Duration Of Gene Expression Research Articles

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

Upstream Processing of Plasmid DNA for Vaccine and Gene Therapy Applications

The demand for plasmid DNA has increased vastly in response to rapid advances in its use in gene therapy and vaccines. These therapies are based on the same principle, i.e. the introduction of nucleic acids in human/non-human cells receptor to restore, cancel, enhance or introduce a biochemical function. Naked plasmid DNA as a vector has attracted a lot of interest since it offers several advantages over a viral vector, especially weak immunogenicity, better safety and easy to manufacture, but low transfection efficacy. Non-viral gene therapy may require considerable amounts (milligram scale) of pharmaceutical-grade pDNA per patient since the efficacy and duration of gene expression is presently relatively low. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of pDNA is becoming more widespread. Through optimization of the biological system, growth environment and the growth mode, improvements can be achieved in biomass productivity, plasmid yield, plasmid quality and production costs. The information on large-scale plasmid production is scarce and usually not available to the scientific community. This review summarizes recent patents and patent applications relating to plasmid upstream processing manufacturing, ranging from plasmid design to growth strategies to produce plasmid-bearing E. coli.

Radiation-Mediated Up-Regulation of Gene Expression from Replication-Defective Adenoviral Vectors: Implications for Sodium Iodide Symporter Gene Therapy

To assess the effects of external beam radiotherapy (EBRT) on adenoviral-mediated transgene expression in vitro and in vivo and to define an optimal strategy for combining sodium iodide symporter (NIS)-mediated (131)I therapy with EBRT. Expression of reporter genes [NIS, green fluorescent protein (GFP), beta-galactosidase (lacZ), and luciferase (Luc)] from replication-deficient adenoviruses was assessed in tumor cell lines under basal conditions and following irradiation. The effects of viral multiplicity of infection (MOI) and EBRT dose on the magnitude and duration of gene expression were determined. In vivo studies were done with Ad-CMV-GFP and Ad-RSV-Luc. EBRT increased NIS, GFP, and beta-galactosidase expression in colorectal, head and neck, and lung cancer cells. Radiation dose and MOI were important determinants of response to EBRT, with greatest effects at higher EBRT doses and lower MOIs. Radiation exerted both transductional (through increased coxsackie-adenoviral receptor and integrin alpha(v)) and nontransductional effects, irrespective of promoter sequence (CMV, RSV, hTR, or hTERT). Analysis of the schedule of EBRT followed by viral infection revealed maximal transduction at 24 hours. Radiation maintained increasing radioiodide uptake from Ad-hTR-NIS over 6 days, in direct contrast to reducing levels in unirradiated cells. The effects of EBRT in increasing and maintaining adenovirus-mediated transgene expression were also seen in vivo using GFP- and luciferase-expressing adenoviral vectors. Radiation increased the magnitude and duration of NIS gene expression from replication-deficient adenoviruses. The transductional effect is maximal at 24 hours, but radioiodide uptake is maintained at an elevated level over 6 days after infection.

Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2

Plant form is shaped by a complex network of intrinsic and extrinsic signals. Light-directed growth of seedlings (photomorphogenesis) depends on the coordination of several hormone signals, including brassinosteroids (BRs) and auxin. Although the close relationship between BRs and auxin has been widely reported, the molecular mechanism for combinatorial control of shared target genes has remained elusive. Here we demonstrate that BRs synergistically increase seedling sensitivity to auxin and show that combined treatment with both hormones can increase the magnitude and duration of gene expression. Moreover, we describe a direct connection between the BR-regulated BIN2 kinase and ARF2, a member of the Auxin Response Factor family of transcriptional regulators. Phosphorylation by BIN2 results in loss of ARF2 DNA binding and repression activities. arf2 mutants are less sensitive to changes in endogenous BR levels, whereas a large proportion of genes affected in an arf2 background are returned to near wild-type levels by altering BR biosynthesis. Together, these data suggest a model where BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs, leading to synergistic increases in transcription.

Sustained GM-CSF and PEI condensed pDNA presentation increases the level and duration of gene expression in dendritic cells

Current techniques to educate dendritic cells (DCs) ex vivo for immunotherapy are plagued by inefficient protocols and DC modifications are often transient and lost upon transplantation. This study investigated the role of sustained presentation of GM-CSF and PEI condensed pDNA (PEI-DNA) on gene transfer and long-term gene expression. Appropriate GM-CSF signaling during DC transfection promoted PEI-DNA uptake, although high cytokine concentrations induced intercellular DNA degradation, indicating the need for controlled presentation. Poly(lactide-co-glycolide) scaffolds that continuously stimulated DCs with both GM-CSF and PEI-DNA led to a 20-fold increase in gene expression, and high levels of expression persisted for a period of 10 days, in vitro. These results encourage the exploitation of biomaterials and GM-CSF to develop novel delivery vectors for genetically modified DCs or to genetically program host DCs in situ for vaccination and the treatment of autoimmunity.

Stem cell therapy for cystic fibrosis: current status and future prospects

Although cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR), seems a good candidate for gene therapy, 15 years of intense investigation and a number of clinical trials have not yet produced a viable clinical gene-therapy strategy. In addition, the duration of gene expression has been shown to be limited, only lasting 1–4 weeks. Therefore, alternative approaches involve the search for, and use of, stem cell populations. Bone marrow contains different stem cell types, including hematopoietic stem cells and multipotent mesenchymal stromal cells. Numerous studies have now demonstrated the ability of hematopoietic stem cells and mesenchymal stromal cells to home to the lung and differentiate into epithelial cells of both the conducting airways and the alveolar region. However, engraftment of bone marrow-derived stem cells into the airways is a very inefficient process. Detailed knowledge of the cellular and molecular determinants governing homing to the lung and transformation of marrow cells into lung epithelial cells would benefit this process. Despite a very low level of engraftment of donor cells into the nose and gut, significant CFTR mRNA expression and a measurable level of correction of the electrophysiological defect were observed after transplantation of wild-type marrow cells into CF mice. It is uncertain whether this effect is due to the presence of CFTR-expressing epithelial cells derived from donor cells or to the immunomodulatory role of transplanted cells. Finally, initial studies on the usefulness of umbilical cord blood and embryonic stem cells in the generation of airway epithelial cells will be discussed in this review.

Modulation of biodistribution and expression of plasmid DNA following mesenchymal progenitor cell-based delivery

Although therapeutic applications of mesenchymal progenitor cells (MPCs) have been studied, the in vivo fate of genes delivered by the MPCs has received little attention. We report here the in vivo kinetics, tissue distribution, and duration of gene expression after systemic administration of plasmid DNA delivered by MPCs. Murine MPCs were isolated from bone marrow, cultured, and transfected with plasmid DNA using polyethylenimine. The gene-modified MPCs or naked plasmid DNA was administered intravenously to mice. Injected MPCs incorporating plasmid DNA yielded elevated serum concentrations when compared with the group treated with plasmid DNA alone, a 280-fold higher level measured at 5-min post-administration. Moreover, plasmid DNA delivered in MPCs was detected in several organs, lymph nodes, and bone marrow. The highest levels of distribution were observed in the liver, followed by lung and spleen at 4 days post-dose. Similar to the distribution of DNA, significant expression levels of the exogenous gene were observed only after delivery of the DNA in MPCs, demonstrating the sustained expression at the liver, lung, and kidney for 4 days after tail vein injection. This study provides perspectives regarding the in vivo fate and target tissue distribution of genes following MPC-based delivery.

342. CpGs Influence the Duration of Gene Expression from Plasmid Vectors after In Vivo Lung Delivery

342. CpGs Influence the Duration of Gene Expression from Plasmid Vectors after In Vivo Lung Delivery

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

730. T Cell Responses to AAV Vector Capsid in Normal Donors and Subjects Who Have Undergone Liver-Directed AAV-Mediated Gene Transfer

