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Generation of Novel Double-Stranded AAV Vectors with an Expanded Packaging Capacity Using Linearized 1-AAV ITR DNA Molecules

Generation of Novel Double-Stranded AAV Vectors with an Expanded Packaging Capacity Using Linearized 1-AAV ITR DNA Molecules

AAV VECTORS: GENOMES AND BIOLOGY not induce the expression of chemokines/cytokines in epitheliumderived cells in vitro. However, in vivo studies showe...

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AAV VECTORS: GENOMES AND BIOLOGY not induce the expression of chemokines/cytokines in epitheliumderived cells in vitro. However, in vivo studies showed that AAV vectors induce transient expression of chemokines/cytokines and leukocyte recruitment to mouse liver. This transient activation of innate immune responses by AAV vectors in vivo might contribute to the generation of an adaptive response; the formation of neutralizing antibodies to AAV capsid proteins. Innate immune responses to AAV vectors in the liver were completely abolished in the absence of Kupffer cells (liver macrophages) suggesting that AAV vectors may interact significantly with macrophages. To test whether AAV can directly infect and activate macrophages, THP-1 cells (human monocytes) were transduced with AAV vectors and chemokine expression was analyzed 6 hours post transduction. In contrast to the response in epithelium-derived cells, AAV vectors carrying different transgenes were able to induce various chemokine and pro-inflammatory cytokine mRNAs, including RANTES, IP10, MIP-1β, MIP-1α, MCP-1 and IL-8 in THP-1 cells in a dose dependent manner. Transductions in the presence of polymyxin B and the use of appropriate vehicles confirmed that the responses were not induced through endotoxin (LPS) or transgene protein contamination. Southern blots indicated internalized vector genomes, suggesting a direct infection and activation of these cells. Furthermore, AAV induced the expression of chemokines in THP-1 cells in the presence or absence of serum suggesting the activation occurs independent of opsonizing proteins or co-factors. Understanding the mechanism of innate immune activation by AAV vectors may allow strategies to minimize humoral adaptive responses permitting the re-administration of these vectors in vivo.

746. Genomic Stability of Self-Complementary Recombinant Adeno-Associated Virus 2 in Mouse Muscle Changchun Ren,1 Sanjay Kumar,1 Selvarangan Ponnazhagan.1 1 Pathology, The University of Alabama at Birmingham, Birmingham, AL.

747. Direct Labeling and Tracking of AdenoAssociated Virus Single Stranded DNA in AAV Transduction Bernd Hauck,1 Wei Zhao,1 Weidong Xiao.1 Division of Hematology, Childrens Hospital of Philadelphia, Philadelphia, PA.

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AAV is a unique gene transfer vector which takes approximately 4-6 week to reach its expression peak. The mechanism for this slow-rise expression profile was proposed to be limited by inefficient second-strand DNA synthesis from single stranded (ss) DNA viral genomes. We generated AAV vectors labeled with BrdU, a nucleotide analog that can be detected by anti-BrdU antibody when the DNA is in ss form, but not in double stranded (ds) form. Vast amounts of ss AAV genome as demonstrated in previous studies should be easily tracked. To our surprise, after AAV transduction, ss AAV genomes were detectable only under denaturing conditions in immunohistochemical analysis or in an ELISA assay. Southern Blot analysis of viral DNA and virion suggested that AAV viral DNA is mostly protected within virions. Extracted cellular fractions demonstrate that viral particles in infected host cells are still infectious. In addition, there is a significant decrease in viral genomes in the first 48 hours post infection. Analysis of the data suggests that free ss DNA seems not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV uncoating before second strand synthesis can occur. AAV single stranded DNA released from viral uncoating is either converted into double stranded DNA efficiently or degraded by cellular repair mechanisms as damaged DNA. This study may provide a tool to track viral DNA and to investigate fate and mechanisms of viral DNA processing.

748. Generation of Novel Double-Stranded AAV Vectors with an Expanded Packaging Capacity Using Linearized 1-AAV ITR DNA Molecules Ognjen Petras,1 Timothy J. Davern,2 Ryan McTaggart.3 Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA; 2Division of Gastroenterology, University of California at San Francisco, San Francisco, CA; 3Surgery, University of California at San Francisco, San Francisco, CA. 1

Recent studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) genome as a self-complementary duplex strand (ds) results in early transgene expression, possibly overcoming the rate-limiting second-strand synthesis. In the present study, we evaluated the molecular organization and stability of the ds-AAV genome in 293 and Hela cells in vitro and in the quadriceps muscle of C57/BL/6J mice in vivo as compare to single-strand (ss) AAV-2 genome. The studies were carried out with rAAV containing GFP gene as either ds or as ss structure encapsidated in AAV-2 capsids. Cohorts of mice were injected with 1x1011 particles of rAAV-GFP (ds/ss) in quadriceps muscle and the injected tissues were harvested 1, 2 and 3 weeks later. The tissue from each animal was cut into two and processed directly for total DNA isolation or paraffin-embedded for immunohistochemistry and in situ hybridization. The total DNA was subjected to Southern blot analysis and PCR using primers specific for the amplification of linear and/or circular concatemers. Immunohistochemistry was performed with anti-GFP antibody and in situ hybridization using a DIG-labeled CMV probe. Results of Southern blot and PCR analysis indicate increased disintegration of ss-AAV vector genome over time in vivo compared to ds-AAV vector genome. Southern blot and PCR analysis also indicated efficient conversion of the vector genome to concatemers in ds vector. Approximately 30-40% of ds-AAV genome was detected as concatemers as early as three weeks. As expected, transgene expression was detected at early time point with ds vector and persisted stably compared to ss vector. These data demonstrate that ds AAV vectors provide better stability for transgene structure and expression and may have better utility in human gene therapy applications. S284

Introduction: The double-stranded AAV (dsAAV) vector is a new entity in the field of AAV based technologies. DsAAVs bypass a key requirement of AAV mediated transduction - conversion of the single stranded AAV genome into a double-stranded DNA template capable of RNA transcription. Currently, the only reported method for efficient and practical synthesis of double-stranded AAV’s involves a standard AAV plasmid that has had one of its two AAV-inverted terminal repeat sequences mutated. Here we report a novel and practical method to synthesize double-stranded AAV vectors with a slightly increased packaging capacity as compared to the recently published technique. Methods: A conventional AAV packaging plasmid was modified by entirely deleting one of its two AAV inverted terminal repeat sequences (ITRs). The resultant 1-ITR molecule was then linearized with common restriction enzymes, such as EcoRI. Subsequently dsAAvs were generated by standard triple transfection of HEK293 cells with two helper plasmids and the linearized 1-ITR molecule. Results: Analysis of dsAAV genomes generated from 1-ITR molecules shows that they consist of a self-complementary single stranded DNA molecule with two flanking wild-type AAV-ITR sequences. The hairpin bend region of the self-complementing genome consists only of the packaged transgene sequence. By contrast, the genome structure of dsAAVs generated from AAV-packaging plasmids with a mutated AAV-ITR contain a mutant AAV-ITR Molecular Therapy Volume 9, Supplement 1, May 2004

Copyright  The American Society of Gene Therapy

AAV VECTORS: GENOMES AND BIOLOGY sequence at their hairpin-bend site. Because dsAAVs generated from 1-ITR molecules lack the acessory AAV-ITR sequence, their efficient packaging capacity is expanded by about 2.5%. We generated several 1-ITR molecules and demonstrated that the specific enzyme used for linearization did not affect the efficiency or purity of dsAAV synthesis. Efficient packaging of dsAAV genomes was demonstrated up to wild-type AAV genome length, and the 1-ITR technique generated greater than 90% pure double stranded genomes. We established that dsAAVs are generated from 1-ITR molecules by means of an intranuclear concatemerization mechanism. Individual 1-ITR molecules are joined intra-nuclearly in HEK293 cells into head-to-head concatemers to form a substrate for synthesis of the novel dsAAV genomes. This mechanism also allows generation of interesting semi-self-complementary genomes with a selfcomplementary region capped at the hairpin bend location by a large loop of single stranded DNA. In vivo, dsAAVs generated by the 1-ITR method show enhanced and accelerated transduction of murine skeletal muscle and hepatocytes. This was demonstrated with AAV serotype 1 encapsidated dsAAVs with a miniature chick beta actin promoter and GFP reporter gene. The dsAAV vector used in these experiments also contained a novel miniaturized bovine growth hormone polyadenylation sequence (BGH polyA) which is only 66% of the wild-type length yet exhibits undiminished performance in vitro and in vivo. Summary:The expansion of packaging capacity afforded by the 1-ITR technique and the modified BGH polyA sequence should be of general interest to investigators who wish to place larger therapeutic transgenes into the restricted confines of dsAAVs.

749. Adeno-Associated Virus Vectors Integrate at Chromosome Breakage Sites Daniel G. Miller,2 Lisa M. Petek,1 David W. Russell.1 1 Hematology, University of Washington, Seattle, WA; 2Pediatrics, Children’s Hospital and Regional Medical Center, Seattle, WA. Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at non-homologous chromosomal locations by an unknown mechanism that does not require viral proteins. We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this by transducing cells containing a specific chromosomal double strand break created by the endonuclease I-SceI. Vector proviruses were frequently found at I-SceI cleavage sites, and sequencing of vector:chromosome junctions revealed micro-homologies, deletions, and insertions that in some cases could be explained by template-directed DNA synthesis prior to ligation. Similar junctions were observed when integration occurred at random locations, and infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration, and suggest that AAV vectors integrate at existing chromosome breaks rather than causing breaks, a finding that has unique implications regarding their clinical use.

750. AAV-2 Vectors and Protease-Activated Receptors: A Potential Interface between Coagulation and Gene Transfer 1

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Jianxiang Zou, Joerg Schuettrumpf, Alexander Schalchterman, Kian W. Tian,1 Jian-Hua Liu,1 Patricia Andrade-Gordon,2 Valder R. Arruda.1 1 Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA; 2R.J. Wood Johnson Pharmaceutical Research Institute, Spring House, PA. Phase I clinical trials demonstrated that AAV based on serotype 2 transduce human skeletal muscle and hepatocytes in vivo . AAV2 is safe and well tolerated at doses tested, and an ongoing liverdirected study resulted in therapeutic FIX levels for hemophilia B. Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

From these studies it is important to determine the minimum safe dose required for therapeutic efficacy. To this end, factors that increase or decrease expression by even modest amounts may become critical in the development of this novel therapeutic. We have uncovered evidence for an unexpected role of specific blood proteases in insuring optimal AAV-2 but not for AAV-5 or AAV-8 vectors. The use of specific inhibitors to thrombin or FXa (common anticoagulant drugs) prior to vector injection in mice resulted in low transgene expression (Blood 102:794a). Proteases trigger cellular responses at least in part through protease-activated receptors (PAR). Recent data show that PAR activation by thrombin enhances αVβ5-dependent (co-receptor for AAV-2) cellular function. Using PAR-1 and -2 knockout mice we sought to determine whether PAR or β5 integrin is required for AAV-2 transduction. Experiments were carried out in adult male C57Bl/6 mice homozygous (-/-) or heterozygous (+/-) for the null alleles of PAR-1, PAR-2 or β5 and compared to wild-type littermate as controls (+/+). A rAAV-2 encoding F.IX gene under the control of liver-specific promoter (hAAT/ApoE) was injected by tail vein at dose of 1x1012 vg/kg and circulating FIX levels were monitored for at least 10 weeks following vector injection. Injection of β5 deficient mice (n=6) resulted in FIX levels of 514 ng/ml which were lower than 3,837± 1,2 ng/ml among 12 controls (p<.0005). These results suggest that β5 co-receptor properties on AAV-2 transduction correlate well with previous in vitro observations (Nat Med 5:78). To test whether PARs have any role independently of β5, we further injected 19 mice of PAR-1 (-/ -) or PAR-1 (+/-) genotype, and F.IX levels were 1,330 ± 376 and 1,782 ± 280 ng/ml, respectively. Among controls (n=10) levels of F.IX were 4,017±1521 ng/ml. These differences were statistically significant when controls were compared to PAR-1 (-/-) or PAR-1 (+/-) at p< .005 and p<.05, respectively. Similarly, among 20 animals of PAR-2 mice (-/-) or (+/-) genotypes, FIX levels were 717±222 ng/ml and 1,197±648 ng/ml, respectively. Injection of controls (n=11) resulted in 3,540±332 ng/ml. Next we injected AAV-2 vector in wild-type animals (n= 4/group) with 10µM/kg of specific activation peptides of PAR-1, PAR-2, or both PAR-1/-2 and compared with injection of scramble peptide as control (LSIGRL). Following simultaneous activation of PAR-1/-2 by SFLLRN, FIX levels were higher when compared to controls (2,654 vs. 1,248 ng/ ml; p<0.005). HepG2 cells, as a model for human hepatocytes, were transduced with AAV-2 following PAR-1/-2 activation and again F.IX secreted in the conditioned media was ∼2-fold higher compared to control peptide. Together these data suggest a novel role of PARs and coagulation on gene transfer by viral vectors. PARs modulation may serve as an alternative strategy to improve viral vectors-cell interaction.

751. Breaking Down AAV: Defining the Minimal Capsid Sequences for AAV Function Yu-Hung Kuo,1 Jill M. Roberts,2 Michael G. Kaplitt.1 1 Neurological Surgery, New York Hospital, Weill Cornell Medical Center, New York, NY; 2Neuroscience, University of Arizona, Tucson, AZ. Adeno-associated virus (AAV) is currently under investigation as a gene therapy vector for treatment of hemophilia, Canavan’s disease and Parkinson’s disease. AAV is particularly attractive for CNS disorders as it is minimally immunogenic, establishes long term expression, and infects nondividing cells including neurons. AAV is a member of the Parvoviridae family and consists of a single stranded DNA packaged within a protein capsid which contains the receptor binding domain and regulates tissue tropism. The capsid is composed of three subunits, all of which share a common C-terminus. All three subunits are encoded by a single gene (cap) through alternative splicing and use of different translation start sites. The major component of the capsid, VP3, is the smallest subunit. The larger S285