111. Post-Transcriptional Gene Silencing of Alpha-1 Antitrypsin by Small Interfering RNAs (siRNA)

AAV VECTORS: BIOLOGY to examine the intracellular trafficking pathways of vectors made with each of these capsids but our results suggest that vector biodistribution and perhaps intracellular transduction pathways are influenced by the presence of the heparin binding motif.

109. Episomal AAV Vector Genome in the Histone-Associated Chromatin Form Is Capable of Superior Transcription with HDAC Inhibitor 1

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Takashi Okada, Ryosuke Uchibori, Mayumi Iwata-Okada, Mutsuko Nonaka-Sarukawa,1 Takayuki Ito,1 Hiroaki Mizukami,1 Akihiro Kume,1 Keiya Ozawa.1,2 1 Division of Genetic Therapeutics, Jichi Medical School, Shimotsuke, Tochigi, Japan; 2Division of Hematology, Jichi Medical School, Shimotsuke, Tochigi, Japan. Background: The transduction of cancer cells using rAAV occurs with low efficiency, which limits its utility in gene therapy. Treatment with a histone deacetylase (HDAC) inhibitor is known to cause the gene expression from a silenced rAAV genome that has been integrated into the host’s genome. However, rAAV exists mostly as an extrachromosomal genome rather than as an integrated genome. Here we show that HDAC inhibitors markedly enhance the rAAVmediated transduction of tumor cells in vitro as well as in vivo with improved therapeutic benefit. Our data also suggest that episomal AAV vector genome in the tumor cells is in the histone-associated chromatin form that is capable of superior transcription. Methods: Cell lines U251MG, 9L, HEp-2, HepG-2 and NKO-1 were transduced with the AAV1-5 vectors expressing eGFP gene at 1 x 10(4) genome copies/cell along with the HDAC inhibitor FK228. Effects on the expression of co-receptors for the AAV were also estimated by the FACS or Q-PCR. Copy number of the rAAV genome in the infected cells was estimated with Q-PCR analysis. Histone association of episomal AAV vector genome or proviral plasmid was characterized by using the chromatin immunoprecipitation (ChIP) assay. To analyze the effect of the FK228 on the transduction efficiency in vivo, U251MG cells were transduced with rAAV2Luciferase at 1 x 10(4) genome copies/cell, and then inoculated subcutaneously into the BALB/c mice along with the inraperitoneal injection of the FK228 at 1 mg/kg. Furthermore, 9L tumor cells were transduced with an AAV5HSV-tk and then inoculated subcutaneously into the BALB/c mice. The tumor-bearing animals received intraperitoneal injection of the FK228 and exposed to GCV for 14 days. Results: The FK228 treatment significantly improved the expression of the transgene in cancer cells in a dose-dependent manner. The highest enhancement was observed in the U-251MG cells with AAV2eGFP. The enhancement of the integrin level as well as coreceptors was limited. Copy number of the rAAV in the transduced cells was also modestly affected by the FK228 treatment. Interestingly, association of the acetylated histone H3 in episomal AAV vector genome was suggested by the ChIP assay. In the analysis using the optical bioluminescence imaging of the subcutaneous tumors, the luciferase expression in animals treated with the FK228 (n=5) was 37.4 fold higher than that in control animals (n=3). Estimation of the tumor growth in the athymic nude mice demonstrated that the combination of AAV-mediated transduction for HSV-tk/GCV therapy and FK228 treatment (n=10) resulted in significant improvement relative to HSV-tk/GCV therapy without FK228 treatment (P < 0.05, n=6). Conclusion: Our study suggests that the improved rAAV-mediated transduction induced by HDAC inhibitor might be related to proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. The use of the HDAC inhibitor would greatly enhance the utility of rAAV-mediated transduction strategies for future cancer gene therapy. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

