Nonviral Gene Transfer Method Research Articles

A Numerical Model of Skin Electropermeabilization Based on In Vivo Experiments

As an alternative to viral methods that are controversial because of their safety issues, chemical and physical methods have been developed to enhance gene expression in tissues. Reversible increase of the cell membrane permeability caused by the electric field--electroporation--is currently one of the most efficient and simple non-viral methods of gene transfer. We performed a series of in vivo experiments, delivering plasmids to rat skin using external plate electrodes. The experiments showed that skin layers below stratum corneum can be permeabilized in this way. In order to study the course of skin tissue permeabilization by means of electric pulses, a numerical model using the finite element method was made. The model is based on the tissue-electrode geometry and electric pulses used in our in vivo experiments. We took into account the layered structure of skin and changes of its bulk electrical properties during electroporation, as observed in the in vivo experiments. We were using tissue conductivity values found in literature and experimentally determined electric field threshold values needed for tissue permeabilization. The results obtained with the model are in good agreement with the in vivo results of gene transfection in rat skin. With the model presented we used the available data to explain the mechanism of the tissue electropermeabilization propagation beyond the initial conditions dictated by the tissue initial conductivities, thus contributing to a more in-depth understanding of this process. Such a model can be used to optimize and develop electrodes and pulse parameters.

Local delivery system of cytotoxic agents to tumors by focused sonoporation

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Non-viral methods for gene transfer towards osteosarcoma therapy

Osteosarcoma, a class of cancer that originates from bone, afflicts mainly young people usually in their teenage years of life. Despite surgery and chemotherapy, the outlook for sufferers is not that positive, with a third of patients with metastatic disease not surviving past the 10-year mark. Like other neoplasms, other forms of therapeutics are being evaluated, and amongst these is gene therapy. This review discusses approaches for gene therapy of osteosarcoma using cationic liposomes and polyethylenimine in vivo. The field is still in its infancy as far as osteosarcoma is concerned and much more needs to be done to test its true potential as a feasible therapeutic modality.

Type I interferon response against viral and non‐viral gene transfer in human tumor and primary cell lines

Type I interferon (IFN-alpha/beta) response is one of the major host defence mechanisms against viruses. Some recent reports suggest that IFNs may interfere with the efficacy of both non-viral and virus-vector-mediated therapeutic gene transfer. The type I IFN response upon different gene transfer methods in human tumor and primary cell lines was studied by analysing IFN-beta mRNA expression, secretion of type I IFNs and accumulation of IFN-alpha/beta-induced MxA protein (myxovirus resistance protein A). Infection with avirulent Semliki Forest virus A7[74] induced MxA protein accumulation and increased the IFN-beta mRNA level, whereas none of the studied virus vectors (adenovirus, CRAd, lentivirus or AAV) induced IFN response. However, plasmid DNA induced the accumulation of MxA protein when transfected with several commercial transfection reagents. RNA transfection appeared to be an efficient inducer of type I IFN response: replicating alphaviral RNA, eukaryotic total RNA, or mRNA all induced both MxA protein accumulation and IFN-beta expression. siRNA transfection failed to induce MxA response. The non-viral gene transfer methods have gained more interest in recent years due to their better safety profiles when compared to their viral counterparts. However, the efficiency of non-viral gene transfer is well below those reached by viral vector systems. The type I interferon response induced by non-viral methods may in part contribute to this inefficiency, while most currently used viral gene transfer vectors fail to induce or are able to suppress type I IFN response.

Non‐viral vectors for cancer therapy

Cancers are diverse and often resistant to therapeutic strategies. Gene therapy has yet to meet the promise of a breakthrough in cancer therapy. There are several barriers to overcome in cancer gene therapy. One of the biggest challenges is the design of appropriate vectors. Numerous viral and non-viral methods for gene transfer have been developed for human gene therapy, but both viral and non-viral vectors have limitations and advantages. In this review article, recent improvements in the development of non-viral vectors for delivering gene therapy for the treatment of cancer will be discussed.