In a Phase I/II study of gene transfer for hemophilia B, an adeno-associated viral vector serotype 2 (AAV-2) was introduced into the liver of human subjects and therapeutic levels of transgene expression (up to 11% of normal) were reached. However, the duration of gene expression was limited; four weeks after infusion, levels of circulating factor IX (F.IX) began to decline, and returned to baseline by week 10. This phenomenon was accompanied by a mild, self-limited, increase in liver enzymes. After a similar phenomenon was observed in another patient, the clinical trial was halted. We hypothesized that T cells directed towards AAV capsid antigens harbored by transduced hepatocytes were activated and these mediated destruction of the transduced hepaotocytes, thereby causing loss of transgene expression and a transient transaminitis. We used IFN|[gamma]| ELISpot analysis of peripheral blood mononuclear cells (PBMCs) from a subject in the clinical study, combined with bioinformatics tools for the prediction of MHC class I binders, to define an MHC Class I-restricted T cell epitope for an HLA type common in the general population (B|[ast]|0702). We then designed pentamers in which five peptide-loaded MHC class I molecules are linked to a fluorochrome to detect and study AAV-specific B|[ast]|0702-restricted CD8+ T cells. Indeed, in the HLA B|[ast]|0702 subject who developed transaminitis after vector infusion, AAV-specific CD8+ T cells were detected by pentamer staining of PBMCs up to two years later. In contrast, we have not detected AAV-specific CD8+ T cells in the peripheral blood of normal donors, though after several rounds of ex vivo stimulation with AAV capsid or peptide epitopes, we have been able to enrich a population of AAV-specific T cells, suggesting that AAV-specific T cells exist in the T cell memory pool. Functional assays on expanded AAV-specific T cells have demonstrated that these T cells specifically produced IFN|[gamma]| upon detection of AAV antigen and specifically lyse target cells presenting antigen in the context of the HLA B|[ast]|0702. These data provide direct evidence that humans mount a cytotoxic immune response to AAV capsid proteins, and that a contracted pool of AAV-specific T cells can remain as memory T cells in normal donors. Thus, re-activation of an AAV-specific T cell response may account for the limited duration of transgene expression that we observed in our clinical study. Moreover, the use of alternate serotypes may not easily avoid this immune response, as T cells from subjects infused with AAV-2 showed functional responses to AAV-1 and AAV-8-derived peptides by both cytokine production and killing assays, and T cells from normal donors that were expanded with an AAV-2 derived epitope also cross-react with the homologous epitope from AAV-5 by pentamer staining. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

367. Lentivirus Pseudotyped with Envelope Proteins F and HN from Sendai Virus Transduce Airway Epithelium Efficiency and Persistently

We have previously shown that recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane. This is in part due to cholesterol and sialic acid in the apical membrane which are respectively utilized by the SeV fusion (F) and hemagluttinin-neuraminidase (HN) proteins for receptor- mediated cell attachment, as well as the capacity of SeV to penetrate mucus efficiently. In contrast to SeV, which leads to transient gene expression, integrating lentiviral vectors have the theoretical advantage of persistent expression for the life-time of the cell, and ifstem cell integration should occur, for the lifetime of the animal. We have recently succeeded in generating a F/HN-pseudotyped Simian immunodeficiency virus (SiVagm) vector at high enough titre for in vivo application (>109 transduction units (TU)/ml). A F/HN-SiV- GFP vector (4x108 TU/mouse in 100 ml) was slowly perfused (over 75 min) onto the nasal epithelium of C57Bl/6 mice and transduced airway epithelial cells efficiently, via the apical membrane without the need for chemical pre-conditioning. We choose transduction of the nasal epithelium as proof-of-principle because we aim to extend these studies into CF knockout mice (only the nasal epithelium shows the CF defect in this model). We previously reported detection of GFP expression at 1, 20, 30, 90 days (n=3 mice/group) and 220 days (n=1) after transduction. Here, we further extended these studies to both include higher n numbers and, to identify transduced cells. Importantly, 180 days after transduction 8 out of 10 (6 histological sections 1 mm apart/mouse) mice still expressed GFP. Preliminary experiments on a single animal also showed persistent expression 360 days after transduction, and further animals transduced 270 and 360 days ago, are currently being analysed (n=12|[ndash]|15/group). This duration of gene expression is far longer than the predicted life-span of airway epithelial cells of approximately 100 days. One explanation is that F/HN-SiV vector has transduced progenitor or stem cells in the mouse nose. Morphological analysis showed that GFP positive cells were ciliated airway epithelial cells, and cells being in the olfactory epithelium which based on morphological criteria, appeared to be sensory neuronal cells.

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.

T Cell Responses to AAV Vector Capsid Limit the Duration of Transgene Expression in Humans after Liver-Directed Gene Therapy.

In a Phase I/II study of gene transfer for hemophilia B, an adeno-associated viral vector serotype 2 (AAV2) was introduced into the liver of human subjects and therapeutic levels of transgene expression (up to 11% of normal) were reached in one subject. However, the duration of gene expression was limited; four weeks after infusion, levels of circulating factor IX (F.IX) began to decline, and returned to baseline by week 10. This phenomenon was accompanied by a mild, self-limited, increase in liver enzymes (transaminitis). After a similar phenomenon was observed in another subject, the clinical trial was halted. One hypothesis for the loss of transgene expression is that immune mediated destruction of transduced hepatocytes caused the delayed rise in transaminases and loss of F.IX expression. IFNγ ELISpot analysis of peripheral blood mononuclear cells (PBMCs) from subjects in the clinical study and from normal donors, and bioinformatics tools for the prediction of MHC class I binders, were used to define two possible MHC Class I-restricted T cell epitopes for two HLA types common in the general population (B*0702 and B*0801). We then designed MHC class I epitope-specific pentamers for the detection and study of CD8+ T cells reacting to the predicted AAV2 capsid epitopes. Indeed, in the two subjects who developed transaminitis after vector infusion, AAV-specific CD8+ T cells were detected by pentamer staining of PBMCs up to two years later. In contrast, normal donors rarely had detectable AAV-specific CD8+ T cells in peripheral blood; even after several rounds of in vitro stimulation with vector capsid antigens, we often did not find AAV-specific CD8+ T cells by pentamer staining. We also performed functional assays, including intracellular cytokine staining (ICCS) and cytotoxic T lymphocyte (CTL) assays, on expanded AAV-specific T cells from one of the subjects in the clinical trial. An Epstein-Barr-virus-transformed lymphoblastoid cell line (LCL) derived from an HLA-matched donor was incubated with an AAV2 epitope, with the homologous peptide from the AAV-8 capsid sequence, or with a known HIV gag that is also HLA B*0702 restricted. Similarly to what observed in mice (Sabatino et al., unpublished), we found that IFNγ was specifically produced when the expanded T cells were stimulated with AAV antigens of both serotype 2 and serotype 8, but not with the HIV gag epitope, in the context of HLA B*0702. Peptide-loaded LCLs were used as targets in a CTL assay and incubated with in vitro expanded T cells (effectors). Specific lysis was observed at an effector-target ratio of 100:1 for the AAV2 epitope and for the homologous peptide from the AAV8 capsid. Together, these data provide direct evidence that humans can mount a cytotoxic immune response to AAV capsid proteins. This T cell response may account for the limited duration of transgene expression that we have observed in our clinical study. Moreover, the use of alternate serotypes may not easily avoid this immune response, as T cells from subjects infused with AAV2 showed functional responses to AAV8-derived peptides by both ICCS and CTL assays. We conclude that the use of immunomodulatory therapy may be a better approach to ensure durable transgene expression in the setting of liver-directed gene therapy.

Factors That Influence Transgene Expression and Cell Viability on DNA–PEI-Seeded Collagen Films

Gene delivery from tissue-engineering devices has the potential to improve healing, but better regulation of the level and duration of gene expression is needed. We hypothesized that transgene expression could be controlled by varying the fabrication and soaking parameters used in making collagen- based gene delivery scaffolds. Collagen films were made from acid-insoluble type I bovine dermal collagen and seeded with plasmid DNA encoding firefly luciferase, complexed with polyethylenimine. By varying the thickness of the films, the volume of the DNA soak solution, and the pH of the DNA soak solution, and by cross-linking the films, we identified variable combinations that produce significantly different levels of cell number and transgene expression in L-929 cells in vitro. Increasing film thickness or soak volume increased overall reporter gene expression. Decreasing film thickness or soak volume decreased cell number but did not significantly change reporter gene expression per cell. Cross-linking by ultraviolet irradiation (before adding the DNA) significantly decreased transgene expression, probably because of decreased swelling of the collagen film. These results suggest that collagen-based biomaterials may be designed and fabricated to induce, in a controlled fashion, various levels of cellularity and transgene expression.

