Duration Of Transgene Expression Research Articles

AAV Vectors Vaccines Against Infectious Diseases

Since their discovery as a tool for gene transfer, vectors derived from the adeno-associated virus (AAV) have been used for gene therapy applications and attracted scientist to this field for their exceptional properties of efficiency of in vivo gene transfer and the level and duration of transgene expression. For many years, AAVs have been considered as low immunogenic vectors due to their ability to induce long-term expression of non-self-proteins in contrast to what has been observed with other viral vectors, such as adenovirus, for which strong immune responses against the same transgene products were documented. The perceived low immunogenicity likely explains why the use of AAV vectors for vaccination was not seriously considered before the early 2000s. Indeed, while analyses conducted using a variety of transgenes and animal species slowly changed the vision of immunological properties of AAVs, an increasing number of studies were also performed in the field of vaccination. Even if the comparison with other modes of vaccination was not systemically performed, the analyses conducted so far in the field of active immunotherapy strongly suggest that AAVs possess some interesting features to be used as tools to produce an efficient and sustained antibody response. In addition, recent studies also highlighted the potential of AAVs for passive immunotherapy. This review summarizes the main studies conducted to evaluate the potential of AAV vectors for vaccination against infectious agents and discusses their advantages and drawbacks. Altogether, the variety of studies conducted in this field contributes to the understanding of the immunological properties of this versatile virus and to the definition of its possible future applications.

In VivoElectroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.

The effect of alginate on DNA delivery from layer-by-layer assembled films

Alginate has been previously utilized as an adjuvant in nanoparticle-mediated gene delivery. In this study, we investigated the extent to which alginate promotes in situ transfection from polyelectrolyte multilayers (PEMs). After spiking free alginate molecules, DNA release from PEMs was increased and transgene expression was enhanced, suggesting that alginate may compete with DNA to weaken electrostatic interaction within PEMs. Consequently, alginate was applied to compose multilayers with DNA and polyethyleneimine (PEI). Interestingly, the incorporated alginate increased not only deposition but also delivery of DNA from PEMs. Quartz crystal microbalance (QCM) analysis suggested that the deposited alginate enhanced PEI adsorption which in turn augmented subsequent DNA deposition. In situ transfection experiments revealed that alginate within PEMs improved not only the level but also the duration of transgene expression. This system should be a potential strategy to regulate gene delivery from scaffolds for tissue engineering application.

Transgene and immune gene expression following intramuscular injection of Atlantic salmon (Salmo salar L.) with DNA-releasing PLGA nano- and microparticles

The use of poly-(D,L-lactic-co-glycolic) acid (PLGA) particles as carriers for DNA delivery has received considerable attention in mammalian studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of PLGA-encapsulated plasmid DNA (pDNA). We injected Atlantic salmon (Salmo salar L.) intramuscularly with a plasmid vector containing a luciferase (Photinus pyralis) reporter gene as a) naked pDNA, b) encapsulated into PLGA nano- (∼320 nm) (NP) or microparticles (∼4 μm) (MP), c) in an oil-based formulation, or with empty particles of both sizes. The ability of the different pDNA-treatments to induce transgene expression was analyzed through a 70-day experimental period. Anatomical distribution patterns and depot effects were determined by tracking isotope labeled pDNA. Muscle, head kidney and spleen from all treatment groups were analyzed for proinflammatory cytokines (TNF-α, IL-1β), antiviral genes (IFN-α, Mx) and cytotoxic T-cell markers (CD8, Eomes) at mRNA transcription levels at days 1, 2, 4 and 7. Histopathological examinations were performed on injection site samples from days 2, 7 and 30. Injection of either naked pDNA or the oil-formulation was superior to particle treatments for inducing transgene expression at early time-points. Empty particles of both sizes were able to induce proinflammatory immune responses as well as degenerative and inflammatory pathology at the injection site. Microparticles demonstrated injection site depots and an inflammatory pathology comparable to the oil-based formulation. In comparison, the distribution of NP-encapsulated pDNA resembled that of naked pDNA, although encapsulation into NPs significantly elevated the expression of antiviral genes in all tissues. Together the results indicate that while naked pDNA is most efficient for inducing transgene expression, the encapsulation of pDNA into NPs up-regulates antiviral responses that could be of benefit to DNA vaccination.

