with low levels of cytotoxicity and hemolysis when compared to a positive control. Biophysical characterization of charge densities of nanocomplexes at various polymer:pDNA weight ratios revealed a positive correlation between surface charge and gene delivery. Nanocomplexes with high surface charge densities were utilized in an in vitro serum gene delivery inhibition assay, and effective gene delivery was observed despite high levels of serum. Overall, these results help to elucidate the influence of charge, size, and PEGylation of nanocomplexes upon the delivery of nucleic acids in physiologically relevant conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    PubMed

    White, J D; Thesier, D M; Swain, J B D; Katz, M G; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, W J; Stedman, H H; Rabinowitz, J; Bridges, C R

    2011-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  17. Human artificial chromosome-based gene delivery vectors for biomedicine and biotechnology.

    PubMed

    Kouprina, Natalay; Tomilin, Alexey N; Masumoto, Hiroshi; Earnshaw, William C; Larionov, Vladimir

    2014-04-01

    Human artificial chromosomes (HACs) have several advantages over viruses as gene delivery vectors, including stable episomal maintenance in a single copy and the ability to carry large gene inserts. In this review, we summarise recent work on gene transfer into mammalian cells using the HACs. HACs allow therapeutic transgenes to be expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Based on the published data, the HAC vectors have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology.

  18. A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

    NASA Astrophysics Data System (ADS)

    Zhang, Zubin; Song, Lina; Dong, Jinlai; Guo, Dawei; Du, Xiaolin; Cao, Biyin; Zhang, Yu; Gu, Ning; Mao, Xinliang

    2013-05-01

    (3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

  19. 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

  20. In vivo rapid gene delivery into postmitotic neocortical neurons using iontoporation.

    PubMed

    De la Rossa, Andres; Jabaudon, Denis

    2015-01-01

    This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, focal and efficient expression of genes in postmitotic neurons. Compared with other gene delivery techniques such as in utero electroporation and viral infection, this method allows rapid (12 h), focal (50-200 μm), mosaic-like (50 to several hundred neurons) targeting of postmitotic neurons within existing circuits. This 'iontoporation' protocol, which can be completed within ∼20 min per mouse, allows straightforward assessment of genetic constructs in postmitotic cortical neurons and subsequent genetic, histological and physiological investigations of gene function.

  1. Lipopolyplex for Therapeutic Gene Delivery and Its Application for the Treatment of Parkinson's Disease.

    PubMed

    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.

  2. Current status of gene delivery: spotlight on nanomaterial-polymer hybrids.

    PubMed

    Park, Juhee; Kim, Won Jong

    2012-09-01

    Gene therapy aims to treat human disorders by introducing genetic materials into specific target cells or tissues. Despite the curability for the origIn of diseases by restoring missing functionalities, no technical feasibility of gene therapy has been established due to the lack of safe and efficient gene delivery systems. The emergence of nanotechnology has provided an opportunity to create nanomaterials that are suitable for the biomedical applications. Nanomaterials integrated with cationic polymers offer novel platforms that allow not only easy incorporation of genetic materials through electrostatic interactions but also further modifications to be upgraded to theranostics. In this article, current status of gene delivery utilizing hybrid nanomaterials that are composed of novel nanoplatforms and cationic polymers are highlighted. In particular, different strategies employed for the construction of nanomaterial-polymer hybrids are described.

  3. In vivo episomal maintenance of a herpesvirus saimiri-based gene delivery vector.

    PubMed

    Smith, P G; Coletta, P L; Markham, A F; Whitehouse, A

    2001-12-01

    Herpesvirus saimiri (HVS) has several properties that make it amenable to development as a gene delivery vector. HVS offers the potential to incorporate large amounts of heterologous DNA and infect a broad range of human cell lines. Upon infection the viral genome can persist by virtue of episomal maintenance and stably maintains heterologous gene expression. Here we report an evaluation of the in vivo properties of HVS, with a view to its development as a gene delivery system. We demonstrate for the first time, the long-term persistence of the HVS genome in tumour xenografts generated from HVS-infected human carcinoma cell lines. The HVS-based vector remained latent in the xenograft without spreading to other organs. Moreover, the long-term in vivo maintenance of the HVS genome, as a nonintegrated circular episome, provided efficient sustained expression of a heterologous transgene. These in vivo results suggest that HVS-based vectors have potential for gene therapy applications.

  4. Intramyocardial BNP gene delivery improves cardiac function through distinct context-dependent mechanisms.

    PubMed

    Moilanen, Anne-Mari; Rysä, Jaana; Mustonen, Erja; Serpi, Raisa; Aro, Jani; Tokola, Heikki; Leskinen, Hanna; Manninen, Aki; Levijoki, Jouko; Vuolteenaho, Olli; Ruskoaho, Heikki

    2011-07-01

    B-type natriuretic peptide (BNP) is an endogenous peptide produced under physiological and pathological conditions mainly by ventricular myocytes. It has natriuretic, diuretic, blood pressure-lowering, and antifibrotic actions that could mediate cardiorenal protection in cardiovascular diseases. In the present study, we used BNP gene transfer to examine functional and structural effects of BNP on left ventricular (LV) remodeling. Human BNP was overexpressed by using adenovirus-mediated gene delivery in normal rat hearts and in hearts during the remodeling process after infarction and in an experimental model of angiotensin II-mediated hypertension. In healthy hearts, BNP gene delivery into the anterior wall of the LV decreased myocardial fibrosis (P<0.01, n=7 to 8) and increased capillary density (P<0.05, n=7 to 8) associated with a 7.3-fold increase in LV BNP peptide levels. Overexpression of BNP improved LV fractional shortening by 22% (P<0.05, n=6 to 7) and ejection fraction by 19% (P<0.05, n=6 to 7) after infarction. The favorable effect of BNP gene delivery on cardiac function after infarction was associated with normalization of cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression and phospholamban Thr17-phosphorylation. BNP gene delivery also improved fractional shortening and ejection fraction in angiotensin II-mediated hypertension as well as decreased myocardial fibrosis and LV collagen III mRNA levels but had no effect on angiogenesis or Ca(2+)-ATPase expression and phospholamban phosphorylation. Local intramyocardial BNP gene delivery improves cardiac function and attenuates adverse postinfarction and angiotensin II-induced remodeling. These results also indicate that myocardial BNP has pleiotropic, context-dependent, favorable actions on cardiac function and suggest that BNP acts locally as a key mechanical load-activated regulator of angiogenesis and fibrosis.

  5. 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.

  6. 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

  7. Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery.

    PubMed

    Ferrara, Katherine; Pollard, Rachel; Borden, Mark

    2007-01-01

    This review offers a critical analysis of the state of the art of medical microbubbles and their application in therapeutic delivery and monitoring. When driven by an ultrasonic pulse, these small gas bubbles oscillate with a wall velocity on the order of tens to hundreds of meters per second and can be deflected to a vessel wall or fragmented into particles on the order of nanometers. While single-session molecular imaging of multiple targets is difficult with affinity-based strategies employed in some other imaging modalities, microbubble fragmentation facilitates such studies. Similarly, a focused ultrasound beam can be used to disrupt delivery vehicles and blood vessel walls, offering the opportunity to locally deliver a drug or gene. Clinical translation of these vehicles will require that current challenges be overcome, where these challenges include rapid clearance and low payload. The technology, early successes with drug and gene delivery, and potential clinical applications are reviewed.

  8. Multilayered Nanoparticles for Gene Delivery Used to Reprogram Human Foreskin Fibroblasts to Neurospheres

    PubMed Central

    Watson, Andre; Ren, Liyun; Mixon, Amanda; Kotha, Shiva P.

    2015-01-01

    Polycationic nanocomplexes are a robust means for achieving nucleic acid condensation and efficient intracellular gene deliveries. To enhance delivery, a multilayered nanoparticle consisting of a core of electrostatically bound elements was used. These included a histone-mimetic peptides, poly-l-arginine and poly-d-glutamic acid was coated with silicate before surface functionalization with poly-l-arginine. Transfection efficiencies and duration of expression were similar when using green fluorescent protein (GFP) plasmid DNA (pDNA) or GFP mRNA. These nanoparticles demonstrated significantly higher (>100%) and significantly longer (15 vs. 4 days) transfection efficiencies in comparison to a commercial transfection agent (Lipofectamine 2000). Reprogramming of human foreskin fibroblasts using mRNA to the Sox2 transcription factor resulted in three-fold higher neurosphere formation in comparison to the commercial reagent. These results demonstrate the potential of these nanoparticles as ideal vectors for gene delivery. PMID:25687130

  9. Systemic gene delivery to the central nervous system using Adeno-associated virus.

    PubMed

    Bourdenx, Mathieu; Dutheil, Nathalie; Bezard, Erwan; Dehay, Benjamin

    2014-01-01

    Adeno-associated virus (AAV)-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  10. 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

  11. 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

  12. Chitosan based oligoamine polymers: synthesis, characterization, and gene delivery.

    PubMed

    Lu, Bo; Wang, Chang-Fang; Wu, De-Qun; Li, Cao; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2009-07-01

    A series of chitosan-based oligoamine polymers was synthesized from N-maleated chitosan (NMC) via Michael addition with diethylenetriamine (DETA), triethylenetetramine (TETA), tetraethylenepentamine (TEPA) and linear polyethylenimine (M(n) 423), respectively. The resulted polymers exhibited well binding ability to condense plasmid DNA to form complexes with size ranging from 200 to 600 nm when the polymer/DNA weight ratio was above 7. The polymer/DNA complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The cytotoxicity assay showed that the synthesized polymers were less toxic than that of PEI(25 K). The gene transfection effect of resulted polymers was evaluated in 293T and HeLa cells, and the results showed that the gene transfection efficiency of these polymers was better than that of chitosan. Moreover, the transfection efficiency was dependent on the length of the oligoamine side chains and the molecular weight of the chitosan derivatives.

  13. 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

  14. Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways

    PubMed Central

    Hwang, Mark E.; Keswani, Rahul K.; Pack, Daniel W.

    2014-01-01

    Purpose Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. Methods We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways. Results PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. Conclusions The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network. PMID:25511918

  15. 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

  16. Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery.

    PubMed

    Roberts, Rebecca; Al-Jamal, Wafa' T; Whelband, Matthew; Thomas, Paul; Jefferson, Matthew; van den Bossche, Jeroen; Powell, Penny P; Kostarelos, Kostas; Wileman, Thomas

    2013-05-01

    Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.

  17. Human erythropoietin gene delivery for cardiac remodeling of myocardial infarction in rats.

    PubMed

    Lee, Youngsook; McGinn, Arlo N; Olsen, Curtis D; Nam, Kihoon; Lee, Minhyung; Shin, Sug Kyun; Kim, Sung Wan

    2013-10-10

    Considerable efforts have been made to exploit cardioprotective drugs and gene delivery systems for myocardial infarction (MI). The promising cardioprotective effects of recombinant human erythropoietin (rHuEPO) protein in animal experiments have not been consistently reproduced in clinical human trials of acute MI; however, the mechanisms underlying the inconsistent discrepancies are not yet fully understood. We hypothesized that the plasmid human erythropoietin gene (phEPO) delivered by our bioreducible polymer might produce cardioprotective effects on post-infarct cardiac remodeling. We demonstrated that intramyocardial delivery of phEPO by an arginine-grafted poly(disulfide amine) (ABP) polymer in infarcted rats preserves cardiac geometry and systolic function. The reduced infarct size of phEPO/ABP delivery was followed by decrease in fibrosis, protection from cardiomyocyte loss, and down-regulation of apoptotic activity. In addition, the increased angiogenesis and decreased myofibroblast density in the border zone of the infarct support the beneficial effects of phEPO/ABP administration. Furthermore, phEPO/ABP delivery induced prominent suppression on Ang II and TGF-β activity in all subdivisions of cardiac tissues except for the central zone of infarct. These results of phEPO gene therapy delivered by a bioreducible ABP polymer provide insight into the lack of phEPO gene therapy translation in the treatment of acute MI to human trials. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Human Erythropoietin Gene Delivery for Cardiac Remodeling of Myocardial Infarction in Rats

    PubMed Central

    Lee, Youngsook; McGinn, Arlo N.; Olsen, Curtis D.; Nam, Kihoon; Lee, Minhyung; Shin, Sug Kyun; Kim, Sung Wan

    2013-01-01

    Considerable efforts have been made to exploit cardioprotective drugs and gene delivery systems for myocardial infarction (MI). The promising cardioprotective effects of recombinant human erythropoietin (rHuEPO) protein in animal experiments have not been consistently reproduced in clinical human trials of acute MI; however, the mechanisms underlying the inconsistent discrepancies are not yet fully understood. We hypothesized that the plasmid human erythropoietin gene (phEPO) delivered by our bioreducible polymer might produce cardioprotective effects on post-infarct cardiac remodeling. We demonstrated that intramyocardial delivery of phEPO by an arginine-grafted poly(disulfide amine) (ABP) polymer in infarcted rats preserves cardiac geometry and systolic function. The reduced infarct size of phEPO/ABP delivery was followed by decrease in fibrosis, protection from cardiomyocyte loss, and down-regulation of apoptotic activity. In addition, the increased angiogenesis and decreased myofibroblast density in the border zone of the infarct support the beneficial effects of phEPO/ABP administration. Furthermore, phEPO/ABP delivery induced prominent suppression on Ang II and TGF-β activity in all subdivisions of cardiac tissues except for the central zone of infarct. These results of phEPO gene therapy delivered by a bioreducible ABP polymer provide insight into the lack of phEPO gene therapy translation in the treatment of acute MI to human trials. PMID:23806842

  19. Recent Advances in Intravesical Drug/Gene Delivery

    PubMed Central

    Tyagi, Pradeep; Wu, Pao-Chu; Chancellor, Michael; Yoshimura, Naoki; Huang, Leaf

    2008-01-01

    Targeting of the drugs administered systemically relies on the higher affinity of ligands for specific receptors to obtain selectivity in drug response. However, achieving the same goal inside the bladder is much easier with an intelligent pharmaceutical approach that restricts drug effects by exploiting the pelvic anatomical architecture of the human body. This regional therapy involves placement of drugs directly into the bladder through a urethral catheter. It is obvious that drug administration by this route holds advantage in chemotherapy of superficial bladder cancer and it has now become the most widely used treatment modality for this ailment. In recent years, the intravesical route has also been exploited either as an adjunct to an oral regimen or as a second-line treatment for neurogenic bladder 1, 2. Instillation of DNA via this route using different vectors has been able to restrict the transgene expression in organs other than bladder. The present review article will discuss the shortcomings of the current options available for intravesical drug delivery (IDD) and lay a perspective for future developments in this field. PMID:16889430

  20. 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

  1. DNA amplification in neutral liposomes for safe and efficient gene delivery.

    PubMed

    Lee, Sangmin; Koo, Heebeom; Na, Jin Hee; Lee, Kyung Eun; Jeong, Seo Young; Choi, Kuiwon; Kim, Sun Hwa; Kwon, Ick Chan; Kim, Kwangmeyung

    2014-05-27

    In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed in vivo gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application.

  2. 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

  3. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins.

    PubMed

    Chen, Qiang; Lai, Huafang; Hurtado, Jonathan; Stahnke, Jake; Leuzinger, Kahlin; Dent, Matthew

    2013-06-01

    Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems as a premier platform for commercial production of pharmaceutical proteins.

  4. Gene Delivery for the Generation of Bioartificial Pacemaker.

    PubMed

    Chan, Patrick K W; Li, Ronald A

    2017-01-01

    Electronic pacemakers have been used in patients with heart rhythm disorders for device-supported pacing. While effective, there are such shortcomings as limited battery life, permanent implantation of catheters, the lack of autonomic neurohumoral responses, and risks of lead dislodging. Here we describe protocols for establishing porcine models of sick sinus syndrome and complete heart block, and the generation of bioartificial pacemaker by delivering a strategically engineered form of hyperpolarization-activated cyclic nucleotide-gated pacemaker channel protein via somatic gene transfer to convert atrial or ventricular muscle cardiomyocytes into nodal-like cells that rhythmically fire action potentials.

  5. CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo.

    PubMed

    Yang, Jing; Hao, Xuefang; Li, Qian; Akpanyung, Mary; Nejjari, Abdelilah; Neve, Agnaldo Luis; Ren, Xiangkui; Guo, Jintang; Feng, Yakai; Shi, Changcan; Zhang, Wencheng

    2017-02-08

    Gene therapy is a promising strategy for angiogenesis, but developing gene carriers with low cytotoxicity and high gene delivery efficiency in vivo is a key issue. In the present study, we synthesized the CAGW peptide- and poly(ethylene glycol) (PEG)-modified amphiphilic copolymers. CAGW peptide serves as a targeting ligand for endothelial cells (ECs). Different amounts of CAGW peptide were effectively conjugated to the amphiphilic copolymer via heterofunctional poly(ethylene glycol). These CAG- and PEG-modified copolymers could form nanoparticles (NPs) by self-assembly method and were used as gene carriers for the pEGFP-ZNF580 (pZNF580) plasmid. CAGW and PEG modification coordinately improved the hemocompatibility and cytocompatibility of NPs. The results of cellular uptake showed significantly enhanced internalization efficiency of pZNF580 after CAGW modification. Gene expression at mRNA and protein levels demonstrated that EC-targeted NPs possessed high gene delivery efficiency, especially the NPs with higher content of CAGW peptide (1.16 wt %). Furthermore, in vitro and in vivo vascularization assays also showed outstanding vascularization ability of human umbilical vein endothelial cells treated by the NP/pZNF580 complexes. This study demonstrates that the CAGW peptide-modified NP is a promising candidate for gene therapy in angiogenesis.

  6. A novel glutathione modified chitosan conjugate for efficient gene delivery.

    PubMed

    Li, Congxin; Guo, Tianying; Zhou, Dezhong; Hu, Yuling; Zhou, Hao; Wang, Shufang; Chen, Jiatong; Zhang, Zhengpu

    2011-09-05

    A novel non-viral gene vector based on poly[poly(ethylene glycol) methacrylate] (PMPEG) and l-glutathione (GSH) grafted chitosan (CS) has been fabricated. First, well-defined brush-like PMPEG living polymers with dithioester residues were prepared by the reversible addition-fragmentation chain transfer (RAFT) polymerization and grafted onto the allylchitosan via radical coupling method. Then, the tripeptide GSH was introduced onto the end of PMPEG chain to give a CS-PMPEG-GSH conjugate. In comparison with pristine chitosan, CS-PMPEG-GSH conjugate could not only condense plasmid DNA (pDNA) and prevent the condensed CS-PMPEG-GSH/pDNA nanoparticle self-aggregation, but also increase the binding ability to cell membrane efficiently and improve decondensed ability of pDNA from the nanoparticles in cytoplasm which thus has resulted in the higher transfection efficiency in mouse embryonic fibroblast cells (NIH3T3). In addition, cytotoxicity assays showed that the conjugate is less cytotoxic than CS, and still retain the cationic polyelectrolyte characteristic as chitosan. These results indicate that the non-viral vector is a promising candidate for gene therapy in clinical application.

  7. Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action.

    PubMed

    Calejo, Maria Teresa; Sande, Sverre Arne; Nyström, Bo

    2013-12-01

    This topic is important as it allows for improved specificity in drug delivery, providing possibilities for reduced side effects, and thereby improved pharmacotherapy. As a wealth of different polymers and mechanisms of action has been suggested, a systematic overview of the field is of current importance. This article presents an overview over a selection of thermoresponsive polymers suitable as excipients in systems for gene and drug delivery with particular emphasis on the influence of polymer structure, composition, molecular weight (MW) and architecture on the responsive mechanisms. Due to the immense number of reports on these increasingly popular materials, focus has been restricted to the use of micelle-forming polymers with a lower critical solution temperature, temperature-responsive hydrogels for drug delivery applications and temperature-sensitive polymers as non-viral vectors for polynucleotide delivery. Specific examples covered are poly-(N-isopropylacrylamide) (PNIPAAM), Pluronics and their derivatives. It is concluded that the studies constitute an excellent platform for development of thermoresponsive systems with improved gene and drug delivery properties. A thorough knowledge of factors important for loading efficiency and drug release is necessary to be able to develop optimal nano-carriers for the future. Other issues that are not fully understood is how small the carriers need to be, and which manufacturing procedures should be used.

  8. Nanoparticle-mediated delivery of therapeutic genes: focus on miRNA therapeutics.

    PubMed

    Muthiah, Muthunarayanan; Park, In-Kyu; Cho, Chong-Su

    2013-09-01

    Micro RNAs (miRNA) are 21 - 23 nucleotides long and regulate the expression of coding genes by binding imperfectly with their 3' UTR region. The miRNA profile is altered in pathological processes, making miRNAs good targets for drug therapy. Restoration of down-regulated miRNA or inhibition of overexpressed miRNA to return miRNA to its normal state is the basis of miRNA-based therapy. This review focuses on nanocarriers used for the delivery of miRNA that confer physical stability to the unstable RNA structure, protect the RNA from nuclease degradation and aid in effective silencing of target genes. The necessity of the nanocarrier for the delivery of the miRNA is emphasized and the recent research on liposome-, metal- and polymer-mediated miRNA delivery for the inhibition or replacement of the disease-related miRNA is summarized. The size, charge and surface properties of nanocarriers have to be tuned to ensure effective and safe delivery of the miRNA in clinical practice. The immune responses related to the nanocarriers and the double-stranded nucleotide delivery remain to be addressed. Also, the binding of miRNAs to non-specific targets has to be studied in more detail because miRNAs have multiple targets due to partial binding unlike siRNA.

  9. Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

    PubMed

    Yokoo, T; Kamimura, K; Suda, T; Kanefuji, T; Oda, M; Zhang, G; Liu, D; Aoyagi, Y

    2013-08-01

    The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time-pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time-pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time-pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml(-1)) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

  10. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination.

    PubMed

    Laube, Beth L

    2005-09-01

    Aerosolized medications have been used for centuries to treat respiratory diseases. Until recently, inhalation therapy focused primarily on the treatment of asthma and chronic obstructive pulmonary disease, and the pressurized metered-dose inhaler was the delivery device of choice. However, the role of aerosol therapy is clearly expanding beyond that initial focus. This expansion has been driven by the Montreal protocol and the need to eliminate chlorofluorocarbons (CFCs) from traditional metered-dose inhalers, by the need for delivery devices and formulations that can efficiently and reproducibly target the systemic circulation for the delivery of proteins and peptides, and by developments in medicine that have made it possible to consider curing lung diseases with aerosolized gene therapy and preventing epidemics of influenza and measles with aerosolized vaccines. Each of these drivers has contributed to a decade or more of unprecedented research and innovation that has altered how we think about aerosol delivery and has expanded the role of aerosol therapy into the fields of systemic drug delivery, gene therapy, and vaccination. During this decade of innovation, we have witnessed the coming of age of dry powder inhalers, the development of new soft mist inhalers, and improved pressurized metered-dose inhaler delivery as a result of the replacement of CFC propellants with hydrofluoroalkane. The continued expansion of the role of aerosol therapy will probably depend on demonstration of the safety of this route of administration for drugs that have their targets outside the lung and are administered long term (eg, insulin aerosol), on the development of new drugs and drug carriers that can efficiently target hard-to-reach cell populations within the lungs of patients with disease (eg, patients with cystic fibrosis or lung cancer), and on the development of devices that improve aerosol delivery to infants, so that early intervention in disease processes with aerosol

  11. [Melanoma: surface markers as the first point of targeted delivery of therapeutic genes in multilevel gene therapy].

    PubMed

    Pleshkan, V V; Zinov'eva, M V; Sverdlov, E D

    2011-01-01

    Melanoma is one of the most malignant tumors, aggressively metastasizing by lymphatic and hematogenous routes. Due to the resistance of melanoma cells to many types of chemotherapy, this disease causes high mortality rate. High hopes are pinned on gene therapeutic approaches to melanoma treatment. At present, one of the main problems of the efficient use of the post-genomic generation therapeutic means is the lack of optimal techniques of delivery of foreign genetic material to the patient's target cells. Surface specific markers of melanoma cells can be considered as promising therapeutic targets. This review describes currently known melanoma specific receptors and its stem cells, as well as contains data on melanoma antigens presented on the cell surface by major histocompatibility complex proteins. The ability of surface proteins to internalize might be successfully used for the development of methods of targeted delivery of gene therapeutic constructs. In conclusion, a concept of multilevel gene therapy and the possible role therein of surface determinants as targets of gene systems delivery to the tumor are discussed.

  12. Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering.

    PubMed

    Ruan, Gui-Xin; Chen, Yu-Zhe; Yao, Xing-Lei; Du, Anariwa; Tang, Gu-Ping; Shen, You-Qing; Tabata, Yasuhiko; Gao, Jian-Qing

    2014-05-01

    Macrophages are the most plastic cells in the hematopoietic system and they exhibit great functional diversity. They have been extensively applied in anti-inflammatory, anti-fibrotic and anti-cancer therapies. However, the application of macrophages is limited by the efficiency of their engineering. The macrophage mannose receptor (MMR, CD206), a C-type lectin receptor, is ubiquitously expressed on macrophages and has a high affinity for mannose oligosaccharides. In the present study, we developed a novel non-viral vehicle with specific affinity for MMR. Mannan was cationized with spermine at a grafted ratio of ∼12% to deliver DNA and was characterized as a stable system for delivery. This spermine-mannan (SM)-based delivery system was evaluated as a biocompatible vehicle with superior transfection efficiency on murine macrophages, up to 28.5-fold higher than spermine-pullulan, 11.5-fold higher than polyethylenimine and 3.0-fold higher than Lipofectamine™ 2000. We confirmed that the SM-based delivery system for macrophages transfection was MMR-specific and we described the intracellular transport of the delivery system. To our knowledge, this is the first study using SM to demonstrate a mannose receptor-specific gene delivery system, thereby highlighting the potential of a novel specific non-viral delivery vehicle for macrophage engineering.

  13. Current strategies in modification of PLGA-based gene delivery system.

    PubMed

    Ramezani, Mohammad; Ebrahimian, Mahboubeh; Hashemi, Maryam

    2016-12-05

    The successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and gene efficiently. This formulation has several advantages in comparison with other formulations including improvement of solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as gene carrier, there exist many challenges. PLGA nanoparticles could protect the encapsulated DNA from in vivo degradation but the DNA release is slowl and their negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce its cytotoxicity, to enhance the delivery efficiency and to target it to specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for modification of PLGA particles applied in gene therapy.

  14. Brain-Targeted Polymers for Gene Delivery in the Treatment of Brain Diseases.

    PubMed

    Lu, Yifei; Jiang, Chen

    2017-04-01

    Gene therapies have become a promising strategy for treating neurological disorders, such as brain cancer and neurodegenerative diseases, with the help of molecular biology interpreting the underlying pathological mechanisms. Successful cellular manipulation against these diseases requires efficient delivery of nucleic acids into brain and further into specific neurons or cancer cells. Compared with viral vectors, non-viral polymeric carriers provide a safer and more flexible way of gene delivery, although suffering from significantly lower transfection efficiency. Researchers have been devoted to solving this defect, which is attributed to the multiple barriers existing for gene therapeutics in vivo, such as systemic degradation, blood-brain barrier, and endosome trapping. This review will be mainly focused on systemically administrated brain-targeted polymers developed so far, including PEI, dendrimers, and synthetic polymers with various functions. We will discuss in detail how they are designed to overcome these barriers and how they efficiently deliver therapeutic nucleic acids into targeted cells.

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

    NASA Astrophysics Data System (ADS)

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

    2017-08-01

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

  16. Multifunctional Spider Silk Polymers for Gene Delivery to Human Mesenchymal Stem Cells

    PubMed Central

    Tokareva, Olena; Glettig, Dean; Abbott, Rosalyn D.; Kaplan, David L.

    2014-01-01

    Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems. PMID:25399785

  17. Multifunctional spider silk polymers for gene delivery to human mesenchymal stem cells.

    PubMed

    Tokareva, Olena S; Glettig, Dean L; Abbott, Rosalyn D; Kaplan, David L

    2015-10-01

    Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40 ), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems. © 2014 Wiley Periodicals, Inc.

  18. Poly(aspartic acid)-based degradable assemblies for highly efficient gene delivery.

    PubMed

    Nie, Jing-Jun; Dou, Xue-Bo; Hu, Hao; Yu, Bingran; Chen, Da-Fu; Wang, Ren-Xian; Xu, Fu-Jian

    2015-01-14

    Due to its good properties such as low cytotoxicity, degradability, and biocompatibility, poly(aspartic acid) (PAsp) is a good candidate for the development of new drug delivery systems. In this work, a series of new PAsp-based degradable supramolecular assemblies were prepared for effective gene therapy via the host-guest interactions between the cyclodextrin (CD)-cored PAsp-based polycations and the pendant benzene group-containing PAsp backbones. Such supramolecular assemblies exhibited good degradability, enhanced pDNA condensation ability, and low cytotoxicity. More importantly, the gene transfection efficiencies of supramolecular assemblies were much higher than those of CD-cored PAsp-based counterparts at various N/P ratios. In addition, the effective antitumor ability of assemblies was demonstrated with a suicide gene therapy system. The present study would provide a new means to produce degradable supramolecular drug delivery systems.

  19. Targeted gene transfection from microbubbles into vascular smooth muscle cells using focused, ultrasound-mediated delivery

    PubMed Central

    Phillips, Linsey C.; Klibanov, Alexander L.; Wamhoff, Brian R.; Hossack, John A.

    2010-01-01

    We investigate a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0–950 kPa) and pulse durations (0–100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA, or ultrasound alone. Microbubbles in combination with ultrasound (500 kPa, 1MHz, 50 cycle bursts at a Pulse Repetition Frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 +/− 27%) and without (19 +/− 8%). Maximal gene transfection (~1% of cells) occurred using 50 cycle, 1 MHz pulses at 300 kPa which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. PMID:20800174

  20. Delivery

    PubMed Central

    Miller, Thomas A

    2013-01-01

    Enthusiasm greeted the development of synthetic organic insecticides in the mid-twentieth century, only to see this give way to dismay and eventually scepticism and outright opposition by some. Regardless of how anyone feels about this issue, insecticides and other pesticides have become indispensable, which creates something of a dilemma. Possibly as a result of the shift in public attitude towards insecticides, genetic engineering of microbes was first met with scepticism and caution among scientists. Later, the development of genetically modified crop plants was met with an attitude that hardened into both acceptance and hard-core resistance. Transgenic insects, which came along at the dawn of the twenty-first century, encountered an entrenched opposition. Those of us responsible for studying the protection of crops have been affected more or less by these protagonist and antagonistic positions, and the experiences have often left one thoughtfully mystified as decisions are made by non-participants. Most of the issues boil down to concerns over delivery mechanisms. © 2013 Society of Chemical Industry PMID:23852646

  1. 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

  2. Preparation and Characterization of Gelatin-Based Mucoadhesive Nanocomposites as Intravesical Gene Delivery Scaffolds

    PubMed Central

    Liu, Ching-Wen; Chang, Li-Ching; Lin, Kai-Jen; Yu, Tsan-Jung; Tsai, Ching-Chung; Wang, Hao-Kuang; Tsai, Tong-Rong

    2014-01-01

    This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays. In vitro lentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cells in vitro and to rat urothelium by intravesical instillation in vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds. PMID:25580433

  3. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

    PubMed Central

    Simeoni, Federica; Morris, May C.; Heitz, Frederic; Divita, Gilles

    2003-01-01

    The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features. PMID:12771197

  4. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells.

    PubMed

    Simeoni, Federica; Morris, May C; Heitz, Frederic; Divita, Gilles

    2003-06-01

    The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.

  5. Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian

    2014-06-01

    Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

  6. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.

    PubMed

    Shen, Jianliang; Xu, Rong; Mai, Junhua; Kim, Han-Cheon; Guo, Xiaojing; Qin, Guoting; Yang, Yong; Wolfram, Joy; Mu, Chaofeng; Xia, Xiaojun; Gu, Jianhua; Liu, Xuewu; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2013-11-26

    Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

  7. Design and Characterization of Novel Recombinant Listeriolysin O–Protamine Fusion Proteins for Enhanced Gene Delivery

    PubMed Central

    2015-01-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO–protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems. PMID:25521817

  8. A Cell-Penetrating Peptide with a Guanidinylethyl Amine Structure Directed to Gene Delivery

    PubMed Central

    Oba, Makoto; Kato, Takuma; Furukawa, Kaori; Tanaka, Masakazu

    2016-01-01

    A peptide composed of lysine with a guanidinylethyl (GEt) amine structure in the side chain [Lys(GEt)] was developed as a cell-penetrating peptide directed to plasmid DNA (pDNA) delivery. The GEt amine adopted a diprotonated form at neutral pH, which may have led to the more efficient cellular uptake of a Lys(GEt)-peptide than an arginine-peptide at a low concentration. Lys(GEt)-peptide/pDNA complexes showed the highest transfection efficiency due to efficient endosomal escape without any cytotoxicity. Lys(GEt)-peptide may be a promising candidate as a gene delivery carrier. PMID:26814673

  9. Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype.

    PubMed

    Delalande, Anthony; Bouakaz, Ayache; Renault, Gilles; Tabareau, Flore; Kotopoulis, Spiros; Midoux, Patrick; Arbeille, Brigitte; Uzbekov, Rustem; Chakravarti, Shukti; Postema, Michiel; Pichon, Chantal

    2011-12-10

    The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Cyclen-Based Cationic Lipids for Highly Efficient Gene Delivery towards Tumor Cells

    PubMed Central

    Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; Zhu, Wen; Yu, Xiao-Qi

    2011-01-01

    Background Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. Methods In this report, we designed and synthesized three amphiphilic molecules (L1–L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. Results Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1–L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. Conclusion Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen. PMID:21887233

  11. Assessment of therapeutic efficacy of liposomal nanoparticles mediated gene delivery by molecular imaging for cancer therapy.

    PubMed

    Zhou, Manqian; Wang, Lina; Su, Weijun; Tong, Lingling; Liu, Yanhua; Fan, Yan; Luo, Na; Zheng, Yizhou; Zhao, Hui; Xiang, Rong; Li, Zongjin

    2012-10-01

    The inadequate treatment efficacy, suboptimal cancer detection and disease monitoring in anticancer therapies have led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable approaches. Molecular imaging technologies with the versatility of liposomal nanoparticles platform offer tangible options to better guide treatment delivery and monitor outcome. In this study, we introduced noninvasive, quantitative and functional imaging techniques with reporter gene methods to probe breast cancer processes with liposomal nanoparticles by bioluminescence imaging (BLI). A breast cancer model was applied for therapy by injecting 5.0 x 10(5) 4T1 cells carrying a reporter system encoding a double fusion reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into BALB/c mice. Liposomal nanoparticles loaded with a triple fusion gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP) were applied by in situ injection for monitoring and evaluating gene therapy. The BALB/c mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by bioluminescence imaging of Fluc and the treatment delivery of liposomal nanoparticle was efficiently tracked by Rluc imaging. In fact, TF plasmids were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. In conclusion, the combination of noninvasive imaging techniques and liposomal nanoparticle can provide a practical and clinically useful way for gene delivery and monitoring the level of gene expression over time and treatment response in patients undergoing gene therapy.

  12. A Photoactivatable AIE Polymer for Light-Controlled Gene Delivery: Concurrent Endo/Lysosomal Escape and DNA Unpacking.

    PubMed

    Yuan, Youyong; Zhang, Chong-Jing; Liu, Bin

    2015-09-21

    Endo/lysosomal escape of gene vectors and the subsequent unpacking of nucleic acids in cytosol are two major challenges for efficient gene delivery. Herein, we report a polymeric gene delivery vector, which consists of a photosensitizer (PS) with aggregation-induced emission (AIE) characteristics and oligoethylenimine (OEI) conjugated via an aminoacrylate (AA) linker that can be cleaved by reactive oxygen species (ROS). In aqueous media, the polymer could self-assemble into bright red fluorescent nanoparticles (NPs), which can efficiently bind to DNA through electrostatic interaction for gene delivery. Upon visible light irradiation, the generated ROS can break the endo/lysosomal membrane and the polymer, resulting in light-controlled endo/lysosomal escape and unpacking of DNA for efficient gene delivery. The smart polymer represents the first successful gene vector to simultaneously address both challenges with a single light excitation process.

  13. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

    NASA Astrophysics Data System (ADS)

    Tiwari, Pawan K.; Soo Lee, Yeon

    2013-08-01

    The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

  14. IONP-PLL: a novel non-viral vector for efficient gene delivery.

    PubMed

    Xiang, Juan-Juan; Tang, Jing-Qun; Zhu, Shi-Guo; Nie, Xin-Min; Lu, Hong-Bin; Shen, Shou-Rong; Li, Xiao-Ling; Tang, Ke; Zhou, Ming; Li, Gui-Yuan

    2003-09-01

    Non-viral methods of gene delivery have been an attractive alternative to virus-based gene therapy. However, the vectors that are currently available have drawbacks limiting their therapeutic application. We have developed a self-assembled non-viral gene carrier, poly-L-lysine modified iron oxide nanoparticles (IONP-PLL), which is formed by modifying poly-L-lysine to the surface of iron oxide nanoparticles. The ability of IONP-PLL to bind DNA was determined by ratio-dependent retardation of DNA in the agarose gel and co-sedimentation assay. In vitro cytotoxic effects were quantified by MTT assay. The transfection efficiency in vitro was evaluated by delivering exogenous DNA to different cell lines using IONP-PLL. Intravenous injection of IONP-PLL/DNA complexes into mice was evaluated as a gene delivery system for gene therapy. The PGL2-control gene encoding firefly luciferase and the EGFP-C2 gene encoding green fluorescent protein were used as marker genes. IONP-PLL could bind and protect DNA. In contrast to PLL and cationic liposomes, IONP-PLL described here was less cytotoxic in a broad range of concentrations. In the current study, we have demonstrated that IONP-PLL can deliver exogenous gene to cells in vitro and in vivo. After intravenous injection, IONP-PLL transferred reporter gene EGFP-C2 to lung, brain, spleen and kidney. Furthermore, we have demonstrated that IONP-PLL transferred exogenous DNA across the blood-brain barrier to the glial cells and neuron of brain. IONP-PLL, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy in vitro and in vivo, especially for gene therapy of CNS disease. Copyright 2003 John Wiley & Sons, Ltd.

  15. Factorial Design and Development of Solid Lipid Nanoparticles (SLN) for Gene Delivery.

    PubMed

    Radaic, Allan; de Paula, Eneida; de Jesus, Marcelo Bispo

    2015-02-01

    Several scientific hurdles still have to be overcome before gene therapy becomes a reality. One of them is the development of safe and efficient gene delivery system. Here, we have employed factorial design to optimize the production of solid lipid nanoparticles (SLN) for gene delivery. A 2 x 3 full-factorial experimental design was used for the optimization of SLNs formulations. The variables were defined by the components of the formulation: concentration of stearic acid, DOTAP, and Pluronic F68 at two levels (-1, 1) and 3 central points (0). Different SNL formulations were prepared by varying the amount of components and several properties were tested, including their capacity to accommodate DNA and protection against DNase degradation, colloidal stability, in vitro cytotoxicity, and transfection efficiency in prostate cancer cells. Finally, response Surface Methodology was used to select the most effective formulation for gene delivery to prostate cancer cells in vitro. In conclusion, this study revealed that stearic acid and Pluronic F68 were determinant to SLN size and stability, respectively, while small amounts of DOTAP are essential for a successful transfection.

  16. Evaluation of Jeffamine®-cored PAMAM dendrimers as an efficient in vitro gene delivery system.

    PubMed

    Aydin, Zeynep; Akbas, Fahri; Senel, Mehmet; Koc, S Naci

    2012-10-01

    In this study, we investigated gene delivery properties of Jeffamine-cored polyamidoamine (PAMAM) dendrimers (JCPDs). The effects of dendrimer concentration, generation, and core size on the gene delivery have been analyzed. The experimental results showed that the JCPD effectively delivered plasmid DNA inside the HeLa cells, and the transfection efficiency improved considerably as the number of generation increased. The cytotoxicity of JCPD in different concentration was tested for HeLa cell line. JCPD was complexed with a lacZ gene carrying plasmid and tested for transfection efficiency using quantitative β-galactosidase expression assay. Additionally, confocal microscopy results revealed that JCPD effectively delivered green fluorescent protein-expressing plasmid into HeLa cells and produced fluorescent signal with satisfactory efficiency. The highest transfection efficiency was obtained from JCPDs G4 and G5, which mixed with expression plasmid vectors at a 10/1 weight ratio. These results indicated that under optimized conditions, JCPD can be considered as an efficient transfection reagent and can be effectively used for gene delivery applications.

  17. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    PubMed Central

    Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

    2012-01-01

    Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

  18. Chitosan-thioglycolic acid conjugate: an alternative carrier for oral nonviral gene delivery?

    PubMed

    Martien, Ronny; Loretz, Brigitta; Thaler, Marlene; Majzoob, Sayeh; Bernkop-Schnürch, Andreas

    2007-07-01

    Regarding safety concerns, nonviral gene delivery vehicles that have the required efficiency and safety for use in human gene therapy are being widely investigated. The aim of this study was to synthesize and evaluate a thiolated chitosan to improve the efficacy of oral gene delivery systems. Thiolated chitosan was synthesized by introducing thioglycolic acid (TGA) to chitosan via amide bond formation mediated by a carbodiimide. Based on this conjugate, nanoparticles with pDNA were generated at pH 4.0 and 5.0. Cytotoxicity of the thiolated chitosan/pDNA nanoparticles on Caco-2 cells was evaluated. The diameter of thiolated chitosan/pDNA nanoparticles was in the range of 100-200 nm. The zeta potential was determined to be 5-6 mV. Due to stability toward nucleases, the transfection rate of thiolated chitosan/pDNA nanoparticles was fivefold higher than that of unmodified chitosan/pDNA nanoparticles. Lactate dehydrogenase tests for thiolated chitosan/pDNA (pH 4.0 and 5.0) showed that (3.79 +/- 0.23)% and (2.9 +/- 0.13)% cell damage. According to these results, thiolated chitosan represents promising excipients for preparation DNA nanoparticles in nonviral gene delivery system.

  19. Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery

    PubMed Central

    Dai, Hui; Jiang, Xuan; Tan, Geoffrey CY; Chen, Yong; Torbenson, Michael; Leong, Kam W; Mao, Hai-Quan

    2006-01-01

    The goal of this study was to examine the efficacy of liver-targeted gene delivery by chitosan-DNA nanoparticles through retrograde intrabiliary infusion (RII). The transfection efficiency of chitosan-DNA nanoparticles, as compared with PEI-DNA nanoparticles or naked DNA, was evaluated in Wistar rats by infusion into the common bile duct, portal vein, or tail vein. Chitosan-DNA nanoparticles administrated through the portal vein or tail vein did not produce detectable luciferase expression. In contrast, rats that received chitosan-DNA nanoparticles showed more than 500 times higher luciferase expression in the liver 3 days after RII; and transgene expression levels decreased gradually over 14 days. Luciferase expression in the kidney, lung, spleen, and heart was negligible compared with that in the liver. RII of chitosan-DNA nanoparticles did not yield significant toxicity and damage to the liver and biliary tree as evidenced by liver function analysis and histopathological examination. Luciferase expression by RII of PEI-DNA nanoparticles was 17-fold lower than that of chitosan-DNA nanoparticles on day 3, but it increased slightly over time. These results suggest that RII is a promising routine to achieve liver-targeted gene delivery by non-viral nanoparticles; and both gene carrier characteristics and mode of administration significantly influence gene delivery efficiency. PMID:17369870

  20. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues

    PubMed Central

    Lai, Samuel K.; Wang, Ying-Ying; Hanes, Justin

    2009-01-01

    Mucus is a viscoelastic and adhesive gel that protects the lung airways, gastrointestinal (GI) tract, vagina, eye and other mucosal surfaces. Most foreign particulates, including conventional particle-based drug delivery systems, are efficiently trapped in human mucus layers by steric obstruction and/or adhesion. Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on anatomical location, thereby strongly limiting the duration of sustained drug delivery locally. A number of debilitating diseases could be treated more effectively and with fewer side effects if drugs and genes could be more efficiently delivered to the underlying mucosal tissues in a controlled manner. This review first describes the tenacious mucus barrier properties that have precluded the efficient penetration of therapeutic particles. It then reviews the design and development of new mucus-penetrating particles that may avoid rapid mucus clearance mechanisms, and thereby provide targeted or sustained drug delivery for localized therapies in mucosal tissues. PMID:19133304

  1. Endothelial lineage cell as a vehicle for systemic delivery of cancer gene therapy.

    PubMed

    Dudek, Arkadiusz Z

    2010-09-01

    A major limitation of cancer gene therapy is the difficulty of delivering a therapeutic gene to distant sites of metastatic disease. A promising strategy to address this difficulty is to use expanded ex vivo cells to produce a therapeutic protein. As with other approaches to gene therapy, this strategy is attractive when the therapeutic protein is unstable ex vivo or has a short circulating half life in vivo. The initial step to develop a cancer gene therapy using autologous cell delivery is the identification of a cell type that migrates to the tumor site, is readily available for harvesting, and is manipulated easily ex vivo. Recent evidence suggests that endothelial progenitor, precursor, and blood outgrowth endothelial cells are attracted to the tumor vasculature by its angiogenic drive. Here, we review recent advances in the study of circulating endothelial cell-mediated tumor vasculogenesis and discuss the advantages and challenges of bringing endothelial lineage-based cancer gene therapy closer to clinical application.

  2. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy.

    PubMed

    Yoon, Ji Young; Yang, Keum-Jin; Park, Shi-Nae; Kim, Dong-Kee; Kim, Jong-Duk

    Dexamethasone (Dex)-loaded PHEA-g-C18-Arg8 (PCA) nanoparticles (PCA/Dex) were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18) core with Dex and an arginine 8 (Arg8) peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA). The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120-140 nm and a zeta potential of 20-25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene) for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr) than did Dex-free PCA nanoparticles and Lipofectamine(®) reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30 h, and its signaling pathway resulted in an increase in phosphorylation of Akt, observed by Western blotting. Thus, Dex within PCA/Dex/gene nanoparticles created an anti-inflammatory effect and enhanced gene expression.

  3. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy

    PubMed Central

    Yoon, Ji Young; Yang, Keum-Jin; Park, Shi-Nae; Kim, Dong-Kee; Kim, Jong-Duk

    2016-01-01

    Dexamethasone (Dex)-loaded PHEA-g-C18-Arg8 (PCA) nanoparticles (PCA/Dex) were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18) core with Dex and an arginine 8 (Arg8) peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA). The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120–140 nm and a zeta potential of 20–25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene) for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr) than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30 h, and its signaling pathway resulted in an increase in phosphorylation of Akt, observed by Western blotting. Thus, Dex within PCA/Dex/gene nanoparticles created an anti-inflammatory effect and enhanced gene expression. PMID:27895484

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

    NASA Astrophysics Data System (ADS)

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

    2005-06-01

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

  5. The potential of adeno-associated viral vectors for gene delivery to muscle tissue

    PubMed Central

    Nahid, M Abu; Gao, Guangping

    2014-01-01

    Introduction Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8+ T-cell response against AAV capsid in humans. Areas covered In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Expert opinion Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application. PMID:24386892

  6. The potential of adeno-associated viral vectors for gene delivery to muscle tissue.

    PubMed

    Wang, Dan; Zhong, Li; Nahid, M Abu; Gao, Guangping

    2014-03-01

    Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8(+) T-cell response against AAV capsid in humans. In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.

  7. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    PubMed Central

    2012-01-01

    Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns. PMID:22853641

  8. Potential for germ line transmission after intramyocardial gene delivery by adeno-associated virus.

    PubMed

    Pachori, Alok S; Melo, Luis G; Zhang, Lunan; Loda, Massimo; Pratt, Richard E; Dzau, Victor J

    2004-01-16

    Intramyocardial injection of adeno-associated virus (AAV) has been shown to be an effective strategy for cardiac gene delivery. This approach leads to long-term gene expression in the heart, offering the possibility of chronic gene therapy. However, the long-term safety of this approach with regard to vector bio-distribution and extracardiac transgene expression has not been evaluated. To examine these issues, 8-week-old male Sprague-Dawley rats were injected intramyocardially with either 4x10(11) particles of AAV-2-lacZ or saline at five locations in the anterioposterior apical region of the left ventricle. Animals were sacrificed at 3 and 6 months after gene transfer, tissues were harvested and analyzed for lacZ expression by semi-quantitative RT-PCR and beta-galactosidase activity using X-gal staining. We observed high level of transgene expression in the myocardium at 3 months after gene transfer, which persisted up to 6 months of follow-up. Also, significantly we detected lacZ expression and beta-galactosidase activity in extracardiac tissues such as liver, kidney, and testes at 6 months. More significantly, late transgene expression was detected in cellular elements of the seminiferous tubule, including Sertoli cells and spermatogonia like cells. These data demonstrate the efficacy of AAV-2 delivery for long-term myocardial gene therapy, but raise concerns about the possibility of ectopic transgene expression and germ cell line infection.

  9. Ligands located within a cholesterol domain enhance gene delivery to the target tissue

    PubMed Central

    Xu, Long; Betker, Jamie; Yin, Hao; Anchordoquy, Thomas J.

    2012-01-01

    Targeted gene delivery provides enormous potential for clinical treatment of many incurable diseases. Liposomes formulated with targeting ligands have been tested extensively both in vitro and in vivo, and many studies have strived to identify more efficacious ligands. However, the environment of the ligand within the delivery vehicle is generally not considered, and this study assesses the effect of ligand micoenvironment by utilizing a lipoplex possessing a cholesterol domain. Our recent work has shown that the presence of the targeting ligand within the cholesterol domain promotes more productive transfection in cultured cells. In the present study, lipoplexes having the identical lipid composition were formulated with different conjugates of the folate ligand such that the ligand was included in, or excluded from, the cholesterol domain. The effect of locating the ligand within the cholesterol domain was then tested in a xenograft tumor model in mice. Lipoplexes that included the ligand within the cholesterol domain showed significantly higher luciferase expression and plasmid accumulation in tumors as compared to lipoplexes in which the ligand was excluded from the domain. These results demonstrate that the microenvironment of the ligand can affect gene delivery to tumors, and show that ligand-mediated delivery can be enhanced by locating targeting ligands within a cholesterol domain. PMID:22440429

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

    PubMed Central

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

    2015-01-01

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

  11. Selective atonal gene delivery improves balance function in a mouse model of vestibular disease.

    PubMed

    Schlecker, C; Praetorius, M; Brough, D E; Presler, R G; Hsu, C; Plinkert, P K; Staecker, H

    2011-09-01

    Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. To more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3'-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored, we delivered atoh1 using a new adenovirus vector, based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. To further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell-specific glial fibrillary acid protein promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance, as measured by time on rotarod compared with untreated controls.

  12. Selective atonal gene delivery improves balance function in a mouse model of vestibular disease

    PubMed Central

    Schlecker, Christina; Praetorius, Mark; Brough, Douglas E.; Presler, Robert G.; Hsu, Chi; Plinkert, Peter K.; Staecker, Hinrich

    2011-01-01

    Loss of balance is often due to loss of vestibular hair cells. In mammals, regeneration of functional hair cells in the mature sensory epithelium is limited; therefore, loss of sensory cells can lead to debilitating balance problems. Delivery of the transcription factor atonal (atoh1) after aminoglycoside ototoxicity has previously been shown to induce the transdifferentiation of supporting cells into new hair cells and restore function. A problem with mouse aminoglycoside models is that the partial loss of hair cells seen in human disease is difficult to establish consistently. In order to more closely mirror human clinical balance dysfunction, we have used systemic application of 3,3’-iminodipropionitrile (IDPN), a vestibulotoxic nitrile compound known to cause vestibular hair cell loss, to induce a consistent partial loss of vestibular hair cells. To determine if balance function could be restored we delivered atoh1 using a new adenovirus vector based on Ad28. The Ad28 adenovector is based on a human serotype with a low seroprevalence that appears to target gene delivery to vestibular supporting cells. In order to further provide cell type selectivity of gene delivery, we expressed atoh1 using the supporting cell specific glial fibrillary acid protein (GFAP) promoter. Delivery of this vector to IDPN-damaged vestibular organs resulted in a significant recovery of vestibular hair cells and restoration of balance as measured by time on rotarod compared to untreated controls. PMID:21472006

  13. F2 Gel Matrix a Novel Delivery System for Immune and Gene Vaccinations.

    PubMed

    Tuorkey, Muobarak J

    2016-01-01

    Exploiting the immune system to abolish cancer growth via vaccination is a promising strategy but that is limited by many clinical issues. For DNA vaccines, viral vectors as a delivery system mediate a strong immune response due to their protein structure, which could afflect the cellular uptake of the genetic vector or even induce cytotoxic immune responses against transfected cells. Recently, synthetic DNA delivery systems have been developed and recommended as much easier and simple approaches for DNA delivery compared with viral vectors. These are based on the attraction of the positively charged cationic transfection reagents to negatively charged DNA molecules, which augments the cellular DNA uptake. In fact, there are three major cellular barriers which hinder successful DNA delivery systems: low uptake across the plasma membrane; inadequate release of DNA molecules with limited stability; and lack of nuclear targeting. Recently, a polysaccharide polymer produced by microalgae has been synthesized in a form of polymeric fiber material polyNacetyl glucosamine (pGlcNAc). Due its unique properties, the F2 gel matrix was suggested as an effective delivery system for immune and gene vaccinations.

  14. Genetic engineering of mesenchymal stem cells by non-viral gene delivery.

    PubMed

    Wang, Weiwei; Xu, Xun; Li, Zhengdong; Lendlein, Andreas; Ma, Nan

    2014-01-01

    Mesenchymal stem cells (MSCs) are an ideal cell source for tissue engineering and regenerative medicine as they possess self-renewal properties and multilineage differentiation potential. They can be isolated from various tissues and expanded easily through normal cell culture techniques. Genetic modifications of MSCs to further improve their therapeutic efficacy have been widely studied and extensively researched. Compared to viral gene delivery methods, non-viral methods generate less toxicity and immunogenicity and thus represent a promising and effective tool for the genetic engineering of MSCs. In the last decades, various non-viral gene delivery strategies have been developed and some of them have been applied for MSC transfection. This paper gives an overview of the techniques, influencing factors and potential applications of non-viral methods used for the genetic engineering of MSCs.

  15. HSV-1 amplicon vectors: a promising and versatile tool for gene delivery.

    PubMed

    Cuchet, Delphine; Potel, Corinne; Thomas, Joélle; Epstein, Alberto L

    2007-07-01

    Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. They carry no virus genes in the vector genome and are, therefore, not toxic to the infected cells or pathogenic for the transduced organisms, making these vectors safe. In addition, the large transgenic capacity of amplicons, which allow delivery of < or = 150 Kbp of foreign DNA, make these vectors one of the most powerful, interesting and versatile gene delivery platforms. Here, the authors present recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review illustrates the many possible applications that are presently being developed with amplicons and discuss the many difficulties still pending to be solved in order to achieve stable and physiologically regulated transgenic expression.

  16. Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection.

    PubMed

    Cervia, Lisa D; Chang, Chun-Chi; Wang, Liangli; Yuan, Fan

    2017-01-01

    A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA.

  17. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

    NASA Astrophysics Data System (ADS)

    Guan, Lingmei; Huang, Saipeng; Chen, Zhao; Li, Yanchao; Liu, Ke; Liu, Yang; Du, Libo

    2015-09-01

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  18. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

    PubMed

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

    2013-03-01

    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  19. Hydrodynamic gene delivery in human skin using a hollow microneedle device.

    PubMed

    Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

    2017-02-27

    Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery.

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

    PubMed

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

    2014-08-01

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

  1. A translatable, closed recirculation system for AAV6 vector-mediated myocardial gene delivery in the large animal.

    PubMed

    Swain, JaBaris D; Katz, Michael G; White, Jennifer D; Thesier, Danielle M; Henderson, Armen; Stedman, Hansell H; Bridges, Charles R

    2011-01-01

    Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvements in cardiac function observed in vector-mediated therapeutic transgene delivery to the murine myocardium. A large animal model validating these results is the likely segue into clinical application. However, controversy still exists regarding a suitable method of vector-mediated cardiac gene delivery that provides for efficient, global gene transfer to the large animal myocardium that is also clinically translatable and practical. Intramyocardial injection and catheter-based coronary delivery techniques are attractive alternatives with respect to their clinical applicability; yet, they are fraught with numerous challenges, including concerns regarding collateral gene expression in other organs, low efficiency of vector delivery to the myocardium, inhomogeneous expression, and untoward immune response secondary to gene delivery. Cardiopulmonary bypass (CPB) delivery with dual systemic and isolated cardiac circuitry precludes these drawbacks and has the added advantage of allowing for control of the pharmacological milieu, multiple pass recirculation through the coronary circulation, the selective addition of endothelial permeabilizing agents, and an increase in vector residence time. Collectively, these mechanics significantly improve the efficiency of global, vector-mediated cardiac gene delivery to the large animal myocardium, highlighting a potential therapeutic strategy to be extended to some heart failure patients.

  2. Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes.

    PubMed

    Ross, Nikki L; Sullivan, Millicent O

    2015-12-07

    For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin postmitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins.

  3. Synthesis of Bisethylnorspermine Lipid Prodrug as Gene Delivery Vector Targeting Polyamine Metabolism in Breast Cancer

    PubMed Central

    Dong, Yanmei; Zhu, Yu; Li, Jing; Zhou, Qing-Hui; Wu, Chao; Oupický, David

    2013-01-01

    Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery. PMID:22545813

  4. Microneedle delivery of plasmid DNA to living human skin: formulation coating, skin insertion and gene expression

    PubMed Central

    Pearton, Marc; Saller, Verena; Coulman, Sion A; Gateley, Chris; Anstey, Alexander V; Zarnitsyn, Vladimir; Birchall, James C

    2012-01-01

    Microneedle delivery of nucleic acids, in particular plasmid DNA (pDNA), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunization. In this study excised human skin explants were used to investigate and optimize key parameters that will determine stable and effective microneedle-facilitated pDNA delivery. These include (i) high dose-loading of pDNA onto microneedle surfaces, (ii) stability and functionality of the coated pDNA, (iii) skin penetration capability of pDNA-coated microneedles, and (iv) efficient gene expression in human skin. Optimization of a dip-coating method enabled significant increases in the loading capacity, up to 100 micrograms of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (<1 Newton) insertion forces. The physical stability of the coated pDNA was partially compromised on storage, although this was improved through the addition of saccharide excipients without detriment to the biological functionality of pDNA. The pDNA-coated microneedles facilitated reporter gene expression in viable human skin. The efficiency of gene expression from coated microneedles will depend upon suitable DNA loading, efficient and reproducible skin puncture and rapid in situ dissolution of the plasmid at the site of delivery. PMID:22516089

  5. Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery.

    PubMed

    Yang, Xinchao; Zhao, Nana; Xu, Fu-Jian

    2014-06-07

    Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

  6. Ultrasound guided site specific gene delivery system using adenoviral vectors and commercial ultrasound contrast agents.

    PubMed

    Howard, Candace M; Forsberg, Flemming; Minimo, Corrado; Liu, Ji-Bin; Merton, Daniel A; Claudio, Pier Paolo

    2006-11-01

    We have evaluated if ultrasound imaging (US) and various commercially available contrast microbubbles can serve as a non-invasive systemically administered delivery vehicle for site-specific adenoviral-mediated gene transfer in vitro and in vivo. The contrast agents were tested for their ability to enclose and to protect an adenoviral vector carrying the GFP marker gene (Ad-GFP) into the microbubbles. We have also evaluated the ability of the innate immune system to inactivate free adenoviruses as well as unenclosed viruses adsorbed on the surface of the contrast agents and in turn the ability of the microbubbles to enclose and to protect the viral vectors from such agents. In vitro as well as in vivo, innate components of the immune system were able to serve as inactivating agents to clear free viral particles and unenclosed adenoviruses adsorbed on the microbubbles' surface. Systemic delivery of Ad-GFP enclosed into microbubbles in the tail vein of nude mice resulted in specific targeting of the GFP transgene. Both fluorescence microscopy and GFP immunohistochemistry demonstrated US guided specific transduction in the targeted cells only, with no uptake in either heart, lungs or liver using complement-pretreated Ad-GFP microbubbles. This approach enhances target specificity of US microbubble destruction as a delivery vehicle for viral-mediated gene transfer.

  7. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

    PubMed Central

    Nitta, Sachiko Kaihara; Numata, Keiji

    2013-01-01

    There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin), protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin). The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed. PMID:23344060

  8. Particle tracking in drug and gene delivery research: State-of-the-art applications and methods.

    PubMed

    Schuster, Benjamin S; Ensign, Laura M; Allan, Daniel B; Suk, Jung Soo; Hanes, Justin

    2015-08-30

    Particle tracking is a powerful microscopy technique to quantify the motion of individual particles at high spatial and temporal resolution in complex fluids and biological specimens. Particle tracking's applications and impact in drug and gene delivery research have greatly increased during the last decade. Thanks to advances in hardware and software, this technique is now more accessible than ever, and can be reliably automated to enable rapid processing of large data sets, thereby further enhancing the role that particle tracking will play in drug and gene delivery studies in the future. We begin this review by discussing particle tracking-based advances in characterizing extracellular and cellular barriers to therapeutic nanoparticles and in characterizing nanoparticle size and stability. To facilitate wider adoption of the technique, we then present a user-friendly review of state-of-the-art automated particle tracking algorithms and methods of analysis. We conclude by reviewing technological developments for next-generation particle tracking methods, and we survey future research directions in drug and gene delivery where particle tracking may be useful.

  9. Nano-biomimetic carriers are implicated in mechanistic evaluation of intracellular gene delivery

    NASA Astrophysics Data System (ADS)

    Alipour, Mohsen; Hosseinkhani, Saman; Sheikhnejad, Reza; Cheraghi, Roya

    2017-01-01

    Several tissue specific non-viral carriers have been developed for gene delivery purposes. However, the inability to escape endosomes, undermines the efficacy of these carriers. Researchers inspired by HIV and influenza virus, have randomly used Gp41 and H5WYG fusogenic peptides in several gene delivery systems without any rational preference. Here for the first time, we have genetically engineered two Nano-biomimetic carriers composed of either HWYG (HNH) or Gp41 (GNH) that precisely provide identical conditions for the study and evaluation of these fusogenic peptides. The luciferase assay demonstrated a two-fold higher transfection efficiency of HNH compared to GNH. These nanocarriers also displayed equivalent properties in terms of DNA binding ability and DNA protection against serum nucleases and formed similar nanoparticles in terms of surface charge and size. Interestingly, hemolysis and cellular analysis demonstrated both of nanoparticles internalized into cells in similar rate and escaped from endosome with different efficiency. Furthermore, the structural analysis revealed the mechanisms responsible for the superior endosomal escaping capability of H5WYG. In conclusion, this study describes the rationale for using H5WYG peptide to deliver nucleic acids and suggests that using nano-biomimetic carriers to screen different endosomal release peptides, improves gene delivery significantly.

  10. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering.

    PubMed

    Nitta, Sachiko Kaihara; Numata, Keiji

    2013-01-14

    There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin), protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin). The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed.

  11. Nano-biomimetic carriers are implicated in mechanistic evaluation of intracellular gene delivery

    PubMed Central

    Alipour, Mohsen; Hosseinkhani, Saman; Sheikhnejad, Reza; Cheraghi, Roya

    2017-01-01

    Several tissue specific non-viral carriers have been developed for gene delivery purposes. However, the inability to escape endosomes, undermines the efficacy of these carriers. Researchers inspired by HIV and influenza virus, have randomly used Gp41 and H5WYG fusogenic peptides in several gene delivery systems without any rational preference. Here for the first time, we have genetically engineered two Nano-biomimetic carriers composed of either HWYG (HNH) or Gp41 (GNH) that precisely provide identical conditions for the study and evaluation of these fusogenic peptides. The luciferase assay demonstrated a two-fold higher transfection efficiency of HNH compared to GNH. These nanocarriers also displayed equivalent properties in terms of DNA binding ability and DNA protection against serum nucleases and formed similar nanoparticles in terms of surface charge and size. Interestingly, hemolysis and cellular analysis demonstrated both of nanoparticles internalized into cells in similar rate and escaped from endosome with different efficiency. Furthermore, the structural analysis revealed the mechanisms responsible for the superior endosomal escaping capability of H5WYG. In conclusion, this study describes the rationale for using H5WYG peptide to deliver nucleic acids and suggests that using nano-biomimetic carriers to screen different endosomal release peptides, improves gene delivery significantly. PMID:28128339

  12. Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes

    PubMed Central

    Ross, Nikki L.; Sullivan, Millicent O.

    2016-01-01

    For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin post-mitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins. PMID:26465823

  13. Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier.

    PubMed

    Kim, Tae-Il; Baek, Jung-Un; Zhe Bai, Cheng; Park, Jong-Sang

    2007-04-01

    We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2-R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200nm at a charge ratio of 150. PPI2-R displayed 80-90% cell viability at even a 150microg/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8-214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10microg pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2-3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier.

  14. In Vivo Fate of Carbon Nanotubes with Different Physicochemical Properties for Gene Delivery Applications.

    PubMed

    Cifuentes-Rius, Anna; Boase, Nathan R B; Font, Ines; Coronas, Nuria; Ramos-Perez, Victor; Thurecht, Kristofer J; Borrós, Salvador

    2017-04-05

    Gene therapy has arisen as a pioneering technique to treat diseases by direct employment of nucleic acids as medicine. The major historical problem is to develop efficient and safe systems for the delivery of therapeutic genes into the target cells. Carbon nanotubes (CNTs) have demonstrated considerable promise as delivery vectors due to their (i) high aspect ratio and (ii) capacity to translocate through plasma membranes, known as the nanoneedle effect. To leverage these advantages, close attention needs to be paid to the physicochemical characteristics of the CNTs used. CNTs with different diameters (thinner and thicker) were treated by chemical oxidation to produce shorter fragments. Rigid (thick) and flexible (thin) CNTs, and their shortened versions, were coated with polyallylamine (ppAA) by plasma-enhanced chemical vapor deposition. The ppAA coating leads to a positively charged CNT surface that is able to electrostatically bind the green fluorescent protein plasmid reporter. This study shows how rigidity and length can affect their (i) behavior in biological media, (ii) ability to transfect in vitro, and (iii) biodistribution in vivo. This study also generates a set of basic design rules for the development of more efficient CNT-based gene-delivery vectors.

  15. Gene delivery to the spinal cord using MRI-guided focused ultrasound

    PubMed Central

    Weber-Adrian, Danielle; Thévenot, Emmanuel; O'Reilly, Meaghan A.; Oakden, Wendy; Akens, Margarete K.; Ellens, Nicholas; Markham-Coultes, Kelly; Burgess, Alison; Finkelstein, Joel; Yee, Albert J.M.; Whyne, Cari M.; Foust, Kevin D.; Kaspar, Brian K.; Stanisz, Greg J.; Chopra, Rajiv; Hynynen, Kullervo; Aubert, Isabelle

    2015-01-01

    Non-invasive gene delivery across the blood-spinal cord barrier (BSCB) remains a challenge for treatment of spinal cord injury or disease. Here, we demonstrate the use of magnetic resonance imaging-guided focused ultrasound (MRIgFUS) to mediate non-surgical gene delivery to the spinal cord, using self-complementary adeno-associated virus serotype 9 (scAAV9). scAAV9 encoding green fluorescent protein (GFP) was injected intravenously in rats. MRIgFUS allows for transient, targeted permeabilization of the BSCB through the interaction of FUS with systemically-injected Definity® lipid-shelled microbubbles. scAAV9-GFP was delivered at 3 dosages: 4×108, 2×109, and 7×109 vector genomes per gram (VG/g). Viral delivery at 2×109 and 7×109 VG/g leads to robust GFP expression in the targeted length and side of the spinal cord. At a dose of 2×109 VG/g, GFP expression was found in 36% of oligodendrocytes, and in 87% of neurons in FUS-treated areas. FUS applications to the spinal cord could address a long-term goal of gene therapy: delivering vectors from the circulation to diseased areas in a noninvasive manner. PMID:25781651

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

    PubMed Central

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

    2016-01-01

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

  17. Prospects for Lentiviral Vector Mediated Prostaglandin F Synthase Gene Delivery in Monkey Eyes In vivo

    PubMed Central

    Lee, Eun Suk; Rasmussen, Carol A.; Filla, Mark S.; Slauson, Sarah R.; Kolb, Aaron W.; Peters, Donna M.; Kaufman, Paul L.; Gabelt, B’Ann True; Brandt, Curtis R.

    2014-01-01

    Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of IOP, may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions. PMID:24559478

  18. Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery

    NASA Astrophysics Data System (ADS)

    Yang, Xinchao; Zhao, Nana; Xu, Fu-Jian

    2014-05-01

    Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

  19. Gene Delivery Mediated by Recombinant Silk Proteins Containing Cationic and Cell Binding Motifs

    PubMed Central

    Numata, Keiji; Hamasaki, Juliana; Subramanian, Balajikarthick; Kaplan, David L

    2010-01-01

    Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems. PMID:20457191

  20. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    PubMed Central

    Burke, Gaelen R.; Strand, Michael R.

    2012-01-01

    Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1) they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2) they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution. PMID:26467950

  1. Instructive nanofibrous scaffold comprising runt-related transcription factor 2 gene delivery for bone tissue engineering.

    PubMed

    Monteiro, Nelson; Ribeiro, Diana; Martins, Albino; Faria, Susana; Fonseca, Nuno A; Moreira, João N; Reis, Rui L; Neves, Nuno M

    2014-08-26

    Inducer molecules capable of regulating mesenchymal stem cell differentiation into specific lineages have proven effective in basic science and in preclinical studies. Runt-related transcription factor 2 (RUNX2) is considered to be the central gene involved in the osteoblast phenotype induction, which may be advantageous for inducing bone tissue regeneration. This work envisions the development of a platform for gene delivery, combining liposomes as gene delivery devices, with electrospun nanofiber mesh (NFM) as a tissue engineering scaffold. pDNA-loaded liposomes were immobilized at the surface of functionalized polycaprolactone (PCL) NFM. Human bone-marrow-derived mesenchymal stem cells (hBMSCs) cultured on RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFM showed enhanced levels of metabolic activity and total protein synthesis. RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFMs induce a long-term gene expression of eGFP and RUNX2 by cultured hBMSCs. Furthermore, osteogenic differentiation of hBMSCs was also achieved by the overexpression of other osteogenic markers in medium free of osteogenic supplementation. These findings demonstrate that surface immobilization of RUNX2 plasmid onto elestrospun PCL NFM can produce long-term gene expression in vitro, which may be employed to enhance the osteoinductive properties of scaffolds used for bone tissue engineering strategies.

  2. Human Artificial Chromosomes for Gene Delivery and the Development of Animal Models

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-01-01

    Random integration of conventional gene delivery vectors such as viruses, plasmids, P1 phage-derived artificial chromosomes, bacterial artificial chromosomes and yeast artificial chromosomes can be associated with transgene silencing. Furthermore, integrated viral sequences can activate oncogenes adjacent to the insertion site resulting in cancer. Various human artificial chromosomes (HACs) exhibit several potential characteristics desired for an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci with their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. HACs have been generated mainly using either a “top-down approach” (engineered chromosomes), or a “bottom-up approach” (de novo artificial chromosomes). The recent emergence of stem cell–based tissue engineering has opened up new avenues for gene and cell therapies. This review describes the lessons learned and prospects identified mainly from studies in the construction of HACs and HAC-mediated gene expression systems in cultured cells, as well as in animals. PMID:21750534

  3. Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example.

    PubMed

    Aissaoui, Abderrahim; Oudrhiri, Noufissa; Petit, Laure; Hauchecorne, Michelle; Kan, Erwan; Sainlos, Matthieu; Julia, Sébastien; Navarro, Jean; Vigneron, Jean-Pierre; Lehn, Jean-Marie; Lehn, Pierre

    2002-02-01

    Artificial self-assembling systems are currently widely investigated as an alternative approach to recombinant viruses for gene transfection in vitro and in vivo. Cationic lipids are particularly attractive, as a great variety of well-characterized reagents can be synthesized from there. Over the last few years, numerous cationic lipid systems have been developed and shown to be efficient for in vitro transfection. However, although some promising results have been reported in the in vivo setting (even in clinical gene therapy trials in man), the in vivo use of cationic lipid-based systems is still problematic, especially when considering the systemic route of administration. Herein, we summarize our own research on a particular class of cationic lipids, cholesterol derivatives characterized by polar headgroups with guanidinium functions, in order to illustrate the basic principles of and the positive results already obtained by cationic lipid-mediated gene delivery as well as the remaining problems that need to be urgently resolved, particularly as regards the systemic administration. In this forward-looking review, we also discuss the present efforts to develop modular systems for improved in vivo transfection. Indeed, lipid-based vectors offer the possibility to create sophisticated modular gene delivery systems capable of self-assembly via hydrophobic interaction between their components, the role of the different functional elements being to help in overcoming the distinct extracellular and cellular barriers to in vivo gene transfection into the various somatic target tissues.

  4. Human artificial chromosomes for gene delivery and the development of animal models.

    PubMed

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-09-01

    Random integration of conventional gene delivery vectors such as viruses, plasmids, P1 phage-derived artificial chromosomes, bacterial artificial chromosomes and yeast artificial chromosomes can be associated with transgene silencing. Furthermore, integrated viral sequences can activate oncogenes adjacent to the insertion site resulting in cancer. Various human artificial chromosomes (HACs) exhibit several potential characteristics desired for an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci with their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. HACs have been generated mainly using either a "top-down approach" (engineered chromosomes), or a "bottom-up approach" (de novo artificial chromosomes). The recent emergence of stem cell-based tissue engineering has opened up new avenues for gene and cell therapies. This review describes the lessons learned and prospects identified mainly from studies in the construction of HACs and HAC-mediated gene expression systems in cultured cells, as well as in animals.

  5. Enhanced gene delivery of low molecular weight PEI by flower-like ZnO microparticles.

    PubMed

    Chen, Ming; Tang, Yaqin; Wang, Tingting; Long, Qipeng; Zeng, Ziying; Chen, Houwen; Feng, Xuli

    2016-12-01

    Low molecular weight (1.8 kDa) branched polyethylenimine (PEI) has been used as non-viral vector for gene delivery because of its low toxicity, however, its further application in biomedical field has been restricted due to its low gene transfection efficiency. Herein, ZnO microflowers were prepared to increase the gene expression level mediated by PEI. Four methods have been applied to tune the shape of ZnO microstructures. Scanning electron microscopy (SEM) demonstrated the successful preparation of four kinds of flower like ZnO microparticles. By loading PEI/pDNA into ZnO microparticles, the formed new complexes showed enhanced gene transfection compared to PEI/pDNA alone. Cell uptaking experiments explained a possible mechanism that the tips of ZnO microflowers penetrated into the surface of cells, thus facilitating the entry of gene cargo into cells. These findings highlight the potential of needle like microstructure as adjuvant for efficient biomacromolecular delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation.

    PubMed

    Molotkov, Dmitry A; Yukin, Alexey Y; Afzalov, Ramil A; Khiroug, Leonard S

    2010-09-17

    Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells. The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

  7. A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery

    NASA Astrophysics Data System (ADS)

    Namiki, Yoshihisa; Namiki, Tamami; Yoshida, Hiroshi; Ishii, Yukiko; Tsubota, Akihito; Koido, Shigeo; Nariai, Kouichi; Mitsunaga, Makoto; Yanagisawa, Satoru; Kashiwagi, Hideyuki; Mabashi, Yasuo; Yumoto, Yoko; Hoshina, Sadayori; Fujise, Kiyotaka; Tada, Norio

    2009-09-01

    Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

  8. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

    PubMed Central

    Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

    2016-01-01

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release. PMID:26883682

  9. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

    NASA Astrophysics Data System (ADS)

    Yang, Yi; Zhao, Hang; Jia, Yanpeng; Guo, Qingfa; Qu, Ying; Su, Jing; Lu, Xiaoling; Zhao, Yongxiang; Qian, Zhiyong

    2016-02-01

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release.

  10. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release.

    PubMed

    Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

    2016-02-17

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release.

  11. Integrated optical transfection system using a microlens fiber combined with microfluidic gene delivery

    PubMed Central

    Ma, N.; Ashok, P. C.; Stevenson, D. J.; Gunn-Moore, F. J.; Dholakia, K.

    2010-01-01

    Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free space bulk optical setup. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. Here we report the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining a microlens fiber based optical transfection system with a micro-capillary based microfluidic system. A fiber based illumination system is also incorporated in the system in order to achieve visual identification of the cell boundaries during transfection. A novel fabrication method is devised to obtain easy and inexpensive fabrication of microlensed fibers, which can be used for femtosecond optical transfection. This fabrication method offers the flexibility to fabricate a microlens which can focus ultra-short laser pulses at a near infrared wavelength to a small focal spot (~3 µm) whilst keeping a relatively large working distance (~20 µm). The transfection efficiency of the integrated system with localized plasmid DNA delivery, is approximately 50%, and is therefore comparable to that of a standard free space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of DNA for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, including the dosing of tissue slices, targeted drug delivery, and targeted gene therapy in vivo. PMID:21258501

  12. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    PubMed

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety.

  13. Rational design of didodecyldimethylammonium bromide-based nanoassemblies for gene delivery.

    PubMed

    Jin, Yiguang; Wang, Shuangmiao; Tong, Li; Du, Lina

    2015-02-01

    Nonviral gene vectors are a hot topic for gene delivery. High cost and low transfection efficiency hinder the application of them. The aim of this study was to find out the optimal gene vectors with lower cost and more effective gene delivery than commonly used gene vectors. A cheap cationic lipid, didodecyldimethylammonium bromide (DDAB) was the basic component and the other components included oleic acid (OA), cholesterol (Chol), cholesteryl succinyl poly(ethylene glycol) 1500 (CHS-PEG), poly(D,L-lactide-co-glycolide)-methoxy-poly(ethylene glycol) (PLGA-PEG). The combinations of DDAB/OA/Chol, DDAB/OA/CHS-PEG and DDAB/PLGA-PEG were adopted to prepare the nanoassemblies named CNA, CPNA and PPNA, respectively. The optimal component ratios were screened out according to their Langmuir monolayer behavior. The optimal preparation method of nanoassemblies involved firstly compressing DNA or siRNA with the cationic lipid (DDAB) and secondly being coated with the helper lipids (OA and CHS-PEG) or the helper polymer (PLGA-PEG). The complexes of genes and cationic lipids were encapsulated into the core of CPNA and PPNA. The optimal gene vectors (CPNA and PPNA) with small sizes, low negative surface charges and non-exposure of cationic lipids were achieved. They had the advantages of no cytotoxicity, high transfection efficiency and low cost. More importantly, CPNA and PPNA were not sensitive to serum and showed the similar or higher transfection efficiency of pDNA and siRNA compared to Lipofectamine 2000. CPNA could mainly enter cell plasma based on endocytosis. The rational design method is useful for the design and optimization of DDAB-based gene carriers and other cationic lipid-based carriers.

  14. Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation.

    PubMed

    Shapiro, Galina; Wong, Andrew W; Bez, Maxim; Yang, Fang; Tam, Sarah; Even, Lisa; Sheyn, Dmitriy; Ben-David, Shiran; Tawackoli, Wafa; Pelled, Gadi; Ferrara, Katherine W; Gazit, Dan

    2016-02-10

    More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble

  15. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    NASA Astrophysics Data System (ADS)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  16. A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors.

    PubMed

    Liu, Yang; Du, Juanjuan; Choi, Jin-sil; Chen, Kuan-Ju; Hou, Shuang; Yan, Ming; Lin, Wei-Yu; Chen, Kevin Sean; Ro, Tracy; Lipshutz, Gerald S; Wu, Lily; Shi, Linqi; Lu, Yunfeng; Tseng, Hsian-Rong; Wang, Hao

    2016-01-04

    Simultaneous delivery of multiple genes and proteins (e.g., transcription factors; TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Human CD64-targeted non-viral siRNA delivery system for blood monocyte gene modulation

    PubMed Central

    Yong, Seok-Beom; Kim, Hyung Jin; Kim, Jang Kyoung; Chung, Jee Young; Kim, Yong-Hee

    2017-01-01

    A subset of phagocytes including inflammatory monocytes in blood migrate and give rise to macrophages in inflammatory tissues which generated the idea that blood monocytes are the therapeutic targets for drug delivery. Fc gamma receptor I (CD64) is a membrane receptor for the Fc region of immunoglobulin G, primarily expressed on monocyte-lineage, and H22 a monoclonal antibody for human CD64 had shown rapid blood monocyte binding and occupation in clinical studies. Small interfering RNA-mediated gene silencing as a therapeutic has been proposed and is a promising strategy in terms of its “knock-down” ability on the target gene prior to translation. However, its instability and off-targeting effect must be overcome for success in clinical studies. In this study, we developed a non-viral delivery system composed of oligo-nona-arginine (9R) and anti-human CD64 single chain antibodies (H22) for human monocyte-specific siRNA delivery. A targeted and efficient siRNA delivery mediated by anti-CD64 scFv-9R was observed in CD64 positive human leukemia cells, THP-1. With primary human blood cells, anti-CD64 scFv-9R mediated gene silencing was quantitatively confirmed representing blood monocyte selective gene delivery. These results demonstrate the potential of anti-CD64 scFv-9R mediated siRNA delivery for the treatment of human inflammatory diseases via blood monocytes gene delivery. PMID:28169353

  18. Extracellular vesicles for nucleic acid delivery: progress and prospects for safe RNA-based gene therapy.

    PubMed

    Jiang, L; Vader, P; Schiffelers, R M

    2017-03-01

    Nucleic acid-based drugs offer a potentially effective tool for treatment of a variety of diseases, including cancer, cardiovascular diseases, neurological disorders and infectious diseases. However, clinical applications are hindered by instability of RNA molecules in the circulation and lack of efficient vectors that can deliver RNAs to target tissues and into diseased target cells. Synthetic polymer and lipids as well as virus-based vectors are among the most widely explored vehicles for RNA delivery, but clinical progress has been limited as a result of issues related to toxicity, immunogenicity and low efficiency. Most recently, the discovery that extracellular vesicles (EVs) are endogenous RNA carriers, which may display better biocompatibility and higher delivery efficiency as compared with the synthetic systems, has provided a ray of hope in coping with the delivery dilemma, and EV-based gene therapy has already sparked general interest both in academia and industry. In this review, the current knowledge on EV biology and their role in cell-cell communication will be summarized. Promises of EVs as drug carriers and recent technologies on tailoring EVs' biological attributes will be included, and preclinical studies in which EVs have shown promise for therapeutic RNA delivery will be discussed.

  19. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    PubMed

    Yan, Ming; Wen, Jing; Liang, Min; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  20. Spider silk-based gene carriers for tumor cell-specific delivery.

    PubMed

    Numata, Keiji; Reagan, Michaela R; Goldstein, Robert H; Rosenblatt, Michael; Kaplan, David L

    2011-08-17

    The present study demonstrates pDNA complexes of recombinant silk proteins containing poly(L-lysine) and tumor-homing peptides (THPs), which are globular and approximately 150-250 nm in diameter, show significant enhancement of target specificity to tumor cells by additions of F3 and CGKRK THPs. We report herein the preparation and study of novel nanoscale silk-based ionic complexes containing pDNA able to home specifically to tumor cells. Particular focus was on how the THP, F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), and CGKRK, enhanced transfection specificity to tumor cells. Genetically engineered silk proteins containing both poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumor cells for target-specific pDNA delivery were prepared using Escherichia coli, followed by in vitro and in vivo transfection experiments into MDA-MB-435 melanoma cells and highly metastatic human breast tumor MDA-MB-231 cells. Non-tumorigenic MCF-10A breast epithelial cells were used as a control cell line for in vitro tumor-specific delivery studies. These results demonstrate that combination of the bioengineered silk delivery systems and THP can serve as a versatile and useful new platform for nonviral gene delivery.

  1. Introduction to Viral Vectors and Other Delivery Methods for Gene Therapy of the Nervous System.

    PubMed

    Manfredsson, Fredric P

    2016-01-01

    The use of gene therapy in neuroscience research has become common place in many laboratories across the world. However, contrary to common belief, the practical application of viral or non-viral gene therapy is not as straightforward as it may seem. All too often investigators see their experiments fail due to low-quality third-party vectors or due to a lack of knowledge regarding the proper use of these tools. For example, researchers often find themselves performing experiments using the wrong methodology (e.g., using the wrong type of vector or mishandling the vector to the point where the efficacy is significantly reduced) resulting in experiments that potentially fail to accurately answer a hypothesis, or the generation of irreproducible data. Thus, it is important for investigators that seek to utilize gene therapy approaches to gain a basic understanding of how to apply this technology. This includes understanding how to appropriately design and execute an experiment, understanding various delivery vehicles (e.g., what virus to use), delivery methods (e.g., systemic versus intracranial injections), what expression system to use, and the time course involved with a particular expression system. This chapter is intended to present an overview of this fundamental knowledge, providing the researcher with a decision tree upon which to build their gene therapy experiment.

  2. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9.

    PubMed

    LaFountaine, Justin S; Fathe, Kristin; Smyth, Hugh D C

    2015-10-15

    In recent years, several new genome editing technologies have been developed. Of these the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.

  3. Efficient Gene Therapy for Parkinson's Disease Using Astrocytes as Hosts for Localized Neurotrophic Factor Delivery

    PubMed Central

    Drinkut, Anja; Tereshchenko, Yuliya; Schulz, Jörg B; Bähr, Mathias; Kügler, Sebastian

    2012-01-01

    Current gene therapy approaches for Parkinson's disease (PD) deliver neurotrophic factors like glial cell line-derived neurotrophic factor (GDNF) or neurturin via neuronal transgene expression. Since these potent signaling-inducing neurotrophic factors can be distributed through long-distance neuronal projections to unaffected brain sites, this mode of delivery may eventually cause side effects. To explore a localized and thus potentially safer alternative for gene therapy of PD, we expressed GDNF exclusively in astrocytes and evaluated the efficacy of this approach in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rat 6-hydroxy-dopamine (6-OHDA) models of PD. In terms of protection of dopaminergic cell bodies and projections, dopamine (DA) synthesis and behaviour, astrocyte-derived GDNF demonstrated the same efficacy as neuron-derived GDNF. In terms of safety, unilateral striatal GDNF expression in astrocytes did not result in delivery of bio-active GDNF to the contralateral hemispheres (potential off-target sites) as happened when GDNF was expressed in neurons. Thus, astrocytic GDNF expression represents a localized but efficient alternative to current gene therapeutic strategies for the treatment of PD, especially if viral vectors with enhanced tissue penetration are considered. Astrocytic neurotrophic factor expression may open new venues for neurotrophic factor-based gene therapy targeting severe diseases of the brain. PMID:22086235

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

    PubMed

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

    2017-03-01

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

  5. Progranulin gene delivery protects dopaminergic neurons in a mouse model of Parkinson's disease.

    PubMed

    Van Kampen, Jackalina M; Baranowski, David; Kay, Denis G

    2014-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity and akinesia/bradykinesia resulting from the progressive loss of nigrostriatal dopaminergic neurons. To date, only symptomatic treatment is available for PD patients, with no effective means of slowing or stopping the progression of the disease. Progranulin (PGRN) is a 593 amino acid multifunction protein that is widely distributed throughout the CNS, localized primarily in neurons and microglia. PGRN has been demonstrated to be a potent regulator of neuroinflammation and also acts as an autocrine neurotrophic factor, important for long-term neuronal survival. Thus, enhancing PGRN expression may strengthen the cells resistance to disease. In the present study, we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of PGRN gene delivery as a therapy for the prevention or treatment of PD. Viral vector delivery of the PGRN gene was an effective means of elevating PGRN expression in nigrostriatal neurons. When PGRN expression was elevated in the SNC, nigrostriatal neurons were protected from MPTP toxicity in mice, along with a preservation of striatal dopamine content and turnover. Further, protection of nigrostriatal neurons by PGRN gene therapy was accompanied by reductions in markers of MPTP-induced inflammation and apoptosis as well as a complete preservation of locomotor function. We conclude that PGRN gene therapy may have beneficial effects in the treatment of PD.

  6. Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

    PubMed

    Gombash, S E; Cowley, C J; Fitzgerald, J A; Lepak, C A; Neides, M G; Hook, K; Todd, L J; Wang, G-D; Mueller, C; Kaspar, B K; Bielefeld, E C; Fischer, A J; Wood, J D; Foust, K D

    2017-08-03

    Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or PBS. Piglets were euthanized three weeks post-injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9 treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.Gene Therapy accepted article preview online, 03 August 2017. doi:10.1038/gt.2017.72.