521. Gene Transfer of an Anti-CCR5 siRNA by Sleeping Beauty (SB) Transposon System

The Sleeping Beauty transposon vector system is a non-viral gene delivery method. It is mediated by a transposase that employs a cut-and-paste mechanism. It is derived by repairing defective copies of Tc1/mariner family transposon elements. The utility of SB transposon system has been demonstrated in various mammalian cell lines for example by the gene transfer of a red fluorescence protein (RFP) in primary human lymphocytes. SiRNAs that mediate RNA interference have also been successfully introduced into cells using this system thus paving the way for permanent gene knock down using this novel non-viral gene transfer method. In our current studies we sought to introduce an anti-CCR5 siRNA into HIV-1 susceptible cells to down regulate this essential coreceptor involved in viral entry. A previously characterized short hairpin siRNA against CCR5 together with an RFP reporter was cloned into the SB gene transfer transposon and introduced into MAGI-CCR5 and GHOST-R3X4R5 cell lines. Since the levels of gene transfer were low, the stable transposed clones were enriched by FACS sorting for RFP expression. The enriched transgenic cells were cultured for many generations and monitored for RFP expression which appeared to be stable. To evaluate the siRNA mediated down regulation of CCR5, the transgenic cells were evaluated by FACS analysis. Both MAGI-CCR5 and GHOST-R3X4R5 cells showed a 90% reduction in CCR5 expression indicating that the siRNA is functional in this system. Stable transposition was also confirmed by southern blot hybridization testing of transposed cells with an RFP derived probe. To evaluate the HIV-1 resistance of the transgenic cells, they were challenged with a CCR5 tropic HIV-1 strain. Viral resistance was monitored by assaying for viral p24 antigen released into the supernatants at different days post viral challenge. Our results have shown significant viral resistance by the anti-CCR5 siRNA transgenic cells. These data indicate that a non-viral SB system can be utilized for transposition of siRNA coding genes to confer resistance against HIV.

High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes.

HVJ-based non-viral gene transfer method: successful gene therapy using HGF and VEGF genes in experimental ischemia

VEGF and HGF are pleiotropic factors that regulate cell growth, cell motility, and morphogenesis of various types of cells. The receptors of these growth factors are expressed in neurons and endothelial cells, and are identified as neurotrophic, neuroprotective, and angiogenic factors. Indeed, gene therapy using viral vectors encoding the VEGF or HGF gene has been reported to be effective for preventing the expansion of ischemic injury. However, the safety issue of viral vectors is a major problem in clinical application. To overcome this problem, we have developed an HVJ-based non-viral vector, which achieves high-efficiency transfection rates of viral vectors with the safety of liposomes. This review discusses the feasibility of gene therapy using an HVJ-based non-viral vector containing the VEGF or HGF gene for cerebral ischemia.

In Vivo Human MCP-1 Transfection in Porcine Arteries by Intravascular Electroporation

The purpose of this study was to develop a nonviral gene transfer method for therapeutic delivery of the human monocyte chemoattractant protein-1 (MCP-1) in patients with peripheral artery disease, using local catheter-mediated electrotransfer of naked plasmid DNA into arteries. Arterial walls of the A. profunda femoris of pigs were transfected either with a human MCP-1 or with a firefly luciferase-encoding DNA construct. The efficacy of electrotransfer of DNA was analyzed after 2 days by quantitative polymerase chain reaction (PCR) or luciferase activity measurements. To optimize MCP-1 gene transfer conditions, a voltage range of 60-150 V was applied as a train of six square pulses of 20 ms each at 1 Hz and was combined with a dose of 150 microg DNA. Subsequently, the optimized voltage was used to test a dose range of 80-300 microg DNA. The voltage optimum for arterial transfection was observed at 80 volts. Using this setting, the dose application of 300 microg MCP-1 plasmid DNA (the maximal dose tested) demonstrated the highest MCP-1 expression signal. The electric pulses and the transfer and expression of human MCP-1 per se did not induce endogenous porcine MCP-1 expression in treated arteries. Interestingly, angioplastic predilation of the artery before gene transfer, which had originally been postulated to enhance transfection by improving access of the plasmid to subendothelial cell layers, resulted in an attenuated transfection efficacy. The present study demonstrates that transluminal catheter-based electroporation provides an efficient technology for nonviral intravascular gene transfer by just applying unformulated DNA.

φC31 Integrase Confers Genomic Integration and Long-Term Transgene Expression in Rat Retina

Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage phiC31 to confer long-term gene expression by means of genomic integration. Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of phiC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with phiC31 integrase showed approximately 85-fold higher long-term transgene expression in the retina than eyes without integrase. Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. phiC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that phiC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.