Systemic Image-Guided Liver Cancer Radiovirotherapy Using Dendrimer-Coated Adenovirus Encoding the Sodium Iodide Symporter as Theranostic Gene

Currently, major limitations for the clinical application of adenovirus-mediated gene therapy are high prevalence of neutralizing antibodies, widespread expression of the coxsackie-adenovirus receptor (CAR), and adenovirus sequestration by the liver. In the current study, we used the sodium iodide symporter (NIS) as a theranostic gene to investigate whether coating of adenovirus with synthetic dendrimers could be useful to overcome these hurdles in order to develop adenoviral vectors for combination of systemic oncolytic virotherapy and NIS-mediated radiotherapy. We coated replication-deficient (Ad5-CMV/NIS) (CMV is cytomegalovirus) and replication-selective (Ad5-E1/AFP-E3/NIS) adenovirus serotype 5 carrying the hNIS gene with poly(amidoamine) dendrimers generation 5 (PAMAM-G5) in order to investigate transduction efficacy and altered tropism of these coated virus particles by (123)I scintigraphy and to evaluate their therapeutic potential for systemic radiovirotherapy in a liver cancer xenograft mouse model. After dendrimer coating, Ad5-CMV/NIS demonstrated partial protection from neutralizing antibodies and enhanced transduction efficacy in CAR-negative cells in vitro. In vivo (123)I scintigraphy of nude mice revealed significantly reduced levels of hepatic transgene expression after intravenous injection of dendrimer-coated Ad5-CMV/NIS (dcAd5-CMV/NIS). Evasion from liver accumulation resulted in significantly reduced liver toxicity and increased transduction efficiency of dcAd5-CMV/NIS in hepatoma xenografts. After PAMAM-G5 coating of the replication-selective Ad5-E1/AFP-E3/NIS, a significantly enhanced oncolytic effect was observed after intravenous application (virotherapy) that was further increased by additional treatment with a therapeutic dose of (131)I (radiovirotherapy) and was associated with markedly improved survival. These results demonstrate efficient liver detargeting and tumor retargeting of adenoviral vectors after coating with synthetic dendrimers, thereby representing a promising innovative strategy for systemic NIS gene therapy. Moreover, our study-based on the function of NIS as a theranostic gene allowing the noninvasive imaging of NIS expression by (123)I scintigraphy-provides detailed characterization of in vivo vector biodistribution and localization, level, and duration of transgene expression, essential prerequisites for exact planning and monitoring of clinical gene therapy trials that aim to individualize the NIS gene therapy concept.

242. A Novel Enhancer Increases the Level and Duration of Transgene Expression in Mice

242. A Novel Enhancer Increases the Level and Duration of Transgene Expression in Mice

Baculovirus as an Ideal Radionuclide Reporter Gene Vector: A New Strategy for Monitoring the Fate of Human Stem Cells In Vivo

PurposeRadionuclide reporter gene imaging holds promise for non-invasive monitoring of transplanted stem cells. Thus, the feasibility of utilizing recombinant baculoviruses carrying the sodium iodide symporter (NIS) reporter gene in monitoring stem cell therapy by radionuclide imaging was explored in this study.MethodsRecombinant baculoviruses carrying NIS and green fluorescent protein (GFP) reporter genes (Bac-NIS and Bac-GFP) were constructed and used to infect human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). Infection efficiency, total fluorescence intensity and duration of transgene expression were determined by flow cytometry. Cytotoxicity/proliferative effects of baculovirus on hUCB-MSCs were assessed using CCK-8 assays. 125I uptake and perchlorate inhibition assays were performed on Bac-NIS-infected hUCB-MSCs. Radionuclide imaging of mice transplanted with Bac-NIS-infected hUCB-MSCs was performed by NanoSPECT/CT imaging.ResultsInfection efficiencies of recombinant baculovirus in hESCs, hiPSCs and hUCB-MSCs increased with increasing MOIs (27.3%, 35.8% and 95.6%, respectively, at MOI = 800). Almost no cytotoxicity and only slight effects on hUCB-MSCs proliferation were observed. Obvious GFP expression (40.6%) remained at 8 days post-infection. The radioiodide was functionally accumulated by NIS gene products and specifically inhibited by perchlorate (ClO4 -). Radioiodide uptake, peaking at 30 min and gradually decreasing over time, significantly correlated with hUCB-MSCs cell number (R2 = 0.994). Finally, radionuclide imaging showed Bac-NIS-infected hUCB-MSCs effectively accumulated radioiodide in vivo, which gradually weakened over time.ConclusionBaculovirus as transgenic vector of radionuclide reporter gene imaging technology is a promising strategy for monitoring stem cell transplantation therapy.

Biomaterial Scaffolds for Controlled, Localized Gene Delivery of Regenerative Factors

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. 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. 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. 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.

Efficacy of MRI visible iron oxide nanoparticles in delivering minicircle DNA into liver via intrabiliary infusion

Gene therapy is a very promising technology for treatment of liver diseases. Minicircle DNA (MCDNA) is a versatile gene vector, which possesses excellent features in bio-safety and duration of transgene expression. However, its application has been hampered by the lack of an efficient gene delivery system. In this study, we developed a magnetic resonance imaging (MRI) visible nanoparticle to monitor MCDNA gene delivery and explore the potential of gene therapy in vivo. The nanoparticle was constructed via self-assembly of stearic acid modified low molecular polyethyleneimine (stPEI) and superparamagnetic iron oxide (SPIO) nanocrystals. The multiple SPIO nanocrystals with a controlled clustering structure in the nanoparticles were designed to achieve high MRI sensitivity. Furthermore, the MCDNA was combined with the nanoparticles via electronic interaction. Through intrabiliary infusion, the stPEI-SPIO/MCDNA nanoparticles were efficiently delivered to liver that was visualized by MRI in vivo and confirmed by histology. Moreover, the MCDNA nanoparticles exhibited non-cytotoxicity with no obvious inflammation in the transfection sites. These results indicate that stPEI-SPIO nanoparticle is able to serve as both an efficient DNA vector delivery system and a sensitive agent for MRI visualization.

Control of Spatiotemporal Distribution of Interferon γ by Genetically Fusing Functional Peptides

Type II interferon (IFNγ) is a representative Th1 cytokine and it possesses a variety of functions, including immune regulation, antiviral and antitumor activity. Because of its multifunctional nature, IFNγ is expected to be applied to the treatment of autoimmune diseases, cancer and viral infection. Although IFNγ has therapeutic potential for such diseases, the clinical use of IFNγ has been limited due to its short in vivo half-life and serious adverse effects. In contrast, gene delivery of IFNγ is an alternative approach to increasing the retention time of IFNγ. To extend transgene expression after plasmid DNA (pDNA) gene transfer, we designed and developed pDNA with varying numbers of CpG motifs. CpG-reduced pDNA resulted in more durable transgene expression than its CpG replete counterpart in mice. Comparison of the effect of promoter/enhancer elements on transgene expression showed that ROSA26 promoter-mediated IFNγ expression was safe because of the lack of an initial surge after hydrodynamic gene transfer. We also designed an IFNγ-mouse serum albumin (MSA) fusion protein, IFNγ-MSA. Gene transfer of this fusion protein resulted in a sustained concentration of IFNγ fusion protein in mouse serum, and inhibited tumor metastasis in mice. These results provide experimental evidence that IFNγ gene therapy can be a useful treatment for a variety of diseases.