  7. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    PubMed Central

    Liu, Yarong; Kim, Young Joo; Ji, Man; Fang, Jinxu; Siriwon, Natnaree; Zhang, Li I; Wang, Pin

    2014-01-01

    Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy. PMID:26015948

  8. Structure and function studies of cationic lipid non-viral gene delivery systems

    NASA Astrophysics Data System (ADS)

    Slack, Nelle Lynn

    Gene and drug delivery systems incorporating lipids mimic the biological environment and thus offer many advantages. In order to design successful gene or drug delivery vehicles based on lipid molecules an understanding of the affects of biopolymers on lipid membranes is necessary. We have examined the structures and interactions involved in two systems based on model biomembrane/biopolymer mixtures that are relevant for gene and drug delivery research. Liposomes incorporating PEG-lipids have shown great promise as drug carriers since they have proven to increase blood circulation times by evading the immune system. Previously, it was found that the addition of single-end-anchored (SEA)-PEG-surfactants to fluid lamellar membrane systems induces a novel hydrogel. As an extension of this work, we examine the affects of adding double-end-anchored (DEA)-PEG-surfactants to fluid membrane systems. The DEA-PEG-surfactants can adopt either a looping or bridging configuration which could deeply alter the microstructure of lamellar phases. We show that the DEA-PEG-surfactants induce gelation of fluid Lalpha phases in a way similar to that previously reported for SEA-PEG-surfactants. We also show, via x-ray diffraction, two striking differences between the SEA-and DEA-PEG-surfactant systems which demonstrate the existence of a large concentration of bridging polymers in the DEA system. The use of cationic liposomes (formed by mixtures of cationic and neutral lipids) as carriers of DNA for delivery in cells is a promising alternative to viral carriers for gene therapy. Using x-ray diffraction and biological assays, we show key parameters for optimizing gene transfer with these systems that are mediated by properties of the lipid. We have found cationic liposomes complexed with supercoiled plasmid DNA in solution self-assemble into a lamellar or an inverted hexagonal structure depending on lipid composition. Transfection efficiencies, determined by X-Gal and Luciferase assays

  9. Robust Cardiomyocyte-Specific Gene Expression Following Systemic Injection of AAV: In Vivo Gene Delivery Follows a Poisson Distribution

    PubMed Central

    Prasad, Konkal-Matt R.; Xu, Yaqin; Yang, Zequan; Acton, Scott T.; French, Brent A

    2010-01-01

    Newly-isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T promoter (cTnT). Using luciferase and eGFP, the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1 week old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared to the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15×1010 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution; requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency. PMID:20703310

  10. Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery.

    PubMed

    Martin, Timothy; Wysocki, Beata; Wysocki, Tadeusz; Pannier, Angela

    2015-01-23

    Nonviral gene delivery systems are a type of nano-communication system that transmit plasmid packets (i.e. pDNA packets) that are programmed at the nanoscale to biological systems at the microscopic cellular level. This engineered nano-communication system suffers large pDNA losses during transmission of the genetically encoded information, preventing its use in biotechnological and medical applications. The pDNA losses largely remain uncharacterized, and the ramifications of reducing pDNA loss from newly designed gene delivery systems remain difficult to predict. Here, the pDNA losses during primary and secondary transmission chains were identified utilizing a MATLAB model employing queuing theory simulating delivery of pEGFPLuc transgene to HeLa cells carried by Lipofectamine 2000 nonviral DNA carrier. Minimizing pDNA loss during endosomal escape of the primary transmission process results in increased number of pDNA in the nucleus with increased transfection, but with increased probability of cell death. The number of pDNA copies in the nucleus and the amount of time the pDNAs are in the nucleus directly correlates to improved transfection efficiency. During secondary transmission, pDNAs are degraded during distribution to daughter cells. Reducing pDNA losses improves transfection, but a balance in quantity of nuclear pDNA, mitosis, and toxicity must be considered in order to achieve therapeutically relevant transfection levels.

  11. Multi-layered nanoparticles for combination gene and drug delivery to tumors

    PubMed Central

    Ediriwickrema, Asiri; Zhou, Jiangbing; Deng, Yang; Saltzman, Mark

    2014-01-01

    Drug resistance and toxicity are major obstacles in cancer chemotherapy. Combination therapies can overcome resistance, and synergies can minimize dosing. Polymer nanocarriers are interesting vehicles for cancer therapeutics for their delivery and tumor targeting abilities. We synthesized a multilayered polymer nanoparticle (MLNP), comprising of poly(lactic-co-glycolic acid) with surface polyethyleneimine and functional peptides, for targeted drug and gene delivery. We confirmed the particle’s ability to inhibit tumor growth through synergistic action of the drug and gene product. MLNPs achieved transfection levels similar to lipofectamine, while maintaining minimal cytotoxicity. The particles delivered camptothecin (CPT), and plasmid encoding TNF related apoptosis inducing ligand (pTRAIL) (CT MLNPs), and synergistically inhibited growth of multiple cancer cells in vitro. The synergy of co-delivering CPT and pTRAIL via CT MLNPs was confirmed using the Chou-Talalay method: the combination index (CI) values at 50% inhibition ranged between 0.31–0.53 for all cell lines. Further, co-delivery with MLNPs resulted in a 3.1–15 fold reduction in CPT and 4.7–8.0 fold reduction in pTRAIL dosing. CT MLNPs obtained significant HCT116 growth inhibition in vivo compared to monotherapy. These results support our hypothesis that MLNPs can deliver both small molecules and genetic agents towards synergistically inhibiting tumor growth. PMID:25112935

  12. Polydioxanone-based bio-materials for tissue engineering and drug/gene delivery applications.

    PubMed

    Goonoo, Nowsheen; Jeetah, Roubeena; Bhaw-Luximon, Archana; Jhurry, Dhanjay

    2015-11-01

    Since the commercialization of polydioxanone (PDX) as a biodegradable monofilament suture by Ethicon in 1981, the polymer has received only limited interest until recently. The limitations of polylactide-co-glycolide (PLGA) coupled with the growing need for materials with enhanced features and the advent of new fabrication techniques such as electrospinning have revived interest for PDX in medical devices, tissue engineering and drug delivery applications. Electrospun PDX mats show comparable mechanical properties as the major structural components of native vascular extracellular matrix (ECM) i.e. collagen and elastin. In addition, PDX's unique shape memory property provides rebound and kink resistance when fabricated into vascular conduits. The synthesis of methyl dioxanone (MeDX) monomer and copolymers of dioxanone (DX) and MeDX have opened up new perspectives for poly(ester-ether)s, enabling the design of the next generation of tissue engineering scaffolds for application in regenerating such tissues as arteries, peripheral nerve and bone. Tailoring of polymer properties and their formulation as nanoparticles, nanomicelles or nanofibers have brought along important developments in the area of controlled drug or gene delivery. This paper reviews the synthesis of PDX and its copolymers and provides for the first time an exhaustive account of its applications in the (bio)medical field with focus on tissue engineering and drug/gene delivery.

  13. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    PubMed Central

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  14. Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration.

    PubMed

    Belmadi, Nawal; Midoux, Patrick; Loyer, Pascal; Passirani, Catherine; Pichon, Chantal; Le Gall, Tony; Jaffres, Paul-Alain; Lehn, Pierre; Montier, Tristan

    2015-09-01

    Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein; the replacement of a missing or inactive gene; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

  15. Gene delivery into mouse retinal ganglion cells by in utero electroporation

    PubMed Central

    Garcia-Frigola, Cristina; Carreres, Maria Isabel; Vegar, Celia; Herrera, Eloisa

    2007-01-01

    Background The neural retina is a highly structured tissue of the central nervous system that is formed by seven different cell types that are arranged in layers. Despite much effort, the genetic mechanisms that underlie retinal development are still poorly understood. In recent years, large-scale genomic analyses have identified candidate genes that may play a role in retinal neurogenesis, axon guidance and other key processes during the development of the visual system. Thus, new and rapid techniques are now required to carry out high-throughput analyses of all these candidate genes in mammals. Gene delivery techniques have been described to express exogenous proteins in the retina of newborn mice but these approaches do not efficiently introduce genes into the only retinal cell type that transmits visual information to the brain, the retinal ganglion cells (RGCs). Results Here we show that RGCs can be targeted for gene expression by in utero electroporation of the eye of mouse embryos. Accordingly, using this technique we have monitored the morphology of electroporated RGCs expressing reporter genes at different developmental stages, as well as their projection to higher visual targets. Conclusion Our method to deliver ectopic genes into mouse embryonic retinas enables us to follow the course of the entire retinofugal pathway by visualizing RGC bodies and axons. Thus, this technique will permit to perform functional studies in vivo focusing on neurogenesis, axon guidance, axon projection patterning or neural connectivity in mammals. PMID:17875204

  16. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

    PubMed

    Lee, Soo-Jin; Yoon, Sung-Hwa; Doh, Kyung-Oh

    2011-08-01

    Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

  17. Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier

    PubMed Central

    Yu, Yanni; Hu, Yongpei; Li, Xiufang; Liu, Yu; Li, Mingzhong; Yang, Jicheng; Sheng, Weihua

    2016-01-01

    The development of a novel cationized polymer used as a gene delivery carrier that can conveniently and effectively transfect cells resulting in a stably expressed target gene remains a challenge. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg–Gly–Asp sequences but a negative charge. In order to render ASF amenable to packaging plasmid DNA (pDNA), spermine was used to modify ASF to synthesize cationized ASF (CASF), which was used as a gene delivery carrier. CASF was characterized using trinitrobenzene sulfonic acid assay, the zeta potential determination, and a Fourier transform infrared analysis, and the results of these characterizations indicated that the –NH2 in spermine effectively reacts with the –COOH in the side chains of ASF. Spermine grafted to the side chains of ASF resulted in the conversion of the negative charge of ASF to a positive charge. CASF packaged pDNA and formed CASF/pDNA complexes, which exhibited spherical morphology with average particle sizes of 215–281 nm and zeta potential of approximately +3.0 mV to +3.2 mV. The results of the MTT assay, confocal laser scanning microscopy, and flow cytometry analysis in a human endothelial cell line revealed that CASF/pDNA complexes exhibited lower cytotoxicity and higher transfection efficiency compared to the pDNA complexes of polyethyleneimine. These results indicate that our synthesized CASF, a cationized polymer, is a potential gene delivery carrier with the advantages of biodegradability and low cytotoxicity. PMID:27042056

  18. Delivery of antioxidant enzyme genes to protect against ischemia/reperfusion-induced injury to retinal microvasculature.

    PubMed

    Chen, Baihua; Caballero, Sergio; Seo, Soojung; Grant, Maria B; Lewin, Alfred S

    2009-12-01

    Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.

  19. Delivery of Antioxidant Enzyme Genes to Protect against Ischemia/Reperfusion-Induced Injury to Retinal Microvasculature

    PubMed Central

    Chen, Baihua; Caballero, Sergio; Seo, Soojung; Grant, Maria B.; Lewin, Alfred S.

    2013-01-01

    Purpose Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. Methods I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Results Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Conclusions Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease. PMID:19628743

  20. Cholesterol-Peptide Hybrids to Form Liposome-Like Vesicles for Gene Delivery

    PubMed Central

    Tang, Qiong; Cao, Bin; Wu, Haiyan; Cheng, Gang

    2013-01-01

    In this paper, four amphiphilic cholesterol-peptide conjugates (Ch-R5H5, Ch-R3H3, Ch-R5 and Ch-R5) were designed and synthesized, and their properties in gene delivery were evaluated in vitro with an aim of developing more efficient gene delivery carriers. These amphiphilic cholesterol-peptide conjugates are composed of hydrophobic cholesterol and positively charged peptides. They were able to self-assemble into micelles at low concentrations and their critical micelle concentrations in phosphate buffered saline (pH 7.4) are ≤85 µg/mL. Amphiphilic cholesterol-peptide conjugates condensed DNA more efficiently than a hydrophilic cationic oligoarginine (R10) peptide with no hydrophobic segment. Their transfection efficiencies were at least two orders of magnitude greater than that of R10 peptide in HEK-293 cells. Moreover, the introduction of histidine residues in cholesterol-peptide conjugates led to higher gene expression efficiency compared with cholesterol-peptides without histidine (Ch-R5 and Ch-R3), and the luciferase expression level was comparable or even higher than that induced by PEI at its optimal N/P ratio. In particular, Ch-R5H5 condensed DNA into smaller nanoparticles than Ch-R3H3 at higher N/P ratios, and the minimum size of Ch-R5H5/DNA complexes was 180 nm with zeta potential of 23 mV, achieved at the N/P ratio of 30. This liposome-like vesicle may be a promising gene delivery carrier for intravenous therapy. PMID:23382899

  1. Optimization of Brush-like Cationic Copolymers for Non-viral Gene Delivery

    PubMed Central

    Wei, Hua; Pahang, JoshuelA; Pun, Suzie H.

    2012-01-01

    Polyethylenimine (PEI) is one of the most broadly used polycations for gene delivery due to its high transfection efficiency and commercial availability but materials are cytotoxic and often polydisperse. The goal of current work is to develop an alternative family of polycations based on controlled living radical polymerization (CLRP) and to optimize the polymer structure for efficient gene delivery. In this study, well-defined poly(glycidyl methacrylate)(P(GMA)) homopolymers were synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization followed by decoration using three different types of oligoamines, i.e., tetraethylenepentamine (TEPA), pentaethylenehexamine (PEHA), and tris(2-aminoethyl)amine (TREN), respectively, to generate various P(GMA-oligoamine) homopolycations. The effect of P(GMA) backbone length and structure of oligoamine on gene transfer efficiency was then determined. The optimal polymer, P(GMA-TEPA)50, provided comparable transfection efficiency but lower cytotoxicity than PEI. P(GMA-TEPA)50 was then used as the cationic block in di-block copolymers containing hydrophilic N-(2-hydroxypropyl) methacrylamide (HPMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA). Polyplexes of block copolymers were stable against aggregation in physiological salt condition and in Opti-MEM due to the shielding effect of P(HPMA) and P(OEGMA). However, the presence of the HPMA/OEGMA block significantly decreased the transfection efficacy of P(GMA-TEPA)50homopolycation. To compensate for reduced cell uptake caused by the hydrophilic shell of polyplex, the integrin-binding peptide, RGD, was conjugated to the hydrophilic chain end of P(OEGMA)15-b-P(GMA-TEPA)50 copolymer by Michael-type addition reaction. At low polymer to DNA ratios, the RGD-functionalized polymer showed increased gene delivery efficiency to HeLa cells compared to analogous polymers lacking RGD. PMID:23240866

  2. In vivo gene targeting of IL-3 into immature hematopoietic cells through CD117 receptor mediated antibody gene delivery

    PubMed Central

    Chapel, Alain; Deas, Olivier; Bensidhoum, Morad; François, Sabine; Mouiseddine, Moubarak; Poncet, Pascal; Dürrbach, Antoine; Aigueperse, Jocelyne; Gourmelon, Patrick; Gorin, Norbert C; Hirsch, François; Thierry, Dominique

    2004-01-01

    Background Targeted gene transfection remains a crucial issue to permit the real development of genetic therapy. As such, in vivo targeted transfection of specific subsets of hematopoietic stem cells might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors. Methods Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates. Results By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals. Conclusions These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells, in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures. PMID:15509303

  3. Chitosan nanoparticles as a potential nonviral gene delivery for HPV-16 E7 into mammalian cells.

    PubMed

    Tahamtan, Alireza; Tabarraei, Alijan; Moradi, Abdolvahab; Dinarvand, Meshkat; Kelishadi, Mishar; Ghaemi, Amir; Atyabi, Fatemeh

    2015-01-01

    Chitosan nanoparticles (CS NPs) were prepared as a carrier for Human papillomavirus type 16 HPV-16) E7 gene and their gene transfection ability were evaluated in vitro. The plasmid expressing green fluorescent protein (pEGFP) was used as a reporter gene. Gel electrophoresis demonstrated full binding of CS NPs with the pDNA. The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. The expression of E7 proteins was confirmed under SDS-PAGE and western blot analysis. As a conclusion CS NPs may serve as an effective nonviral carrier for delivery of nucleotides into eukaryotic cells.

  4. Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

    PubMed

    Zorzi, Giovanni K; Párraga, Jenny E; Seijo, Begoña; Sanchez, Alejandro

    2015-11-01

    Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations.

  5. Real-Time Imaging of Gene Delivery and Expression with DNA Nanoparticle Technologies

    NASA Astrophysics Data System (ADS)

    Sun, Wenchao; Ziady, Assem G.

    The construction of safe, efficient, and modifiable synthetic DNA nanoparticles is an emerging technology that has achieved important milestones of success in the past 5 years. Advances in chemical conjugation, purification, and controlled synthesis have allowed researchers to produce uniform and stable particles, whose physical characteristics can be well characterized and monitored. As a result of these improvements, DNA nanoparticles have now been cleared for clinical testing, and show good potential for human gene therapy. A very important recent development in the study of DNA nanoparticles is the use of small-animal imaging. Real-time imaging has become a valuable technique for tracking particle biodistribution and gene transfer efficacy. In this chapter, we discuss how bioluminescent, positron emission tomography, and magnetic resonance imaging can be used separately or in concert to study particle delivery, localization, and magnitude of gene expression in vivo.

  6. Mesenchymal stem cells as therapeutics and vehicles for gene and drug delivery.

    PubMed

    Porada, Christopher D; Almeida-Porada, Graça

    2010-09-30

    Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they produce immunosuppression upon transplantation; 5) their pronounced anti-inflammatory properties; and 6) their ability to home to damaged tissues, tumors, and metastases following in vivo administration. In this review, we summarize the latest research in the use of mesenchymal stem cells in regenerative medicine, as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Mesenchymal Stem Cells as Therapeutics and Vehicles for Gene and Drug Delivery

    PubMed Central

    Porada, Christopher D.; Almeida-Porada, Graça

    2010-01-01

    Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they produce immunosuppression upon transplantation; 5) their pronounced anti-inflammatory properties; and 6) their ability to home to damaged tissues, tumors, and metastases following in vivo administration. In this review, we summarize the latest research in the use of mesenchymal stem cells in regenerative medicine, as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells. PMID:20828588

  8. Effect of adenoviral delivery of prodynorphin gene on experimental inflammatory pain induced by formalin in rats

    PubMed Central

    Chen, Xionggang; Wang, Tingting; Lin, Caizhu; Chen, Baihong

    2014-01-01

    Circumstantial evidences suggest that dynorphins and their common precursor prodynorphin (PDYN) are involved in antinociception and neuroendocrine signaling. DREAM knockout mice had increased levels of PDYN and dynorphin expression, and reduced sensitivity to painful stimuli. However, some data support the notion that the up-regulation of spinal dynorphin expression is a common critical feature in neuropathic pain. It is not clear whether the production of dynorphin A can be increased when more PDYN is present. In this study we investigated the changes in pain behaviors, spinal PDYN mRNA expression and dynorphin A production on formalin-induced pain in rats receiving the pretreatment of adenoviral delivery of PDYN. Our results showed that the adenoviral transfer of PDYN gene was sufficient to reduce pain behaviors resulting from formalin injection, and the antinociceptive effect after receiving the pretreatment of adenoviral delivery of PDYN was mediated at the level of the spinal cord via KOR. PMID:25663984

  9. Electrotriggered, spatioselective, quantitative gene delivery into a single cell nucleus by Au nanowire nanoinjector.

    PubMed

    Yoo, Seung Min; Kang, Mijeong; Kang, Taejoon; Kim, Dong Min; Lee, Sang Yup; Kim, Bongsoo

    2013-06-12

    Delivery of bioactive materials into a cell is highly important in the study of cell biology and medical treatments. Ideal nanoinjectors should be able to deliver biomaterials with high spatial resolution while causing minimum cell damage. We developed a Au nanowire (NW) nanoinjector that has the thinnest diameter (100–150 nm) among the DNA delivering devices as well as optimum mechanical properties, minimizing cell damage. Well-defined (111) single-crystalline Au surface and high electric conductivity of a Au NW nanoinjector allow precisely timed and efficient electrochemical release of DNA molecules attached on a Au NW surface. Both linear DNA and plasmid DNA were delivered separately and showed successful expression. The Au NW nanoinjector would find important biomedical applications in the fields such as gene therapy, DNA vaccination, targeted drug delivery, and probe/control of cell signaling events.

  10. Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery.

    PubMed

    Numata, Keiji; Mieszawska-Czajkowska, Aneta J; Kvenvold, Laura A; Kaplan, David L

    2012-01-01

    Nanoscale complexes of recombinant silk molecules containing THPs with DNA are designed as less cytotoxic and highly target-specific gene carriers. Genetically engineered silk proteins containing poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumorigenic cells for target-specific pDNA delivery are prepared, followed by in vitro transfection into MDA-MB-435 melanoma cells, highly metastatic human breast tumor MDA-MB-231 cells, and non-tumorigenic MCF-10A breast epithelial cells. The silk/poly(L-lysine) block copolymer containing Lyp1 (ML-Lyp1) shows significant differences from silk/poly(L-lysine) block copolymer containing F3 (ML-F3) in cytotoxicity to MCF10A cells. ML-F3 is the most promising candidate for target delivery into tumorigenic cells.

  11. Topical Non-Invasive Gene Delivery using Gemini Nanoparticles in Interferon-gamma-deficient Mice

    SciTech Connect

    Badea,I.; Wettig, S.; Verrall, R.; Foldvari, M.

    2007-01-01

    Cutaneous gene therapy, although a promising approach for many dermatologic diseases, has not progressed to the stage of clinical trials, mainly due to the lack of an effective gene delivery system. The main objective of this study was to construct and evaluate gemini nanoparticles as a topical formulation for the interferon gamma (IFN-{gamma}) gene in an IFN-{gamma}-deficient mouse model. Nanoparticles based on the gemini surfactant 16-3-16 (NP16-DNA) and another cationic lipid cholesteryl 3{beta}-(-N-[dimethylamino-ethyl] carbamate) [Dc-chol] (NPDc-DNA) were prepared and characterized. Zetasizer measurement indicated a bimodal distribution of 146 and 468 nm average particle sizes for the NP16-DNA ({zeta}-potential +51 mV) nanoparticles and monomodal distribution of 625 nm ({zeta}-potential +44 mV) for the NPDc-DNA. Circular dichroism studies showed that the gemini surfactant compacted the plasmid more efficiently compared to the Dc-chol. Small-angle X-ray scattering measurements revealed structural polymorphism in the NP16-DNA nanoparticles, with lamellar and Fd3m cubic phases present, while for the NPDc-DNA two lamellar phases could be distinguished. In vivo, both topically applied nanoparticles induced higher gene expression compared to untreated control and naked DNA (means of 0.480 and 0.398 ng/cm{sup 2} vs 0.067 and 0.167 ng/cm{sup 2}). However, treatment with NPDc-DNA caused skin irritation, and skin damage, whereas NP16-DNA showed no skin toxicity. In this study, we demonstrated that topical cutaneous gene delivery using gemini surfactant-based nanoparticles in IFN-{gamma}-deficient mice was safe and may provide increased gene expression in the skin due to structural complexity of NP16 nanoparticles (lamellar-cubic phases).

  12. Short-term efficiency and safety of gene delivery into canine kidneys.

    PubMed

    Chetboul, V; Klonjkowski, B; Lefebvre, H P; Desvaux, D; Laroute, V; Rosenberg, D; Maurey, C; Crespeau, F; Adam, M; Adnot, S; Eloit, M; Pouchelon, J L

    2001-03-01

    Gene delivery of biologically active molecules to the kidney may have potential therapeutic applications in renal and cardiovascular diseases. Recombinant adenovirus is one of the most efficient vectors for in vivo gene delivery. However, in vivo toxicity at the site of administration has to be evaluated for the successful use of adenovirus-mediated gene transfer. The aim of this study was to document precisely the short-term safety of different routes of intra-renal adenoviral administration and to compare their transduction efficiency. Dog puppies were injected with an adenoviral vector expressing the beta-galactosidase reporter gene in both kidneys via three different routes, i.e. intra-renal-ureteral route (IU) and intra-renal-arterial route with (IAC) or without (IA) clamping of the renal vein. Toxicity of viral administration was assayed on day 4 at both physiological and histological levels. Renal samples were monitored for the presence of nuclear beta-galactosidase-expressing cells. All renal physiological parameters (glomerular filtration rate, effective renal plasma flow, and electrolyte excretion fractions) remained stable whatever the route of viral administration. No histological lesion was detected in any of the haematoxylin-eosin-stained kidney sections, and there was no evidence of ischaemia-reperfusion injury in the kidneys subjected to venous clamping. Efficient transgene expression was obtained in dog kidneys following IAC and IU injection of adenoviral vectors. Gene transfer via the IAC route induced gene expression predominantly in the cortical interstitial cells. Retrograde IU adenoviral injection resulted in reduced transduction efficiency compared with the IAC route, with transgene expression occurring mainly in the distal tubular and pyelic epithelial cells. The two major findings of this study were (i) the absence of acute histological and functional renal alteration following intra-arterial and intra-ureteral injections of adenoviral

  13. Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols.

    PubMed

    Vulugundam, Gururaja; Kumar, Krishan; Kondaiah, Paturu; Bhattacharya, Santanu

    2015-04-14

    Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene

  14. Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis.

    PubMed

    Lu, Hua-Ding; Zhao, Hui-Qing; Wang, Kun; Lv, Lu-Lu

    2011-11-28

    Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.

  15. Catheter-based antegrade intracoronary viral gene delivery with coronary venous blockade

    PubMed Central

    Hayase, Motoya; Monte, Federica del; Kawase, Yoshiaki; MacNeill, Briain D.; McGregor, Jennifer; Yoneyama, Ryuichi; Hoshino, Kozo; Tsuji, Tsuyoshi; De Grand, Alec M.; Gwathmey, Judith K.; Frangioni, John V.; Hajjar, Roger J.

    2005-01-01

    Hayase, Motoya, Federica del Monte, Yoshiaki Kawase, Brian D. MacNeill, Jennifer McGregor, Ryuichi Yoneyama, Kozo Hoshino, Tsuyoshi Tsuji, Alec M. De Grand, Judith K. Gwathmey, John V. Frangioni, and Roger J. Hajjar. Catheter-based antegrade intracoronary viral gene delivery with coronary venous blockade. Am J Physiol Heart Circ Physiol 288: H2995–H3000, 2005; doi:10.1152/ajpheart.00703.2004.—The purpose of this study is to evaluate the feasibility of percutaneous antegrade myocardial gene transfer (PAMGT). A consistent and safe technique for in vivo gene transfer is required for clinical application of myocardial gene therapy. PAMGT with concomitant coronary venous blockade was performed in 12 swine. The myocardium was preconditioned with 1 min of occlusion of the left anterior descending and left circumflex arteries. The anterior interventricular vein was occluded during left anterior descending artery delivery, and the great cardiac vein at the entrance of the middle cardiac vein was occluded during left circumflex artery delivery. With arterial and venous balloons inflated (3 min) and after adenosine (25 μg) injection, PAMGT was performed by antegrade injection of an adenoviral solution (1 ml of 1011 plaque-forming units in each coronary artery) carrying β-galactosidase or saline through the center lumen of the angioplasty balloon. In one set of animals, PAMGT was performed with selective coronary vein blockade (n = 9); in another set of animals, PAMGT was performed without coronary vein blockade (n = 5). At 1 wk after gene delivery, the animals were killed. Quantitative β-galactosidase analysis was performed in the left and right ventricular walls. PAMGT was successfully performed in all animals with and without concomitant occlusion of the coronary veins. Quantitative β-galactosidase analysis showed that PAMGT with coronary blockade was superior to PAMGT without coronary blockade. β-Galactosidase activity increased significantly in the

  16. A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

    PubMed

    Haitao, Pan; Qixin, Zheng; Xiaodong, Guo

    2007-03-01

    A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.

  17. A NOVEL GENE DELIVERY METHOD TRANSDUCES PORCINE PANCREATIC DUCT EPITHELIAL CELLS

    PubMed Central

    Griffin, Michelle A.; Restrepo, M. Santiago; Abu-El-Haija, Marwa; Wallen, Tanner; Buchanan, Elizabeth; Rokhlina, Tatiana; Chen, Yong Hong; McCray, Paul B.; Davidson, Beverly L.; Divekar, Abhay; Uc, Aliye

    2014-01-01

    Gene therapy offers the possibility to treat pancreatic disease in Cystic Fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF, thus CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via umbilical artery in newborns and via femoral artery at older ages; delivery approaches which can be translated to humans. PMID:24257348

  18. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors.

    PubMed

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-09-14

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote the DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing the cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity. © 2017 IOP Publishing Ltd.

  19. A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

    PubMed

    Griffin, M A; Restrepo, M S; Abu-El-Haija, M; Wallen, T; Buchanan, E; Rokhlina, T; Chen, Y H; McCray, P B; Davidson, B L; Divekar, A; Uc, A

    2014-02-01

    Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

  20. Macrophages in gene therapy: cellular delivery vehicles and in vivo targets.

    PubMed

    Burke, B; Sumner, S; Maitland, N; Lewis, C E

    2002-09-01

    The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.

  1. Development of TMTP-1 targeted designer biopolymers for gene delivery to prostate cancer.

    PubMed

    McBride, John W; Massey, Ashley S; McCaffrey, J; McCrudden, Cian M; Coulter, Jonathan A; Dunne, Nicholas J; Robson, Tracy; McCarthy, Helen O

    2016-03-16

    Designer biopolymers (DBPs) represent state of the art genetically engineered biomacromolecules designed to condense plasmid DNA, and overcome intra- and extra- cellular barriers to gene delivery. Three DBPs were synthesized, each with the tumor molecular targeting peptide-1 (TMTP-1) motif to specifically target metastases. Each DBP was complexed with a pEGFP-N1 reporter plasmid to permit physiochemical and biological assay analysis. Results indicated that two of the biopolymers (RMHT and RM3GT) effectively condensed pEGFP-N1 into cationic nanoparticles <100 nm and were capable of transfecting PC-3 metastatic prostate cancer cells. Conversely the anionic RMGT DBP nanoparticles could not transfect PC-3 cells. RMHT and RM3GT nanoparticles were stable in the presence of serum and protected the cargo from degradation. Additionally it was concluded that cell viability could recover post-transfection with these DBPs, which were less toxic than the commercially available transfection reagent Lipofectamine(®) 2000. With both DBPs, a higher transfection efficacy was observed in PC-3 cells than in the moderately metastatic, DU145, and normal, PNT2-C2, cell lines. Blocking of the TMTP-1 receptors inhibited gene transfer indicating internalization via this receptor. In conclusion RMHT and RM3GT are fully functional DBPs that address major obstacles to gene delivery and target metastatic cells expressing the TMTP-1 receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear.

    PubMed

    Pinyon, Jeremy L; Tadros, Sherif F; Froud, Kristina E; Y Wong, Ann C; Tompson, Isabella T; Crawford, Edward N; Ko, Myungseo; Morris, Renée; Klugmann, Matthias; Housley, Gary D

    2014-04-23

    The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

  3. A top-down approach for construction of hybrid polymer-virus gene delivery vectors.

    PubMed

    Ramsey, Joshua D; Vu, Halong N; Pack, Daniel W

    2010-05-21

    Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

  4. Novel Cholesterol-Based Cationic Lipids as Transfecting Agents of DNA for Efficient Gene Delivery

    PubMed Central

    Ju, Jia; Huan, Meng-Lei; Wan, Ning; Qiu, Hai; Zhou, Si-Yuan; Zhang, Bang-Le

    2015-01-01

    The design, synthesis and biological evaluation of the cationic lipid gene delivery vectors based on cholesterol and natural amino acids lysine or histidine are described. Cationic liposomes composed of the newly synthesized cationic lipids 1a or 1b and neutral lipid DOPE (1,2-dioleoyl-l-α-glycero-3-phosphatidyl-ethanolamine) exhibited good transfection efficiency. pEGFP-N1 plasmid DNA was transferred into 293T cells by cationic liposomes formed from cationic lipids 1a and 1b, and the transfection activity of the cationic lipids was superior (1a) or parallel (1b) to that of the commercially available 3β-[N-(N',N'-dimethylaminoethyl)-carbamoyl] cholesterol (DC-Chol) derived from the same cholesterol backbone with different head groups. Combined with the results of agarose gel electrophoresis, transfection experiments with various molar ratios of the cationic lipids and DOPE and N/P (+/−) molar charge ratios, a more effective formulation was formed, which could lead to relatively high transfection efficiency. Cationic lipid 1a represents a potential agent for the liposome used in gene delivery due to low cytotoxicity and impressive gene transfection activity. PMID:25768346

  5. Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

    PubMed

    Ju, Jia; Huan, Meng-Lei; Wan, Ning; Hou, Yi-Lin; Ma, Xi-Xi; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2016-05-15

    Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Novel cholesterol-based cationic lipids as transfecting agents of DNA for efficient gene delivery.

    PubMed

    Ju, Jia; Huan, Meng-Lei; Wan, Ning; Qiu, Hai; Zhou, Si-Yuan; Zhang, Bang-Le

    2015-03-11

    The design, synthesis and biological evaluation of the cationic lipid gene delivery vectors based on cholesterol and natural amino acids lysine or histidine are described. Cationic liposomes composed of the newly synthesized cationic lipids 1a or 1b and neutral lipid DOPE (1,2-dioleoyl-L-α-glycero-3-phosphatidyl-ethanolamine) exhibited good transfection efficiency. pEGFP-N1 plasmid DNA was transferred into 293T cells by cationic liposomes formed from cationic lipids 1a and 1b, and the transfection activity of the cationic lipids was superior (1a) or parallel (1b) to that of the commercially available 3β-[N-(N',N'-dimethylaminoethyl)-carbamoyl] cholesterol (DC-Chol) derived from the same cholesterol backbone with different head groups. Combined with the results of agarose gel electrophoresis, transfection experiments with various molar ratios of the cationic lipids and DOPE and N/P (+/-) molar charge ratios, a more effective formulation was formed, which could lead to relatively high transfection efficiency. Cationic lipid 1a represents a potential agent for the liposome used in gene delivery due to low cytotoxicity and impressive gene transfection activity.

  7. Melittin-grafted HPMA-Oligolysine Based Copolymers for Improved Gene Delivery

    PubMed Central

    Schellinger, Joan G.; Pahang, Joshuel A.; Johnson, Russell N.; Chu, David S.H.; Sellers, Drew L.; Maris, Don O.; Convertine, Anthony J.; Stayton, Patrick S.; Horner, Philip J.; Pun, Suzie H.

    2012-01-01

    Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-L-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendant groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~ 100–200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain. PMID:23261217

  8. Efficient gene delivery with osmotically active and hyperbranched poly(ester amine)s.

    PubMed

    Arote, Rohidas B; Lee, Eun-Sun; Jiang, Hu-Lin; Kim, You-Kyoung; Choi, Yun-Jaie; Cho, Myung-Haing; Cho, Chong-Su

    2009-12-01

    Degradable and hyperbranched poly (ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between hydrophilic glycerol triacrylate (GTA) and low-molecular-weight polyethylenimine (LMW-PEI) and evaluated as nonviral gene carriers. PEAs effectively condensed DNA with particle sizes below 200 nm and suitable surface charges (15-45 mV), suitable for intracellular delivery. PEAs degraded in a controlled fashion showing half-lives of more than 12 days and were essentially nontoxic in three different cell lines. Elevated transfection levels by luciferase assay revealed the superiority of PEAs over PEI 25K and Lipofectamine. PEAs synthesized using 1:4 mol ratio of GTA to PEI [GTA/PEI-1.2(1:4)] showed highest transfection efficiency in HepG2 cells. PEAs showed significant gene expression in vitro as well as in vivo through aerosol administration. Reduction in packed cell volume (PCV) of cells when treated with polyplexes supported the hyperosmotic effect of PEAs. Effect of bafilomycin A1 on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. High transfection efficiency was attributed to the synergism from hyperosmotic glycerol backbone in the PEAs and endosomal buffering capacity of PEI amine groups. Therefore, this convergence of osmotically active biodegradable PEAs suggests their potential as a safe and efficient gene delivery vector.

  9. Artificial virus as trump-card to resolve exigencies in targeted gene delivery.

    PubMed

    Ajithkumar, K C; Pramod, K

    2017-05-28

    Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized for successfully deliver the genetic material. However, the therapeutic use of viruses for gene delivery is controversial because, the potentially replicable genomes recombine or integrates with the cell DNA resulted with immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective nonviral gene therapy vehicles arises and the non-viral vectors, protein only particles and virus like particles (VLP) can be engineered which contain all the required functional modules in single chain molecules. These are resembles to viruses called artificial virus. The artificial virus eliminates the disadvantages of the viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because independent elements such as cell ligands, membrane active peptides, etc can be incorporated in to the protein. The protein-DNA complexes appeared as tight and monodisperse spherical nanoparticles of nearly 80 nm size resemble bacterial inclusion bodies. The nucleic acids act as compacting, molecular glue that affects the conformation of the protein units, altering the α- helix structure, minimizing their aggregation tendency and promoting receptor mediated cell uptake and proper intracellular trafficking to the cell nucleus. Such tunable system mimics the activities of infected viruses and used for the safe and effective delivery of drugs and genetic material in gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis.

    PubMed

    Pike, L S; Tannous, B A; Deliolanis, N C; Hsich, G; Morse, D; Tung, C-H; Sena-Esteves, M; Breakefield, X O

    2011-12-01

    Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.

  11. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity

    SciTech Connect

    Yan, Chen; Jie, Leng; Yongqi, Wang; Weiming, Xiao; Juqun, Xi; Yanbing, Ding; Li, Qian; Xingyuan, Pan; Mingchun, Ji; Weijuan, Gong

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.

  12. Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats.

    PubMed Central

    Wang, C; Chao, L; Chao, J

    1995-01-01

    Hypertension is a multigene and multifactorial disorder affecting approximately 25% of the population. To demonstrate potential therapeutic effects of human tissue kallikrein in hypertension, spontaneously hypertensive rats were subjected to somatic gene therapy. Two human tissue kallikrein DNA constructs, one under the promoter control of the metallothionein metal response element and the other under the control of the Rous sarcoma virus 3'-LTR, were generated. We delivered naked DNA constructs into spontaneously hypertensive rats via intravenous injection. The expression of human tissue kallikrein in rats was identified in the heart, lung, and kidney by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. A single injection of both human kallikrein plasmid DNA constructs caused a sustained reduction of blood pressure which began 1 wk after injection and continued for 6 wk. A maximal effect of blood pressure reduction of 46 mmHg in rats was observed 2-3 wk after injection with kallikrein DNA as compared to rats with vector DNA (n = 6, P < 0.05). The hypotensive effect caused by somatic gene delivery of human tissue kallikrein in hypertensive rats is reversed by subcutaneous injection of aprotinin, a potent tissue kallikrein inhibitor. No antibodies to either human tissue kallikrein or kallikrein DNA were detected in rat sera after injection of the human kallikrein gene. These results show that direct gene delivery of human tissue kallikrein causes a sustained reduction in systolic blood pressure in genetically hypertensive rats and indicate that the feasibility of kallikrein gene therapy for treating human hypertension should be studied. Images PMID:7535795

  13. In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina.

    PubMed

    Lee, Joo Yong; Hwang, Yoonha; Kim, Ji Hyun; Kim, Yu Sun; Jung, Bok Kyoung; Kim, Pilhan; Lee, Heuiran

    2016-06-01

    The purpose of this study was to evaluate longitudinal gene expression patterns by retinal imaging using a modified custom-built confocal laser-scanning microscope in experimental rats after intravitreal injection of recombinant adeno-associated virus 2 (rAAV2-green fluorescent protein [GFP]). Ten 9-week-old Wistar rats were divided into two groups: experimental group (group 1) that received a rAAV2-GFP intravitreal injection and control group (group 2) that received a vehicle. After anesthesia using a Zoletil intraperitoneal injection, 8 μL rAAV2-GFP in group 1 or vehicle in group 2 was injected intravitreally using a 33-G Hamilton syringe. In vivo fluorescence retinal images were acquired under anesthesia at 2, 4, 6, and 13 days after rAAV or vehicle delivery. Differences in GFP fluorescence were identified starting from day 2 after the intravitreal injection of rAAV2-GFP in group 1. Between days 4 and 6, the intensity and area of fluorescence in the retina began to increase and peaked at day 13. Based on the pattern of GFP expression, the axon of the nerve fiber layer ganglion cell was identified. In group 2, eyes treated with the vehicle showed a small amount of autofluorescence in a limited area for up to 2 weeks, with no increase in intensity during this period. In vivo retinal imaging confirmed gene expression within 2 weeks after the intravitreal injection of rAAV2-GFP. Gene transfer and expression in the rat retina occurs quickly in 2 days and appears to peak within 2 weeks of gene delivery. In vivo retinal imaging may be a useful noninvasive tool to continuously monitor gene expression in the retina over time.

  14. An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

    PubMed

    Walsh, D P; Heise, A; O'Brien, F J; Cryan, S-A

    2017-09-14

    Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (Superfect(TM)) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.Gene Therapy advance online publication, 14 September 2017; doi:10.1038/gt.2017.58.

  15. Myocardial β2-adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure

    PubMed Central

    Rengo, G; Zincarelli, C; Femminella, GD; Liccardo, D; Pagano, G; de Lucia, C; Altobelli, GG; Cimini, V; Ruggiero, D; Perrone-Filardi, P; Gao, E; Ferrara, N; Lymperopoulos, A; Koch, WJ; Leosco, D

    2012-01-01

    BACKGROUND AND PURPOSE We investigated whether β2-adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart. EXPERIMENTAL APPROACH We explored the angiogenic effects of β2-adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β2-adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β2-adrenoceptor −/− mice undergoing MI. KEY RESULTS Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β2-adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-β2-adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, β2-adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β2-adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In β2-adrenoceptor−/− mice, we found a ∼25% reduction in cardiac capillary density compared with β2-adrenoceptor+/+ mice. The lack of β2-adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls. CONCLUSIONS AND IMPLICATION β2-Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism. PMID:22452704

  16. Sonochemical preparation of silica nanorods for gene delivery using single-walled carbon nanotubes as templates.

    PubMed

    Lee, Kyoung G; Min, Jung Sun; Wi, Rinbok; Kim, Jin Chul; Ahn, Jeong Keun; Kim, Do Hyun

    2011-01-01

    Silica nanorods were fabricated with single-walled carbon nanotubes (SWCNTs) via ultrasound. The diameter of the resulting SWCNT-silica particles ranged from 60 to 70 nm. The morphology of this composite material was investigated via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The individual SWCNTs are uniformly coated with silica and formed a unique nanocomposite material. The important role of ultrasound and the mechanism of silica layer formation on SWCNTs were explained via the hydrolysis of the silica source and the adsorption of the siloxane groups on the SWCNT surfaces under ultrasound irradiation. The amino-functionalized silica nanorods were demonstrated as non-viral vectors for gene delivery.

  17. Gene-environment interaction in preterm delivery with special reference to organochlorine pesticides.

    PubMed

    Mustafa, M D; Banerjee, B D; Ahmed, Rafat S; Tripathi, A K; Guleria, Kiran

    2013-01-01

    We investigated the association between glutathione S-transferases mu1 (GSTM1), theta 1 (GSTT1), Cytochrome P450IA1-T6235C (rs4646903, CYP1A1m1) and CYP1A1-1462V (rs1048943, CYP1A1m2) gene polymorphisms, and organochlorine pesticides (OCPs) level with risk of preterm delivery (PTD). Maternal and cord blood samples of PTD (n = 156) cases and subjects of full-term delivery (FTD, n = 151) were collected at the time of delivery/after delivery. Women occupationally exposed to OCPs and other high-risk factors such as anemia, hypertension and dietary habit were excluded. The OCP levels were estimated by gas chromatography, and polymorphic analysis of GSTM1/GSTT1 and CYP450 genes was carried out using multiplex PCR and PCR-restriction fragment length polymorphism, respectively. The frequency of GSTM1/GSTT1 (null) genotype was significantly higher in PTD cases than in the controls. Significantly high levels of α-hexachlorocyclohexane (HCH), γ-HCH and Dichlorodiphenyldichloroethylene (p'p'-DDE) were observed in maternal blood, while significantly high levels of p,p'-dichlorodiphenyltrichloroethane and p'p'-DDE were found in the cord blood of PTD cases compared with the controls. A significant association was seen between β-HCH and GSTM1 genotype when interaction between GSTM1 gene polymorphism, maternal blood OCP levels and period of gestation (POG) was ascertained. A significant reduction in POG was observed. Similarly, cord blood dieldrin levels were significantly associated with CYP1A1m2 (Aa/aa) with reduction in POG. Our observations indicate that higher levels of OCPs in pregnant women may be associated with increased risk of 'idiopathic' PTD. Furthermore, this study shows that the interaction between high OCPs levels and polymorphism in CYP1A1m2 and GSTM1 null genotypes may magnify the risk of PTD, thus providing evidence for a gene-environment interaction in pregnant women.

  18. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

    PubMed

    Miao, Carol H

    2016-01-01

    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  19. Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability.

    PubMed

    Eltoukhy, Ahmed A; Chen, Delai; Alabi, Christopher A; Langer, Robert; Anderson, Daniel G

    2013-03-13

    Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy

    DTIC Science & Technology

    2015-09-01

    in Intractable Epilepsy PRINCIPAL INVESTIGATOR: Michael A. King, Ph.D. CONTRACTING ORGANIZATION: University of Florida REPORT DATE: September 2015...efficacy against seizures. 15. SUBJECT TERMS Epilepsy ; seizure; kindling; somatatostatin; traumatic brain injury; gene delivery; adeno­ associated viral...Disease Modification and Cognitive Sparing in Intractable Epilepsy Table of Contents Page 1. Introduction………………………………………………………….4 2. Keywords

  1. Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo.

    PubMed

    Lilley, C E; Groutsi, F; Han, Z; Palmer, J A; Anderson, P N; Latchman, D S; Coffin, R S

    2001-05-01

    Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604-5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

  2. CRISPR genome engineering and viral gene delivery: a case of mutual attraction.

    PubMed

    Schmidt, Florian; Grimm, Dirk

    2015-02-01

    The adaptation of the CRISPR/Cas9 DNA engineering machinery for mammalian cells has revolutionized our approaches to low- or high-throughput genome annotation and paved the way for conceptually novel therapeutic strategies. A large part of the attraction of CRISPR stems from the small size of its two core components--Cas9 and gRNA--and hence its compatibility with virtually any available viral vector delivery system. As a result, over the past two years, four major classes of viral vectors have already been engineered and applied as CRISPR delivery tools--retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs). The juxtaposition of these two technologies reflects a case of tremendous mutual attraction and holds unprecedented promises for biology and medicine. Here, we provide an overview of the state-of-the-art of this rapidly emerging field, from a comparative description of the principal vector designs, to a synopsis of some of the most exciting applications that were reported to date, including the use of viral CRISPR vectors for genome-wide loss-of-function screens, multiplexed gene editing or disease modeling in animals. Once specificity and safety have been improved further, viral vector-mediated in vitro/in vivo CRISPR delivery and expression promise to radically transform basic and applied biomedical research.

  3. Integration of drug, protein, and gene delivery systems with regenerative medicine.

    PubMed

    Lorden, Elizabeth R; Levinson, Howard M; Leong, Kam W

    2015-04-01

    Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g., hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and microparticle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein, and gene delivery in the context of regenerative medicine. Next, we will present case examples of how delivery technologies are being applied to promote angiogenesis in nonhealing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench to bedside for the clinical translation of these novel therapeutics.

  4. Integration of Drug, Protein, and Gene Delivery Systems with Regenerative Medicine

    PubMed Central

    Lorden, Elizabeth R.; Levinson, Howard M.; Leong, Kam W.

    2013-01-01

    Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g. hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and micro-particle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein and gene delivery in the context of regenerative medicine. Next we will present case examples of how delivery technologies are being applied to promote angiogenesis in non-healing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench-to-bedside for the clinical translation of these novel therapeutics. PMID:25787742

  5. DNA-templated semiconductor nanocrystal growth for controlled DNA packing and gene delivery.

    PubMed

    Gao, Li; Ma, Nan

    2012-01-24

    DNA-templated semiconductor nanocrystal (SNC) growth represents a facile means to generate bioactive hybrid nanostructures by directly integrating DNA molecules and luminescent SNCs together via a one-step synthesis, which has been applied to biosensing and cell imaging. In this study we for the first time demonstrated that DNA-templated CdS SNC growth could also be used to rationally tune the structures and activities of large DNA molecules. We explored the synergistic effects of nanocrystal growth on the sizes and charges of DNA molecules and demonstrate that the CdS growth-induced DNA packing could be used as a smart gene delivery system. Herein we used DNA plasmids encoding intact enhanced green fluorescence protein (EGFP) genes as templates to grow CdS SNCs and found that the stepwise growth of CdS nanocrystals can spontaneously induce DNA condensation and negative charge shielding in a synergistic manner. The condensed DNA plasmids exhibited efficient cellular uptake and a relative gene transfection efficiency of 32%. The transfection efficiency can be further doubled in the presence of chloroquine. We elucidated that the gene transfection and expression is controlled by reversible DNA packing, where ligand exchange of DNA with intracellular glutathione molecules plays a critical role in the recovery of DNA plasmids for gene expression. © 2011 American Chemical Society

  6. Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

    PubMed

    Colella, P; Trapani, I; Cesi, G; Sommella, A; Manfredi, A; Puppo, A; Iodice, C; Rossi, S; Simonelli, F; Giunti, M; Bacci, M L; Auricchio, A

    2014-04-01

    Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

  7. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  8. Elastin-like recombinamers with acquired functionalities for gene-delivery applications.

    PubMed

    Piña, Maria J; Alex, Susan M; Arias, Francisco J; Santos, Mercedes; Rodriguez-Cabello, Jose C; Ramesan, Rekha M; Sharma, Chandra P

    2015-10-01

    In this work, well-defined elastin-like recombinamers (ELRs) were studied as a choice to the existing nonviral vectors due to their biocompatibility and ease of scale-up. Functional motifs, namely penetratin and LAEL fusogenic peptides were incorporated into a basic ELR sequence, and imidazole groups were subsequently covalently bound obtaining ELRs with new functionalities. Stable polyplexes composed of plasmid DNA and ELRs were formed. A particle size around 200 nm and a zeta potential up to nearly +24 mV made them suitable for gene delivery purposes. Additionally, viability and transfection assays with C6 rat glioma cell line showed an increase in the cellular uptake and transfection levels for the construction containing the LAEL motif. This study highlights the importance of controlling the polymer functionality using recombinant techniques and establishes the utility of ELRs as biocompatible nonviral systems for gene-therapy applications.

  9. Gene delivery techniques for adult stem cell-based regenerative therapy.

    PubMed

    Seo, Seog-Jin; Kim, Tae-Hyun; Choi, Seong-Jun; Park, Jeong-Hui; Wall, Ivan B; Kim, Hae-Won

    2013-11-01

    Over the past decade, stem cells have been considered to be a promising resource to cure and regenerate damaged or diseased tissues with research extending from basic studies to clinical application. Furthermore, genetically modified stem cells have the potential to reduce tumorigenic risks and achieve safe tissue formation. Recent advances in genetic modification of stem cells have rendered these cells more accessible and stable. The successful genetic modification of stem cells relies heavily on designing vector systems, either viral or nonviral vectors, which can efficiently deliver therapeutic genes to the cells with minimum toxicity. Currently, viral vectors showing high transfection efficiencies still raise safety issues, whereas safer nonviral vectors exhibit extremely poor transfection in stem cells. Here, we attempt to review and discuss the main factors raising concern in previous reports, and devise strategies to solve the issues in gene delivery systems for successful stem cell-targeting regenerative therapy.

  10. Targeted Drug Delivery to the Peripheral Nervous System using Gene Therapy

    PubMed Central

    Wolfe, Darren; Mata, Marina; Fink, David J.

    2012-01-01

    Gene transfer to target delivery of neurotrophic factors to the primary sensory afferent for treatment of polyneuropathy, or of inhibitory neurotransmitters for relief of chronic pain, offers the possibility of a highly selective targeted release of bioactive molecules within the nervous system. Preclinical studies with non-replicating herpes simplex virus (HSV)-based vectors injected into the skin to transduce neurons in the dorsal root ganglion have demonstrated efficacy in reducing-pain related behaviors in animal models of inflammatory pain, neuropathic pain, and pain caused by cancer, and in preventing progression of sensory neuropathy caused by toxins, chemotherapeutic drugs or resulting from diabetes. Successful completion of the first phase 1 clinical trial of HSV-mediated gene transfer in patients with intractable pain from cancer has set the stage for further clinical trials of this approach. PMID:22565023

  11. Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration

    SciTech Connect

    Liang, Yanran; Liu, Zhonglin; Shuai, Xintao; Wang, Weiwei; Liu, Jun; Bi, Wei; Wang, Chuanming; Jing, Xiuna; Liu, Yunyun; Tao, Enxiang

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Nogo receptor can inhibit growth of injured axons, thus affecting neural regeneration. Black-Right-Pointing-Pointer The delivery of siRNA is crucial to inhibit NgR expression in NSCs. Black-Right-Pointing-Pointer Non-viral vector PEG-PEI condensed siRNA targeting NgR into nanoscale particles. Black-Right-Pointing-Pointer PEG-PEI/siRNA at N/P = 15 displayed high transfection efficiency and low cytotoxicity. Black-Right-Pointing-Pointer PEG-PEI has great potential in carrying siRNA to diminish the gene expression in NSCs. -- Abstract: The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P = 15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P < 0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P < 0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that

  12. Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery.

    PubMed

    LaManna, Caroline M; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J; Barthélémy, Philippe; Grinstaff, Mark W

    2012-07-17

    Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a

  13. Charge-reversal Lipids, Peptide-based Lipids, and Nucleoside-based Lipids for Gene Delivery

    PubMed Central

    LaManna, Caroline M.; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J.; Barthélémy, Philippe; Grinstaff, Mark W.

    2013-01-01

    Conspectus Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy’s appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to a myriad of diseases by tailoring the treatment to a specific individual’s genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when utilizing non-viral vectors have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: 1) that releasing the nucleic acid from the carrier will increase gene transfection; 2) that utilizing biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery; and 3) that mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will give unique supramo