AAV VECTORS: VECTOR BIOLOGY 516 Analysis of AAV Serotype Vector Integration in Normal and DNA-PKcs-Deficient Scid Mice by a Novel Strategy Katsuya Inagaki,1 Xiaolin Wu,2 Sally Fuess,1 Theresa A Storm,1 Mark A Kay,1 Hiroyuki Nakai.1 Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA; 2Laboratory of Molecular Technology, SAIC-Frederick, Inc, National Cancer Institute at Frederick, Frederick, MD rAAV vectors integrate into host chromosomal DNA infrequently However, we still may need to consider the possibility of adverse effects caused by rAAV vector integration because 1) rAAV2 vector integrations in animals accompany host chromosomal deletions or translocations, and occur preferentially in or near gene regulatory sequences; and 2) to % integrations occur in or near cancerrelated genes In order to further address this issue and understand the biology of rAAV vectors in vivo, we investigated rAAV8 vector integration in normal and DNA-PKcs-deficient scid mice Scid mice were chosen because DNA-PKcs has been shown to be involved in rAAV vector genome processing Here, we developed a novel strategy for isolation of a large number of rAAV proviruses directly from vector-transduced animal tissues with high efficiency and reliability, and without any selective pressure The salient features of the strategy are: 1) the use of a shuttle rAAV8 vector carrying an ISceI site and AmpR/Ori (AO) cassette, 2) physical separation of integrated and extrachromosomal vector genomes following ISceI digestion of tissue DNA; 3) removal of unwanted genomic DNA by digestion with a DNA conformation-dependent nuclease; 4) mixing the tissue DNA with yeast genomic DNA at 1:1 ratio at the beginning of the procedure, with yeast DNA serving as a tag sequence to monitor for undesired intermolecular recombination that may arise during the procedure We injected normal and scid mice with 7.2x10e12 vg of this rAAV8 shuttle vector via the tail vein, and made rAAV8 integration provirus libraries from transduced mouse liver DNA Transformation of bacteria with ∼0.3 µg liver DNA generated over a thousand colonies in each library Initial sequencing results of 121 and 69 clones from normal and scid mouse libraries demonstrated that 23 (19%) and 52 (75%) of the sequenced clones carried rAAV8 vector-cellular DNA junctions, respectively Importantly, we have not seen any intermolecular recombination between rAAV8 vector and yeast genomes, establishing the high reliability of this strategy A preliminary analysis suggested that integration sites in scid mice are more dispersed than in normal mice In addition, we found that a majority of rAAV8 genomes in scid mouse liver form extrachromosomal double-stranded “dog bone” structures, i.e., with both terminal hairpin loops closed Such dog bone forms are present at undetectable levels in rAAV2-injected mouse livers, but may represent intermediates toward various vector forms Thus the isolation of a large number of rAAV proviral genomes from quiescent somatic cells in animals is feasible by our novel strategy, and highthroughput rAAV integration site analyses in normal and scid mouse tissues and detailed characterization of the dog bone forms will provide new insights into the mechanisms of rAAV vector transduction and integration in animal tissues 517 Type Rep52 Is Superior to Authentic Rep52 for Producing Recombinant AdenoAssociated Virus Type in Insect Cells Masashi Urabe,1 Takayo Nakakura,1 Hiroaki Mizukami,1 Akihiro Kume,1 Robert M Kotin,2 Keiya Ozawa.1 Div Genet Ther, Jichi Med Sch, Minami-kawachi, Tochigi, Japan; Lab Biochem Genet, NHLBI, NIH, Bethesda, MD The recently described method for producing recombinant adenoassociated virus (rAAV) type in insect cells facilitates scale-up by S200 using suspension cell cultures and baculovirus expression vectors (Hum Gene Ther 13:1935-1943, 2002) AAV5 is one of the most divergent dependovirus characterized and has been shown to utilize different receptors than AAV2 for example, and has demonstrated different tissue tropism from other serotype rAAVs In addition to vectors derived from serotype 2, other serotypes constitute a vector set from which an optimal one can be selected for specific applications Thus, we established a method to generate rAAV5 in insect cells The current process requires triple infection with recombinant baculoviruses that provide the following: AAV structural proteins that form the virus capsid (VP 1, 2, 3); two of the AAV non-structural proteins for replication and encapsidation (Rep 78 and Rep 52); and the AAV vector DNA which contain the gene of interest flanked by the AAV origins of replication (ITRs) Compared to other rAAV serotypes produced in insect cells, the rAAV5 yield per cell was substantially lower and we tested some modifications of the system The initial Rep baculovirus drove type Rep72 expression with a truncated promoter for the immediateearly gene of Orgyia pseudotsugata nuclear polyhedrosis virus The titers of the Rep baculovirus were relatively lower than others We constructed Rep baculoviruses with a series of truncated p10 promoters for Rep78 and selected one that could produce rAAV5 at a high titer and propagate well Rep52 or small Rep protein packages AAV genome into preformed empty capsids We examined Rep52 of other serotypes, 1, 2, 3, for generation of rAAV5 particles The titer of rAAV5-GFP produced with type 1, 2, 3, or small Rep was 56,000±3,200, 41,000±18,900, 42,000±7,300, or 39,000±3,500 particles per Sf9 cell while rAAV5-GFP produced by authentic Rep52 was 13,500±3,200 The rAAV5-GFP produced with either serotype small Rep has an equal ratio of nucleic acid to capsid protein assessed by real-time PCR quantification and silver staining of purified rAAV5 particles Also, rAAV5-GFP produced with either combination of large and small Rep proteins, transduced the simian Cos cell line with similar efficiency The analysis by cesium density gradient of insect-cell lysates indicated that approximately 50% of all capsids contained the vector genome These results indicate that heteroserotypic small Rep polypeptide is able to substitute for AAV5 small Rep and package AAV vector genome with type in Sf9 cells and the new Rep baculovirus expressing type Rep78 under the control of a truncated p10 promoter and type Rep52 will contribute to the development of more efficient production of rAAV5 in insect cells 518 Production of Recombinant Type AdenoAssociated Virus in Bioreactors Yves Durocher,1 Phuong Lan Pham,1 Gilles St-Laurent,1 Danielle Jacob,1 Brian Cass,1 Josephine Nalbantoglu,2 Amine Kamen.1 Animal Cell Technology, Biotechnology Research Institute, Montreal, QC, Canada; 2Departement of Neurology and Neurosurgery, McGill University and Montreal Neurological Institute, Montreal, QC, Canada Recombinant adeno-associated viruses (rAAV) represent a promising gene therapy vector that may be used in the treatment of diverse human diseases The major obstacle to broaden their usage is the availability of a production process able to provide sufficient quantities for preclinical and human trials We present here a successful process for rAAV-2 production in low-serum and serumfree medium performed in a 3L stirred-tank bioreactor The process is based on the triple transfection of suspension-growing HEK293 cells employing polyethylenimine (PEI) as the DNA carrier Production of AAV-GFP in a 3L serum-free medium bioreactor yielded titers of 5.1 x 10 11 infectious viral particles (IVP), corresponding to x 1012 viral genome (VG) or 6.8 x 1012 viral particles (VP) The cell-specific and total viral titers obtained in suspension culture were about three-fold higher to those obtained Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy AAV VECTORS: VECTOR BIOLOGY with adherent cells The process is very simple and robust as it does not require a medium exchange, either before or after transfection, making it particularly attractive for the generation of large and homogeneous stocks of rAAV vectors 519 Selective Inactivation of Helper Adenovirus with High Hydrostatic Pressure for AAV-2 Vector Production Joshua N Leonard,1 David V Schaffer,1 Peter Ferstl,2 Antonio Delgado.2 Department of Chemical Engineering and Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA; 2Deparment of Fluid Mechanics and Process Automation, Technical University of Munich, Freising, Germany Recombinant adeno-associated virus (AAV) is a highly promising gene therapy vector for numerous reasons, including its nonpathogenicity and its ability to induce long-term expression of a transgene in multiple target cell types However, the large scale production of AAV is complex, either involving transient plasmid transfection or co-infection with a helper virus (such as adenovirus), which must eventually be removed from the product to avoid helperinduced pathogenicity The helper virus approach offers some advantages for large scale production, though adenovirus must then be eliminated during AAV purification One widely used procedure for inactivating adenovirus following the production phase involves heating to 55 °C However, this procedure results in the concomitant loss of ∼50% of the active AAV particles Therefore, it may be desirable to develop alternate, rapid, robust, and economical methods that selectively inactivate undesired viruses while leaving AAV particles intact We have developed a method for using high hydrostatic pressure to selectively inactivate adenovirus without causing any significant loss of AAV serotype (AAV-2) infectivity High hydrostatic pressure has previously been shown to effectively inactivate microorganisms, including viruses such as HIV-1, poliovirus, and adenovirus but to our knowledge, ours is the first study to evaluate the stability of AAV-2 under high pressure Towards this end, we determined the inactivation kinetics of AAV-2 under a range of pressurization conditions, including precise control for incidental thermal effects We thereby determined a range of high-pressure treatment conditions that leave AAV-2 fully active while rendering adenovirus essentially fully inactivated AAV-2 inactivation kinetics would also be useful in determining whether high pressure might be used to inactivate other undesired viruses, such as those that might contaminate the producer cell lines, during the production of clinicalgrade viral therapeutics Moreover, this procedure might also yield biological insights into the structural stability of AAV-2, and we are investigating the mechanisms by which this virus is inactivated under high pressure 520 The Effects of Transgene and Cassette Size on Recombinant AAV2 Production and Expression Corinne M Goldsmith,1 Antonis Voutetakis,1 Bruce J Baum.1 NIDCR/GTTB, National Institutes of Health, Bethesda, MD Background: Recombinant Adeno-asssociated virus serotype two (rAAV2) vectors are strictly limited with regard to the size of the transgene cassette that can be accommodated (²4.7 kb) Within this strict size limitation, there may be preferential sizes for the inserted cassette that can affect optimal packaging efficiency and transgene expression (Dong et al, Hum Gene Ther, 1996) In the present study, we examined if the transgene itself will affect the optimal packaging size and have constructed two series of five different-sized rAAV2 vectors, encoding either human erythropoietin Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy (hEpo) or growth hormone (hGH) Methods: The two transgenes chosen encode secretory proteins with cDNAs of similar size (hGH2 and Epo; 666 bp and 579 bp, respectively) These cDNAs were cloned into pDT1.1 (termed pAAV-MCS2.7 in Braddon et al, Hum Gene Ther, 1998.), which is a rAAV2 plasmid containing the CMV promoter/enhancer, SV40 polyadenylation sequence and ampicillin resistance gene In addition to each cDNA, a stuffer sequence (derived from the hGH gene obtained from pOGH), of either 598, 1268, 1750 or 2002 bp, was added This resulted in constructs with final ITR-ITR sizes of approximately1.8 (no stuffer), 2.4, 3.1, 3.6, and 3.8 kb Co-transfection of each construct with the pDG helper plasmid (a generous gift of Prof J.A Kleinschmidt), in 293T cells (9 x 150 mm plates) yielded rAAV2 vectors The vectors were purified using CsCl gradients as described (Kok et al, Hum Gene Ther, 2003), and fractions with a refractive index (RI) from 1.368 1.374 were retained for characterization Vector particles were quantified by QPCR, and infectious units determined by infection of 293 cells at a MOI = 13 for 40 hours in the absence of wild type adenovirus We determined transgene expression in culture media using a specific ELISA Results and Discussion: Generally similar results were obtained with vectors encoding both transgenes The rAAV2 vectors made in the absence of any stuffer sequence (∼1.8 kb) resulted in the highest particle titers in CsCl gradient fractions and exhibited the greatest transducing activity in vitro For example, with the hEpo vectors, particle titers in the optimal CsCl fraction ranged from a low of 2.7x10e9/mL (1268kb stuffer; 3.1 kb vector) to a high of 1.5x10e11 particles/mL (no stuffer sequence; 1.8 kb vector) Similarly, when tested at comparable MOI in 293 cells, the 1.8 kb vector resulted in the greatest hEpo production (∼45mU/mL), while the 3.1 kb vector yielded ∼3.5% that level (∼1.6mU/mL) The other vectors generated all showed relatively weak transducing activity (