110. Investigation of Biochemical Factors That May Influence Immunogencity of AAV2 Vectors Bernd Hauck,1 Federico Mingozzi,1 Valder Arruda,1 Katherine A. High,1 J. Fraser Wright.1 1 Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA. Recombinant AAV2 expressing human coagulation factor IX has been demonstrated to efficiently transduce human hepatocytes leading to expression of therapeutic levels of human factor IX in a human subject. However a T cell response directed to the AAV2 capsid was reported that resulted in elimination of transduced cells (Manno et al, Nat. Med. 2006). In order to better understand determinants of immunogenicity, we report here preliminary assessment of biochemical features that may influence vector immunogenicity. We tested whether purposely induced vector aggregation or oxidation could lead to increased immune responses in a mouse model. To induce aggregation, a common concentrated stock (E14 particles / mL) of highly purified, non aggregated AAV2 empty capsids in PBS was subjected to conditions of reduced ionic strength (60min) followed by readjustment to physiological ionic strength. A control sample was adjusted to the same final volume and concentration by addition of PBS only. To induce oxidation, capsid was treated with 1µM Cu2+ (60min). Modified and control capsid preparations were administered via portal vein injecftion to C57/BL6 mice previouly immunized with AAV2 empty capsids. Assessment of anti-AAV2 IgG antibody titers post administration showed no significant difference between mice receiving control, aggregated or oxidized vector. Similarly T lymphocytes isolated from spleens of animals in each group did not secrete IFNgamma in response to AAV2 capsid-derived peptides as assessed by ELISPOT and intracellular cytokine staining. A third biochemical feature that we begun to investigate is structure and provenance of the nucleic acids packaged by AAV vectors. The bacterial plasmid DNA used as a template for the transgene cassette in AAV vectors produced using transient transfection may be incorporated into vector particles. If so, a subpopulation of vector particles would be predicted to contain pathogen associated molecular patterns such as non-methylated CpG motifs. We looked for evidence of packaging of bacterial DNA by AAV2 by producing AAV2 vectors (GFP) by transient transfection using a vector plasmid prepared using the thymidine analogue BrdU. The resulting vector was purified and fractionated by CsCl gradient ultracentrifugation, and fractions were tested for transduction on HEK293 cells. We observed the major (expected) vector band at approx 1.39 g/mL, and a minor higher density band at approx. 1.44 g/mL. These data support that a subpopulation of AAV2 vectors produced using transient transfection contains bacterial plasmid DNA, which may contribute to activation of innate immune responses following in vivo administration. In vivo testing is required to further assess this parameter.

111. Post-Transcriptional Gene Silencing of Alpha-1 Antitrypsin by Small Interfering RNAs (siRNA) Pedro E. Cruz,1 Alexandra Golant,2 Terence R. Flotte.1 Department of Pediatrics, Powell Gene Therapy Center and Genetics Institute, University of Florida, Gainesville, FL; 2College of Human Ecology, Division of Nutritional Sciences, Cornell University, Ithaca, NY.

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Alpha-1 antitrypsin (AAT) is a serum serine protease inhibitor that counteracts the effects of neutrophil-derived proinflammatory proteins. Several mutant AAT alleles result in AAT deficiency with either chronic obstructive lung disease, chronic liver disease, or both. PiZ is the most common mutant allele of AAT. A Gln to Lys change S45

AAV VECTORS: BIOLOGY in exon V at position 342 results in a propensity for polymerization of the protein and impaired secretion from hepatocytes. The lack of secreted AAT leads to lung disease in a majority of patients, which is primarily due to the unopposed action of neutrophil products on the extracellular matrix in the lung. Meanwhile, the accumulation of Z mutant AAT in hepatocytes contributes to chronic liver disease in a smaller percentage of patients with this allele. Thus, replacement of the wild-type (M) AAT is the primary strategy to treat the lung disease, while down-regulation of mutant (Z) AAT would likely be needed to ameliorate the liver disease. We designed three siRNAs to target the AAT gene (siRNA-AAT1, siRNA-AAT33, and siRNA-AAT55), targeting the 5’ end, the center, or the 3’ end of the coding region, respectively. siRNAs were transfected into HEK-293 cells in the context of a hairpin-forming RNA molecule expressed from a plasmid. HEK-293 cells were transfected with an AAT-expressing plasmid to produce the target mRNA. In addition, siRNAs have been delivered by transducing HepG2 cells, using a recombinant adeno-associated virus (rAAV) vector. HepG2 cells normal secrete super-physiologic levels of AAT, so no additional AAT expression plasmid was required in that case. AAT secretion was measured by ELISA after the transfection or transduction of the cells. Measurement of AAT by ELISA showed that both Z-AAT and M-AAT could be down-regulated by 3- to 4fold after transfection or transduction of the siRNA expressing vectors. These changes were sustained over 72 hours in culture. MAAT appeared to be a better target than Z-AAT for siRNA-mediated downregulation. This suggests that an siRNA-mediated strategy for treatment of AAT liver disease might optimally be combined with a strategy for M-AAT augmentation that employed an ectopic site of delivery. Future plans include the use of rAAV serotype 8 vectors to deliver AAT siRNA to murine hepatocytes expression human ZAAT in vivo. Supported by NIH and the Alpha One Foundation grants.

112. Overexpression of the Coxsackie Adenovirus Receptor (CAR) Gene Influences AAV2 Transduction in Human Trabecular Meshwork Cells Guorong Li,1 Teresa Borras.1 Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

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Glaucoma is a chronic disease and the second leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), created by the resistance to aqueous humor flow exerted by the trabecular meshwork tissue, is the major risk factor for the development of glaucoma. A dysfunctional trabecular meshwork leads to an increased resistance and elevated IOP. Because of their long-term expression and low immune response, AAV vectors would be ideal to treat a chronic disease such as glaucoma. However, all attempts to transduce the trabecular meshwork tissue with AAV have failed. Previous studies showed that lack of AAV transduction to the trabecular meshwork was not due to failure of viral entry. Transduction was then obtained upon coinfection of rAAV2 with ∆E1/∆E3 Adenoviruses. In the present study, we used this observation to study mechanisms for the lack of transduction of rAAV2 in the glaucoma-associated human trabecular meshwork cells. Microarray analysis, TaqMan real time PCR and western blots demonstrated that the Coxsackie Adenovirus Receptor (CAR) gene was highly induced during the transduction enhancement observed upon coinfection. Expression of CAR was induced 9-fold on human trabecular meshwork coinfected cells (rAAV2.GFP:∆E1/∆E3 Ad.LacZ; VP ratio 1:0.05), while it was induced 3-fold in cells infected with the same VP of ∆E1/∆E3Ad.LacZ alone. Infection with rAAV2 alone had no effect on CAR expression. As a consequence of CAR increase, significant induction of the Ad transgene (LacZ) expression was S46

also observed. This Ad.LacZ induction was mostly blocked by pretreatment of the cells with CAR antibody, indicating that the presence of rAAV2 facilitated Ad entry through the coinfectioninduced expression of CAR. Silencing CAR expression with siRNA 24 h prior to coinfection significantly reduced mRNA and protein of the rAAV2.GFP and Ad.LacZ transgenes. When treatment with CAR siRNA was performed 3 h after coinfection (and extensive media washing), rAAV2.GFP transduction was reduced while Ad.LacZ was not. This result suggested that CAR not only serves as an Ad receptor but influences rAAV2 transduction independently of Ad entry. To further test this independent CAR effect on rAAV2 transduction, the 1102 bp & 844 bp CAR splice variants from human trabecular meshwork cells (full-length and exons 1-4&7, respectively ) were cloned into expression plasmid pcDNA3.1D/V5-His-TOPO under the control of the CMV promoter (pGL3 and pGL4). Transfection of both plasmids 48 h before rAAV2 infection increased GFP transgene mRNA 6-fold, and fluorescence intensity by 35%, while transfection by the empty control plasmid did not. In summary, our results demonstrate that the CAR gene is highly induced only after coinfection of AAV/Ad and that its overexpression not only facilitates further Ad infection but acts independently to enhance rAAV2 transduction. This study offers alternative methods to transduce the trabecular meshwork with long-term, non immunogenic AAV for the treatment of glaucoma and provides new insights on the interaction of AAV with its helper Ad.

113. Transduction of Neural Cells in Primate Brain Using Adeno-Associated Virus (AAV) Serotypes 2 and 5 Eleni A. Markakis,1 Csaba Leranth,2 Jeremy Bober,2 R. Jude Samulski,3 J. E. Rabinowitz,3 X. Zhou,3 Kenneth Vives,4 D. Eugene Redmond, Jr.1,4 1 Psychiatry, Yale University School of Medicine, New Haven, CT; 2 Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT; 3Pharmacology, University of North Carolina, Chapel Hill, Chapel Hill, NC; 4 Neurosurgery, Yale University School of Medicine, New Haven, CT. There are currently eleven identified serotypes of the adenoassociated virus (AAV), a single stranded DNA parvovirus used extensively for gene delivery in animals; yet AAV serotype 2, the first to be characterized, is the most widely used for gene delivery. While homology exists among most serotypes, considerable differences in the viral capsid surface have been described for AAV4, and AAV5, which are believed to have greater tropism for cells in the central nervous system than other serotypes of AAV. To test the relative efficiency with which vectors made from AAV2, and AAV5 can transduce cells of the primate central nervous system, we injected the brains of St. Kitts green monkeys with viral vectors of both serotypes, each expressing the reporter gene green fluorescent protein (GFP). The vectors were all of comparable titer, injected in equal amounts in positive pressure, stereotaxic injections using the same rate of infusion into the substantia nigra. Animals were perfused after a one month survival, and their brains removed and sectioned. One series of sections was stained with the antibody to GFP using a diaminobenzidine (DAB) secondary reaction. These DAB sections were counted under a light microscope using a computer equipped with software for unbiased stereology, and counts for labeled cells were made. Remaining adjacent series of sections were processed for immunohistochemistry for glial fibrillary acidic protein (GFAP), an intermediate filament protein that labels cells of astroglial lineage, or neurofilament (NF), three intermediate filament proteins that are structural components of neurons and their axons. Sections were reacted with fluorescent secondary antibodies, the inherent fluorescence of GFP remaining unreacted, and the sections were Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy