DNA Delivery Methods Research Articles

Gene transfer to skeletal muscle by site-specific delivery of electroporation and ultrasound

Transfecting foreign genes into target cells require certain vectors and specific techniques. With more new genes and gene targets for gene therapy are continually being discovered, which provide useful clues for the study of gene function and gene therapy for human disease. However, there still remain a number of important unresolved problems associated with the use of viral and non viral vectors or techniques, such as secondary toxicity, immune response, or low gene transfer efficiency. Therefore, efficient and safe approaches of gene delivery in vivo still need to be found for medical applications. Electroporation or ultrasound (US), which involving electrical pulses or a US field to increase cell membrane permeability, has shown to be an efficient, safe and simple non viral physical method of DNA delivery in vivo and alternative technique in the field of gene therapy. However, the high field strength or energy often required for electroporation or US can result in tissue damage, thus limit their widely clinical applications. In recent years, site-specific gene delivery by electroporation or US has aroused much attention, because of optimized protocols and novel devices using a lower field strength than conventional methods, which has shown high transfection efficiency with minimal tissue damage. In this paper, we reviewed the advancement in the field of electroporation and US for gene delivery, particularly by site-specific delivery into skeletal muscle for gene therapy and their applications in Alzheimer’s disease (AD)’s treatment.

Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.

From Gene to Epigene-Based Therapies Targeting the Vascular Endothelium

Vascular endothelial dysfunction is a key biological process underlying the development of cardiovascular disease and therefore of potential importance as a target for gene-based therapy. Modification of nitric oxide bioavailability through gene therapy is possible in animal studies and of clinical relevance because of the central role for nitric oxide in vascular homeostasis. However, most clinical trials have so far focused on endothelial-related pathways, in particular, vascular endothelial growth factor, to induce angiogenesis, with variable results. The slow progress of the development of gene-therapy targeted at the endothelium relates to a range of complexities of design of therapy including mode of gene delivery. This is usually achieved through the use of viral or non-viral vectors but the best physical and vector methods for delivery of complimentary DNA to the vascular endothelium remains under investigation. More recently there has been emerging interest into other genome-based methods to alter vascular phenotype, in particular, gene-based modification of endothelial progenitor cell function and whether gene function might be modifiable through induced epigenetic changes.

The therapeutic potential of phiC31 integrase as a gene therapy system

Introduction: The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells.Areas covered: The φC31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells. We focus on work with the φC31 integrase system during the past 12 – 18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells.Expert opinion: We conclude that the φC31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The φC31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.

DNA vaccines delivered by human papillomavirus pseudovirions as a promising approach for generating antigen-specific CD8+ T cell immunity

BackgroundHuman papillomavirus (HPV) pseudovirions have recently been shown to deliver DNA efficiently in vivo, resulting in the priming of antigen-specific CD8+ T cells in vaccinated mice. In the current study, we compare the different preparation methods for the generation of HPV pseudovirions for their ability to efficiently infect cells. We also compare the antigen-specific CD8+ T cell immune responses generated by different DNA delivery methods and several commonly used forms of vaccination with that of HPV pseudovirions.ResultsWe found that the preparation method of pseudovirions is important for the efficient delivery of encapsidated DNA. We have shown that vaccination with DNA encoding model antigen ovalbumin (OVA) delivered by HPV-16 pseudovirions was capable of generating therapeutic antitumor effects against OVA-expressing tumor. In addition, vaccination with DNA encoding OVA delivered by HPV-16 pseudovirions generated the highest number of OVA-specific CD8+ T cells in mice in our system compared to DNA delivered by other delivery methods. We also found that vaccination with OVA DNA delivered by HPV-16 pseudovirions generated the highest number of OVA-specific CD8+ T cells in mice compared to other forms of antigen-specific vaccines. Furthermore, HPV-16 pseudovirions were capable of carrying DNA vaccine encoding clinically relevant antigen, telomerase reverse transcriptase, to generate antigen-specific CD8+ T cell immune responses.ConclusionsOur data suggest that DNA vaccines delivered by HPV-16 pseudovirions may be advantageous compared to other delivery methods and other forms of antigen-specific vaccines for application to antigen-specific immunotherapy.

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Biolistic maize transformation: Improving and simplifying the protocol efficiency

Two decades have passed since the first maize transformation protocol. Genetic breeding has been decisive and essential to transform maize a major food crop worldwide. Biotechnology plays an important role in plant genetic breeding, particularly for the introduction of novel traits in order to improve agronomic performance, medical and industrial applications and food quality. In recent years, the development of efficient plant regeneration systems in cereal crops and the field of biotechnology have opened up new opportunities for genetic transformation of crop plants. Some monocot plants were initially considered difficult for genetic engineering, primarily due to their recalcitrance to in vitro regeneration and their resistance to Agrobacterium. Continuous efforts and studies of different tissues for regeneration potential, development of various DNA delivery methods, and optimization of gene expression cassettes have led to the development of reliable transformation protocols for major cereals, including maize. Consequently, this research group has focused its attention on maize transformation mediated by microprojectile bombardment as a device of DNA delivery into maize cells. This method offers a rapid and simple way of introgression of candidate genes into cells. However, there are some points that still need to be studied and improved in order to achieve appropriate transformation efficiency to optimize the processing conditions to obtain fertile plants. Key words: Biolistic, transient expression, reporter genes, plant genetic improvement.

A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria

Using food and commensal lactic acid bacteria (LAB) as vehicles for DNA delivery into epithelial cells is a new strategy for vaccine delivery or gene therapy. However, present methods for DNA delivery with LAB have suffered low efficiency. Our goal was to develop a new system to deliver DNA into epithelial cells with high efficiency using food and commensal LAB. An Escherichia coli-LAB shuttle plasmid, pLKV1, for DNA delivery into eukaryotic cells was constructed. Two reporter plasmids with green and red fluorescent protein genes were also constructed to monitor the uptake of protein and DNA, respectively. Bacteria delivering these reporter plasmids into Caco-2 cells were monitored by fluorescence microscopy. Several methods that weaken the bacterial cell wall prior to co-culture with Caco-2 cells were evaluated for their role in the improvement of gene transfer efficiency. Treating Streptococcus gordonii with penicillin and lysozyme greatly increased its rate of gene delivery to mammalian cells compared to untreated control bacteria, while glycine pretreatment promoted the highest gene transfer rate for Lactococcus lactis. Uptake of green fluorescent bacteria by Caco-2 cells showed that the cell wall-weakening treatment promoted the internalization of the noninvasive bacteria into Caco-2 cells. In conclusion, we have developed a noninvasive system using LAB as a vehicle for vaccine delivery or gene therapy, and tested this system in vitro with Caco-2 cells.

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

BackgroundThermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes.Methodology/Principal FindingsHere we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic.Conclusions/SignificanceIn this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically.

Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma

BackgroundHydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, ϕC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with ϕC31 integrase expression in tissue culture, leading to questions about safety.Methodology/Principal FindingsTo study whether hydrodynamic delivery alone, or in conjunction with delivery of ϕC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive ϕC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection.Conclusions/SignificanceOur data suggest that ϕC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking ϕC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.

Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair

Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders.

In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection

DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream. A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution. We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing. The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.

Abstract 2183: High-throughput screening, identification and characterization of novel kinases involved in chemosensitization of breast cancer cells

Abstract Background - Development of drug resistance is considered to be one of the most challenging issues in chemotherapy for cancer patients. As one of the conventional chemotherapeutic agent, paclitaxel was originally extracted from Pacific yew tree and has been applied widely in ovarian cancer, breast cancer and lung cancer. Resistance to paclitaxel has posed severe problems in cancer treatment. We are therefore interested in identifying the genes that render paclitaxel-resistant breast cancer cells to become more sensitive with the drug treatment. The mechanisms and signaling pathways of these kinases in relations to paclitaxel resistance and their molecular targets have been investigated. Methods - We have screened a shRNA library containing 592 novel kinase genes with 1306 clones for the chemosensitizer loci. DNA was extracted in a high throughput manner, and cell transfection/transduction was carried out on 384 well plates using a unique solid-phase DNA delivery method. The positive hits were selected according to the alteration on their IC50 values for the drug using cell viability assays. Quantitative PCR, Western blot, and caspase assays have been employed to elucidate the signaling pathways of these kinases in response to paclitaxel. Protein purification and interaction studies have also been carried out in attempt to elucidate the putative substrates for the kinases and their interaction partners. Results and Conclusions - We have established a high throughput screening platform for identification of the chemosensitizer loci. High transfection/transduction efficiencies can be achieved using our solid-phase gene delivery method. Several novel unknown kinase genes that render drug-resistant cancer cells to become sensitive have been selected. The positive shRNA hits were further verified by quantitative real-time PCR. Among the 20 hits that showed diminished IC50 values of paclitaxel in breast cancer cells, we have selected two candidates for further studies. Knocking down of TAOK3 or VRK3 in breast cancer cells resulted in reduction of IC50 of paclitaxel to half of the original value. Furthermore, we have investigated the signaling pathways of these 2 kinases in relation to paclitaxel resistance. The different signaling pathways elucidated have revealed the complex nature of drug resistance development in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2183.

Site-specific gene integration technologies for crop improvement

Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the use of site-specific recombination systems has been developed which enables integration into previously engineered genomic sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology in generating transgenic events with a predictable range and stability of expression through successive generations, which are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly used method. The present article discusses barriers and solutions for making it readily available to both academic research and applicative use.

DNA Vaccines against Infectious Diseases and Cancer

Progress in the development of DNA vaccines and their delivery strategies has been made since their initial concept as a next generation vaccine. Since DNA vaccine includes non-infectious DNA parts of pathogens, it can't cause disease yet it closely mimic the natural process of infection and immune responses. Despite their early promising results of controlling infectious diseases and cancer in small animal models, DNA vaccines failed to display a level of immunogenicity required for combating these diseases in humans, possibly due to their lower protein expression levels. However, increasing evidence has shown that DNA vaccines are clinically well-tolerated and safe. Furthermore, one notable advantage of DNA vaccines includes convenient utilities of plasmid DNAs coding for antigens. For instance, any emerging pathogens could be prevented easily and timely by allowing the simple exchange of antigen-encoding genes. In this review, newly developed DNA vaccine strategies, including electroporation, which has emerged as a potent method for DNA delivery, targeting infectious diseases and cancer will be discussed with a focus on any on-going DNA vaccine trials or progress made pre-clinically and in clinics.

Electroporation: A promising method for the nonviral delivery of DNA vaccines in humans?

The without a doubt major obstacle for making DNA vaccines a commercial success is delivery. If delivery cannot be made simple, cheap and effective, DNA vaccines may not become a viable option for human use. Numerous clinical trials have confirmed that a standard needle and syringe simply do not do the job, i.e., delivering the DNA payload inside the cell. Having recognized this shortcoming, investigators have developed several new approaches for DNA vaccine delivery. In particular, new types of delivery devices, originally intended for in vitro use, have been applied for in vivo delivery. These include particle bombardment or biolistic delivery, and in vivo electroporation (EP). Importantly, both techniques seem to overcome the size barrier, meaning that they work in both mice and larger animals. In vivo EP has the key features of improved DNA uptake, increased antigen expression and a local inflammation. These factors are essential to make DNA vaccines effective in a larger host. Early data from clinical trials with DNA vaccines delivered by in vivo EP are cautiously promising. Thus, we may be entering a new era of DNA vaccination where we start to see clinical effects in humans; however, these may also be accompanied by side effects, as the vaccines become more effective.

Shock waves for ballistic delivery of DNA droplets into living cells

The biolistic approach of DNA/drug delivery was applied to deliver liquid DNA into living cells. The liquid DNA to be delivered was deposited as a drop on a thin aluminum foil and the posterior surface of the foil was ablated using an Nd:YAG laser. The ablation launched a shock wave through the foil. A part of this shock wave was transmitted to the drop, and a part was reflected back into the foil as an expansion wave. The wave motions caused the drug droplet to accelerate and acquire a sufficiently high velocity in the forward direction. A part of the propelled DNA droplet, on impacting the surface of a soft, living target, entered the target cells, accomplishing the drug delivery. The technique was tested on E-coli bacteria by delivering a plasmid DNA pUC119 into the bacterial cells. A few bacterial colonies could be transformed by this method of DNA delivery.

224 Persistent virological benefit in SIV-infected macaques upon therapeutic vaccination with DNA vectors by in vivo constant-current electroporation

Background: We have previously shown that SIV-infected macaques immunized during ART by intramuscular injection of optimized plasmid DNA mount an immune response able to reduce viremia in both prophylactic and therapeutic interventions against SIV infection. Here, we tested the more efficient in vivo constant-current electroporation as DNA delivery method. Methods: Rhesus macaques chronically infected with SIVmac251 were treated with combination antiretroviral therapy (ART). During this time, the animals were vaccinated by in vivo constant-current electroporation with optimized forms of DNA vectors expressing the majority of SIV proteins and then released from ART. The animals were monitored for rebounding virus and changes in SIV specific immune responses during and after termination of therapy. Cellular immune responses were monitored in peptide-stimulated PBMC by multiparametric flow cytometry combining cell surface and intracellular cytokine staining. Results: DNA vaccination by in vivo electroporation was very effective in inducing recall immune responses. These responses were between 5-10-fold higher that those previously observed by IM injection. Animals treated with a 2nd cycle of ART and therapeutic vaccination showed additional persistent virological benefit (>1 log10), as measured by decreased viremia after release from ART. Control of viremia was associated with a very strong induction of SIV-specific responses, and was characterized by the production of IFN-γ, IL−2, and TNFα either alone or in combination as double or triple cytokine positive multifunctional T cells. A significant induction of CD4+ T cell responses, mainly targeting Gag, Nef, and Pol, as well as of CD8+ T cells, mainly targeting Env, was found in both T cells with central memory and effector memory markers. Conclusions: In vivo electroporation of optimized forms of DNA vaccines during ART induced immune responses able to persistently decrease viremia after ART release. DNA vaccination has many advantages, including the possibility to administer the vaccine several times, and was shown to induce potent, efficacious, and long-lasting recall immune responses. Therefore, these data support the concept of adding DNA therapeutic vaccination to the HAART regimen to boost the HIV-specific immune responses.

24. Towards Reversible Packaging Methods for DNA and RNA Delivery

24. Towards Reversible Packaging Methods for DNA and RNA Delivery