Direct FGF-2 Gene Transfer via Recombinant Adeno-Associated Virus Vectors Stimulates Cell Proliferation, Collagen Production, and the Repair of Experimental Lesions in the Human ACL

Background: Basic fibroblast growth factor (FGF-2) is a powerful stimulator of fibroblast proliferation and type I/III collagen production. Hypothesis: Overexpression of FGF-2 via direct recombinant adeno-associated virus (rAAV) vector–mediated gene transfer enhances the healing of experimental lesions to the human anterior cruciate ligament (ACL). Study Design: Controlled laboratory study. Methods: rAAV vectors carrying a human FGF-2 sequence or the lacZ marker gene were applied to primary human ACL fibroblasts in vitro and to intact or experimentally injured human ACL explants in situ to evaluate the efficacy and duration of transgene expression and the potential effects of FGF-2 treatment upon the proliferative, metabolic, and regenerative activities in these systems. Results: Sustained, effective dose-dependent lacZ expression was achieved in all systems tested (up to 96% ± 2% in vitro and 80%-85% in situ for at least 30 days). rAAV allowed for continuous FGF-2 production both in vitro and in the intact ACL in situ (32.7 ± 1.4 and 33.1 ± 0.8 pg/mL/24 h, respectively, ie, up to 41-fold more than in the controls at day 30; always P ≤ .001), leading to significantly and durably enhanced levels of proliferation and type I/III collagen production vis-à-vis lacZ (at least 3- and 4-fold increases at day 30, respectively; always P ≤ .001). Most notably, rAAV FGF-2 promoted a significant, long-term production of the factor in experimental ACL lesions (92.7 ± 3.9 pg/mL/24 h, ie, about 5-fold more than in the controls; P ≤ .001) associated with enhanced levels of proliferation and type I/III collagen synthesis (at least 2- and 4-fold increases at day 30, respectively; always P ≤ .001). Remarkably, the FGF-2 treatment allowed for a decrease in the amplitude of such lesions possibly because of the increased expression in contractile α–smooth muscle actin, ligament-specific transcription factor scleraxis, and nuclear factor–κB for proliferation and collagen deposition, which are all markers commonly induced in response to injury. Conclusion: Efficient, stable FGF-2 expression via rAAV enhances the healing of experimental human ACL lesions by activating key cellular and metabolic processes. Clinical Relevance: This approach has potential value for the development of novel, effective treatments for ligament reconstruction.

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

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

Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression

Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.

Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation

Gene therapy holds great potential for the treatment of various acquired and inherited pulmonary diseases. Among various viral vectors, adeno-associated viral (AAV) vectors have been most frequently used in different clinical trials of pulmonary gene therapy. In the present study, we examined the kinetics and duration of transgene expression, vector biodistribution and development of neutralizing antibodies (NAB) in mice after pulmonary application of AAV2/9 vector. The pulmonary route of application did not affect any of the measured parameters. Transgene expression and biodistribution analysis at day 450 post-application confirmed the systemic spread of the vector after pulmonary delivery. Using SPB(-/-) mice, the study shows that AAV2/9-mediated gene expression is influenced by animal gender but not mouse genotype and is insensitive to the presence of lung inflammation. Lower expression levels were observed in male compared with female mice, and transient immunosuppression with dexamethasone significantly reduced the development of NAB in both genders of mice. The study thus advances this serotype for further development and use as a therapeutic vector.

Helper-Dependent Adenoviral Vector Achieves Prolonged, Stable Expression of Interleukin-10 in Rabbit Carotid Arteries but Does Not Limit Early Atherogenesis

Vascular gene therapy could potentially complement or replace current therapies for human atherosclerosis, while avoiding their side effects. However, development of vascular gene therapy is limited by lack of a useful vector. Helper-dependent adenovirus (HDAd) shows promise to overcome this barrier because, unlike first-generation adenovirus, HDAd achieves durable transgene expression in the artery wall with minimal inflammation. To begin to test whether HDAd, delivered to the artery wall, can limit atherosclerosis we constructed HDAd that expresses rabbit interleukin (IL)-10, a potent atheroprotective cytokine, and tested its activity in a rabbit model of early carotid atherogenesis. HDAd expressed immunoreactive, active IL-10 in vitro. In contrast to other HDAd-expressed transgenes, IL-10 expression from HDAd increased significantly between 3 days and 2 weeks after infusion and remained stable for at least 8 weeks. Rising, persistent IL-10 expression was associated with relative persistence of HDAdIL-10 genomes 4 weeks after infusion, compared with HDAdNull genomes. Surprisingly, IL-10 expression had no significant effects on atherosclerotic lesion size, macrophage content, or expression of either adhesion molecules or atherogenic cytokines. These results might be due to inadequate protein expression in vivo or lack of suitability of this rabbit model to reveal IL-10 therapeutic effects. IL-10 remains a promising agent for vascular gene therapy and HDAd remains a promising vector; however, proof of efficacy of HDAdIL-10 is elusive. Future preclinical studies will be aimed at increasing IL-10 expression levels and improving the sensitivity of this animal model to detect atheroprotective effects.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Rubbing Gastric Serosal Surface Enhances Naked Plasmid DNA Transfer in Rats and Mice

We have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in mice resulted in effective but transient transgene expression. Here, we developed a simple method to improve not only the transfection efficiency but also the duration of transgene expression. Rubbing the gastric serosal surface using a medical spoon immediately after instillation of naked plasmid DNA onto the gastric serosal surface resulted in 59-fold higher transgene expression 24 h after administration in rats. Without rubbing, transgene expression decreased under the detection limit 7 d after administration. On the other hand, rubbing the gastric serosal surface with a medical spoon after instillation of plasmid DNA prolonged transgene expression for one month. Mechanistic study in mice revealed that improved transfection should not be due to stimulation of cell function such as macropinocytosis by rubbing because rubbing before instillation of plasmid DNA did not improve transfection. Plasmid DNA should enter effectively into cells during rubbing. These findings are valuable to develop an effective method of in vivo gene transfer into peritoneal organs.

An Update on Canine Adenovirus Type 2 and Its Vectors

Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors.

Gene Transfer to the Cerebellum

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Use of MGMT Resistance to Enrich for Transduced Hematopoietic Cells in Gene Therapy for Fabry Disease.

Abstract 3584Poster Board III-521Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A (α-gal A) activity that results in the widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction, and stroke occur in patients with this condition due to deposition of globotriaosylceramide (Gb3) in vascular endothelial cells. Gb3 primarily originates from the breakdown of red blood cells. Currently, Fabry disease is mainly treated by enzyme replacement therapy (ERT). This treatment represents only a short-term therapeutic strategy and does not solve the basic genetic defect of the disorder. Fabry patients have also shown variable and muted response to ERT. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression, however lower transduction efficiencies may be an obstacle to attainment of successful therapy. Therefore, our goal is to design and implement a recombinant lentiviral vector (LV) to confer drug tolerance to modified Fabry patient HSCs in order to enrich them in vivo. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression provides cellular resistance to alkylating agents, which can be administered to kill residual untransduced HSCs while modified cells are protected. To this end, we have developed two different LV constructs. The first iteration was represented by a bicistronic LV encoding the human α-gal A cDNA followed by an EMCV-IRES element and a mutated, but functional, form of the selectable marker (MGMT-P140K). Next, a second iteration has been developed with the order of the transgenes reversed and carrying a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of both cassettes were tested in the human erythroleukaemic cell line (K562) by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that the second construct allowed sufficient MGMT expression for selection of transduced cells while maintaining α-gal A expression at appreciable levels. Flow cytometry analysis revealed that one drug treatment was sufficient to increase the MGMT-positive cell population from 15% to 90%, which resulted in a 2-fold increase in the enzyme activity. However, two treatments were required for achieving a similar result with the LV of the first iteration. The second LV also facilitated enrichment of transduced Fabry B cells in vitro. Future directions on this project include the modification of Fabry patient bone marrow cells, transplantation into a novel xenograft mouse model that we have recently created, and selection and amplification of the modified cells in vivo. Considering that clinical evidence suggests that even a slight increase in α-gal A activity may help to correct the disease, these results suggest that the MGMT-based drug selection system holds promise for providing a cure for Fabry Disease with a single gene therapy treatment. Disclosures:No relevant conflicts of interest to declare.