Effect of Adenovirus-Mediated Transfer of the CTLA4IG Gene in Hamster-to-Rat Xenotransplantation

Costimulatory molecules such as CD28 and B7 are essential for T cell activation, as well as playing a role in the T cell receptor and major histocompatibility complex pathway. It is well known that rejection in allotransplantation is diminished by treatment with CTLA4Ig, but whether a similar effect occurs in xenotransplantation remains to be determined. In this study, we investigated whether adenovirus-mediated gene transfer with CTLA4Ig cDNA by intravenous injection to the recipient is effective in suppression in hamster-to-rat cardiac xenotransplantation. With CTLA4Ig gene transfer, the duration of gene expression was clearly prolonged, based on reduced production of antiadenovirus antibody and shrinkage of the spleen. The survival of cardiac xenografts was significantly prolonged with CTLA4Ig gene transfer compared to the control graft, and survival with combination use of FK506 and CTLA4Ig gene transfer in xenotransplantation was also significantly prolonged compared to that with CTLA4Ig gene transfer alone. Cessation of the cardiac graft in the combination treatment occurred in parallel with the elevation of antihamster IgM antibodies in rat sera. These results suggest that adenovirus-mediated CTLA4Ig gene transfer is effective for immunosuppression in hamster-to-rat xenotransplantation.

Gene therapy for ischemic neuronal injury

Advances in the area of stroke and other related disorders have identified a variety of molecular targets for potential therapeutic intervention. The use of modified viral vectors has now made it possible to introduce foreign DNA into central nervous system cells, permitting overexpression of a protein of interest. A particular advantage of the herpes simplex system is that the virus is neurotropic, and is therefore suited for relatively selective gene therapy to neurons. The vectors used by our group to date utilize an amplicon based bipromoter system which permits expression of both the gene of interest as well as a reporter gene. Using this strategy, we have shown that potentially neuroprotective genes can be transferred to individual central nervous system cells, and can confer a relative resistance to cerebral ischemic and excitotoxic insults. We previously reported that gene therapy using a neurotropic herpes simplex viral (HSV) vector system containing bipromoter vectors to transfer various protective genes to neurons. Using this system in experimental models of stroke, cardiac arrest and excitotoxicity, we have found that it is possible to enhance neuron survival against such cerebral insults by overexpressing genes that target various facets of injury. Of the genes studied by our labs, we have shown that glucose transporter (GLUT-1), the calcium binding protein calbindin D28 K (CaBP), anti-oxidant genes glutathione peroxidase (GPx) and catalase (CAT), the anti-apoptotic protein Bcl-2 and the 70 kDa heat shock protein (HSP70) improve neuron survival after ischemia and excitotoxicity. Bcl-2, GPx and HSP70 also appear to protect when administered post insult. Because the extent of vector uptake is limited, it is not generally possible to affect overall infarct size or behavior. Regardless, we have demonstrated that for some cases, gene transfer may also improve cell function. Gene transfer can also be used in combination with other potential neuroprotective strategies with synergistic effects. For instance, mild hypothermia is well known to protect the brain from experimental brain ischemia provided brain cooling begins within hours of ischemia onset. This hypothermic neuroprotection is also associated with Bcl-2 upregulation in some instances, and hypothermia suppresses many aspects of apoptotic death. Our recent work has shown that two different kinds of protective therapies, Bcl-2 overexpression and hypothermia, both inhibit aspects of apoptotic cell death cascades, and combination of hypothermia with Bcl-2 gene transfer will prolong the temporal therapeutic window for Bcl-2 gene therapy. Some limitations of this technique exist, the main ones being that of delivery and extent and duration of transfection. Part of the limited duration of gene expression may be due to a localized tissue inflammatory response to the vectors themselves. This problem may be partially surmounted by the coadministration of anti-inflammatory treatments to prolong survival of transfected cells. While neuronal gene therapy for cerebral ischemia is still limited by the numbers of cells which vectors can transfect, it provides a powerful tool to understand mechanisms of cell death and identify potential molecular targets for therapy.

IFN-α gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes

Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.

742. Human Artificial Chromosome (HAC) Vector Provides Long-Term Therapeutic Transgene Expression in Normal Human Primary Fibroblasts

Top of pageAbstract Human artificial chromosomes (HACs) are segregating freely from host chromosomes through a set of cell divisions. Accordingly, the host genome is not disrupted and the expression of the transgene could be sustained for a prolonged period without suffering the effects of its surrounding sequence on the host genome. These are the ideal properties required for vectors in gene therapy, and therefore HACs are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. We constructed a novel human chromosome 21-derived HAC (21|[Delta]|pqHAC) vector, which devoid of most expressed genes by telomere truncation at the genomic regions proximal to the centromere in both p and q arms, and inserted human erythropoietin (EPO) gene, which is a growth factor for erythroid cells and widely used for the clinical treatment of anemia in renal insufficiency, into the HAC (EPO-21|[Delta]|pqHAC) as a model of therapeutic transgenes. Although low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy, we successfully demonstrated the introduction of EPO-21|[Delta]|pqHAC vector into normal primary human fibroblasts (hPFs) with micro cell-mediated chromosome transfer (MMCT). However, the mitotic stability of EPO-21|[Delta]|pqHAC vector in normal hPFs and the growth ability of the resultant hPF clones to obtain graft cells for transplantation remained to be elucidated. Here, we studied the hPF clones harboring the EPO-21|[Delta]|pqHAC vector cytogenetically and demonstrated that the structurally defined, extra artificial chromosome was highly maintained in normal hPFs under non-selective condition at a constant copy number mostly in a single copy per cell without accompanying aberration of host chromosomes. Mitotic-loss rates were determined, and the level of stability of EPO-21|[Delta]|pqHAC vector was comparable with those reported for the other top-down and bottom-up artificial chromosomes in human fibro-sarcoma cell line HT1080 cells. These hPF clones proliferated from a single colony up to 3.8x107 cells and then their growth stopped, suggesting that cell senescence occurred and the introduction of the EPO-21|[Delta]|pqHAC vector did not cause immortalization. We also showed the long-term expression of human epo gene in the hPFs carrying the EPO-21|[Delta]|pqHAC vector. In conclusion, this study shows the potential application of the 21|[Delta]|pqHAC vector in hormone-replacement gene therapy, which might ensure safety with no lesion of host chromosomes and provides long-term therapeutic transgene expression. This also implies that utilization of the HAC vector offers novel options for ex vivo gene therapy.

976. IFNα Gene Therapy of Woodchuck Hepatitis with Adeno-Associated Virus: Differences in Duration of Gene Expression and Antiviral Activity Using Intraportal or Intramuscular Routes

976. IFNα Gene Therapy of Woodchuck Hepatitis with Adeno-Associated Virus: Differences in Duration of Gene Expression and Antiviral Activity Using Intraportal or Intramuscular Routes

Angiogenesis is confined to the transient period of VEGF expression that follows adenoviral gene delivery to ischemic muscle

Therapeutic angiogenesis involves the introduction of exogenous growth factor proteins and genes into ischemic tissues to augment endogenous factors and promote new vessel growth. Positive results from studies in animal models of peripheral arterial disease (PAD) and coronary artery disease over the past decade have supported the implementation of clinical trials testing vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and genes. Although several clinical trials reported positive results, others have been disappointing and results of a recent Phase II trial of VEGF delivered by adenovirus (the RAVE trial) were negative. It has been suggested that the duration of gene expression following delivery by adenovirus may be insufficient to produce stable vessels. Here we present direct evidence in support of this using the rabbit ischemic hindlimb model injected with adenovirus encoding VEGF165. Immunohistology indicated an activation of endothelial cell cycling and proliferation 2-3 days after VEGF delivery that coincided closely with transient VEGF expression. Ki-67-positive endothelial nuclei were evident at high levels in capillaries and large vessels in muscles from treated animals. Angiography indicated increased density of both large and small vessels in Ad-VEGF-treated muscle at 1 week, but no significant differences thereafter. The early burst of endothelial proliferation was accompanied by increased nuclear fragmentation and condensation in VEGF-treated muscles, suggesting coincident apoptosis. No further endothelial cell proliferation took place after 1 week although there was still evidence of apoptosis. The results suggest that angiogenesis is confined to the short period of VEGF expression produced by adenovirus and early gains in collateralization rapidly regress to control levels when VEGF production ceases.