Method — A nonviral gene transfer method for transfecting multipotent adult progenitor cells (MAPC)

Multipotent adult progenitor cells (MAPC) are stem cells isolated primarily from adult bone marrow that have the ability to differentiate in vitro into cells with phenotypic and functional characteristics of cells from the three germ layers, namely endoderm, mesoderm and neuroectoderm. In addition, MAPCs proliferate for extended periods of time without obvious senescence. Hence, MAPCs may be ideal cells for therapy of genetic disorders, provided that the main impediment to gene therapy, namely efficient gene transfer and persistent gene expression, can be overcome. Most commercially available lipid-based methods that very highly efficiently transfect cell lines and primary cells, fail to transfect MAPC. However, 10–15% transfection of MAPC can be achieved using Superfect. However, this approach requires high density culture of MAPCs which subsequently differentiate. Between 12 and 15% transfection is achieved in MAPCs using electroporation, but this is highly toxic to MAPCs. Here, using transient expression of an enhanced green fluorescent protein (EGFP) gene, we report that nucleofection results in a transfection efficiency of over 25% with mouse MAPC without perturbing the conditions suitable for MAPC growth and maintenance, and with significantly less toxicity than electroporation. Similar results were seen for rat and human MAPC. Efficient transfection of MAPC by nucleofection offers an appealing non-viral mode of gene delivery and can be used to overexpress genes of interest in these cells. In addition, it should easily accomodate emerging site-specific integration technology, thereby culminating eventually in MAPC-mediated long-term gene therapy for genetic disorders.

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney

Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.

Polyfection as Nonviral Gene Transfer Method — Design of Novel Nonviral Vector Using α‐Cyclodextrin

AbstractFor Abstract see ChemInform Abstract in Full Text.

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.

Muscle-targetedinterleukin-12gene therapy of orthotopic hepatocellular carcinoma in mice usingin vivoelectrosonoporation

AbstractWe developed a new potent nonviral gene transfer method into mouse muscles in vivo named “electrosonoporation.” We tried in this report to treat murine orthotopic hepatocellular carcinoma (HCC) by muscle-targeted mouse interleukin-12 (mIL-12) gene transfer using in vivo electrosonoporation. I.m. administration of the mIL-12 gene with electrosonoporation elevated serum IL-12 and IFN-γ and significantly prolonged the survival periods with both growth inhibition of orthotopic HCC and inhibition of spontaneous lung metastasis. The IL-12 gene therapy reduced the number of microvessels and induced more Mac-1-positive cells into HCC. These results show that muscle-targeted mIL-12 gene therapy for orthotopic HCC using in vivo electrosonoporation is very efficient and is thus promising for further clinical trial.

Polyfection as nonviral gene transfer method -design of novel nonviral vector using alpha-cyclodextrin

Due to the growing concerns over the toxicity and immunogenicity of viral DNA delivery systems, DNA delivery via nonviral routes has become more desirable and advantageous. In particular, polycation complexes with DNA (polyplex) are attractive nonviral vectors. To design novel polycationic vectors, we prepared polyamidoamine starburst dendrimer (dendrimer) conjugates with three cyclodextrins (CDE conjugates) and three generations (G2, G3, and G4) of dendrimers. Of seven CDE conjugates, an alpha-CDE conjugate (G3) with an average degree of substitution (DS) of alpha-CyD of 2.4 [alpha-CDE conjugate (G3, DS 2.4)] showed greater gene transfer activity than dendrimers and other alpha-CDE conjugates with less cytotoxicity. These results suggest the potential use of alpha-CDE conjugate (G3, DS 2.4) as a polycationic vector in vitro and in vivo. Herein, I review a recent polyfection method, with special focus on alpha-CDE conjugate (G3, DS 2.4).

Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver.

We developed a nonviral gene transfer method using rapid injection of naked DNA targeting the liver and applied it in a rat model of liver transplantation. Inbred Dark Agouti and Lewis rats were used. To test the efficacy and adverse effects of systemic or local (catheter-based) injection, different volumes of phosphate-buffered saline containing naked DNA encoding beta-galactosidase (lacZ) were injected. Luciferase expression was followed by non-invasive imaging, and a cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig) protein was tested functionally by allogenic heart transplantation. Gene transfer was then tested in rat auxiliary liver transplantation (ALT) and orthotopic liver transplantation (OLT). The timing of gene transfer was evaluated in the auxiliary liver transplantation model, and OLT was performed using a liver graft to which luciferase or the CTLA4Ig gene was transferred 2 days before. LacZ was expressed extensively in a volume-dependent manner; however, a large volume often induced recipient death. After local delivery of CTLA4Ig cDNA to the liver, survival of Dark Agouti heart grafts lengthened with increased CTLA4Ig serum levels. Liver grafts injected with naked DNA at the time of donation did not survive, but livers grafted 2 days after gene transfer survived. Successful expression of luciferase and production of CTLA4Ig were finally confirmed in the rat that underwent OLT. We successfully applied a nonviral hydrodynamic gene transfer method to the rat liver and showed its potential in liver grafting. The high incidence of graft failure when this procedure is performed on the day of organ donation is a potential limitation that needs to be overcome in clinical application.

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice.

The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.

New non-viral method for gene transfer into primary cells

The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons.