101. Molecular Mechanism Responsible for High ...

AAV VECTORS: BIOLOGY AAV VECTORS: BIOLOGY 101. Molecular Mechanism Responsible for High Level Transgene Expression in Murine and Nonhuman Primate Liver with the Second Generation of Novel AAV Serotype Vectors You Lu,1 Xun Sun,1 Larry T. Bish,2 Julie C. Johnston,1 Roberto Calcedo,1 Rebecca L. Grant,1 Gaung-Ping Gao,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Department of Physiology, University of Pennsylvania, Philadelphia, PA. AAV vectors are being considered for in vivo application of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second generation vectors based on the novel serotypes AAV7 and 8 containing self-complimentary (sc) genomes in both murine nonhuman primate livers. Superior murine liver transduction by sc over the traditional single stranded (ss) vector genomes was confirmed for all serotypes examined. Stable levels of transgene expression were achieved in cynomolgus macaques with efficiencies at least 2 logs higher than what was achieved with AAV2 vectors using ss genomes. In an attempt to understand molecular mechanisms responsible for the higher level gene transfer, we performed a series of detailed molecular studies of the vector genomes. This included treatment with exonuclease with specific substrate preferences, single cutter restriction enzyme digestion mediated and locus specific hybridization based vector genome mapping, bacteriophage Phi29 DNA polymerase mediated double stranded circular template specific isothermal rolling circle linear amplification for rescue, cloning and characterization of persisted circular genomes, and differential quantification of different populations of vector genomes by real time PCR. Our data indicated that the higher level of transgene expression achieved with the sc vectors was due to both increased numbers of persisting genomes and increased activity of these genomes. Study of the molecular dynamics of vector genome processing in murine liver revealed that both ss and sc vectors persisted as episomal circular forms but sc vectors were more quickly and more efficiently converted into circular forms. The structure and formation of concatemers were similar with the types of genomes. The molecular structures of AAV genomes in macaques were more heterogeneous consisting of primarily circular monomer and concatemers in different configurations, and some unexpected molecular entities. More extensive rearrangements and deletions were observed in macaque than in mouse. It will be useful to further understand differences in the molecular processing of AAV genomes in mice and macaques as human applications are being considered.

retroviral vector insertion into the first intron of the LMO2 gene that occurred in one of the SCID patients in a human lympholeukemia cell line (Jurkat) using recombinant adeno-associated viral (rAAV) vector mediated gene targeting. The rAAV-LMO2 vector was constructed as follows. First, 1.3kb fragments containing LMO2 intron sequences from upstream and downstream of the retroviral insertion site in the SCID patient were amplified by PCR from human genomic DNA and sequenced to confirm identity to the genomic locus. Next, a gene cassette was designed to have the GFP coding sequences under the control of a single retroviral LTR that was used in the gene therapy trial followed by a polyadenylation signal. Then this 1.5kb single LTR-GFP cassette was placed in a reverse orientation between the 5’ and 3’ segments from LMO2 intron 1 and the assembled three fragments were cloned between AAV ITRs to derive the single stranded rAAV targeting vector. After packaging into serotype 1 capsid proteins, particles were purified by iodixanol gradient and gel filtration chromatography followed by ultrafiltration concentration. The Jurkat T cell line was transduced with rAAV-LMO2 particles at three multiplicities of infection (MOI). The percentage of GFP expressing cells was maximal (77%) at 3 days post-transduction and gradually decreased to stabilize at 2.2% at the highest MOI after 21 days. GFP expressing cells were selected by FACS from this cell population at 9 days post-transduction and clones derived from single cells were recovered. We initially screened 60 GFP-positive clones for targeting into the LMO2 locus and nine clones were positive by PCR using one primer in the GFP expression cassette and the other in the LMO2 intron just 3’ to the region of homology with the targeting vector. These clones were analyzed by Southern hybridization using BglII or DraIII digestion with GFP and LMO2 probes. Four out of nine clones showed the predicted bands in addition to wildtype bands indicating successful gene targeting without rearrangement into one of the LMO2 alleles. Next, LMO2 expression was compared to that in control Jurkat cells by real-time quantitative RT-PCR using the comparative CT method. Expression of LMO2 mRNA was at a very low level in untransduced Jurkat cells but was highly upregulated in all 4 clones ranging from an 1100 to 3500-fold increase. Thus we have reproduced an LTR insertion at the exact location as occurred in one of the SCID patients and showed that a single LTR is sufficient to both maintain GFP expression and activate the upstream LMO2 promoter. This system should prove useful for evaluating the relative safety of specific retroviral vector designs with respect to the potential for protooncogene activation.

103. A Screen for Host Cellular Proteins That Interact with Adeno-Associated Virus Capsid Proteins Reveals Proteins Involved in AAV8 Transduction Bassel Akache,1 Sally Fuess,1 Dirk Grimm,1 Mark A. Kay.1 Pediatrics, Stanford University Medical Center, Stanford, CA.

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102. Activation of the LMO2 Gene in Human Lymphoid Cells by rAAV-Mediated Targeted Insertion of a Single LTR Expression Cassette into the First Intron Byoung Y. Ryu,1 John T. Gray,1 David M. Bodine,2 Arthur W. Nienhuis.1 1 Hematology/Oncology, St. Jude Children’s Research Hospital, Memphis, TN; 2Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD. The pathogenic activation of the LMO2 proto-oncogene contributed to leukemia in two children otherwise successfully treated for Severe Combined Immunodeficiency (SCID) in a gene therapy trial (Science 302: 415-419, 2003). We have reproduced the S42

Vectors from adeno-associated virus (AAV) appear very promising for use in some gene therapy applications, due to their relative safety and sustained gene expression. Different serotypes of AAV have been isolated, each having different properties. For example, it has been shown that AAV-8 uncoats more readily than AAV-2 in the nuclei of hepatocytes in mouse liver, possibly explaining the more robust transduction efficiencies of AAV-8 (Thomas et al., Journal of Virology, 2004, 78 (6) p. 3110-3224). Our goal is to better understand the mechanisms behind AAV-8 transduction by identifying cellular proteins that interact with the capsid, and that are necessary for optimal transduction. Towards this end, we used a yeast two-hybrid screen to identify cellular proteins that interact with different domains of the AAV-8 capsid. Using a portion of the AAV capsid

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

AAV VECTORS: BIOLOGY protein VP3 as bait against a mouse liver cDNA library, we recovered around 700 clones. Analysis of the sequence of these clones revealed that over 100 genes had multiple hits, indicating that the interactions might be genuine. These included genes encoding many different types of proteins that may be involved in viral binding, entry and trafficking. In particular, some clones contained portions of cathepsins B and L, lysosomal proteases shown to be important in the uncoating of reovirus and the Ebola virus. These clones did interact with the corresponding portion of the AAV-2 capsid, but not AAV-5, when using the same two-hybrid system. Infection of murine fibroblast NIH-3T3 cells by AAV-2 and AAV-8 was severely inhibited by treating the cells with the cathepsin B inhibitor Ca074Me. In addition, capsid proteins from assembled AAV-2 and AAV-8, but not AAV-5, were cleaved specifically when incubated with purified cathepsin B protein, which was consistent with our two-hybrid data. Cathepsin B seems to be involved in the uncoating of both AAV-2 and 8. This data confirms that some of the proteins isolated in the screen are indeed involved in AAV transduction, and further analysis of the other genes recovered in our screen may help reveal other cellular proteins important for AAV8-mediated gene transfer.

104. Role of the HI Loop in the AdenoAssociated Virus (AAV) Life Cycle Nina DiPrimio, Aravind Asokan, Richard J. Samulski. 1 Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC. The fivefold axis of symmetry associated with pore region in icosahedral AAV capsids has been extensively studied in regard to its role in deploying the phospholipase A2 domain and packaging of the 4.7kb AAV genome. However, the role of the HI loops surrounding the fivefold pore, which appear to vary significantly in amino acid sequence between AAV serotypes has not been defined thus far. The HI loop is a surface-displayed peptide motif of 10-15 amino acid residues connecting the H and I beta-strands of the VP3 subunit of AAV and extends to interact with a fivefold-related neighboring subunit. In order to understand the role of this capsid region on the AAV life cycle, we substituted the HI loop in the VP3 subunit of the AAV2 capsid with corresponding regions from the VP3 subunits of AAV1, AAV4, AAV5 or AAV8. In addition, we created a deletion mutant lacking the HI loop and an AAV2 mutant substituted with a loop containing ten glycine residues. Determination of the mutant virus titers by dot blot analysis revealed that vector genome titers of AAV2HI1 (AAV1 HI loop substituted in AAV2) and AAV2HI8 displayed a two-fold decrease in titer, while AAV2HI4 displayed a ten-fold decrease in titer when compared to parental AAV2. On the other end of this spectrum, AAV2HI5, AAV2HIdel (deletion mutant), and AAV2HIpolyG (poly-glycine loop) were unable to package vector genomes. Further western and dot blot western analysis revealed that AAV2HI5 and AAV2HIdel mutants were able to synthesize capsid subunits, but unable to assemble into capsids. Interestingly, the AAV2HIpolyG mutant assembled into empty capsids highlighting the plasticity of this loop region. Further characterization of AAV HI loop mutants using transduction assays, heparin binding assays and electron microscopy are currently in progress. Our studies implicate a potential role for the HI loop in AAV genome packaging and capsid assembly.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

105. Interactions between Modified AAV Vectors and Alternative Cell Surface Receptors Define Intercellular Trafficking Pathways and Tranduction in Human Vascular Endothelial Cells Matthew D. Stachler,1,2 Jeffrey S. Bartlett.1,2 Gene Therapy Center, Childrens Research Institute, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH.

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Adeno-associated virus (AAV) vectors transduce a wide variety of cell types. However, cells of the vascular system remain refractive to AAV-mediated gene transduction. AAV1-based vectors have shown the most promise in the vasculature. To improve upon AAV1’s vascular tropism, peptides targeted to receptors expressed on the vasculature were incorporated into the AAV1 capsid. These included peptides targeted to vascular endothelial growth factor receptor 2 (VEGFR2), Tie2, and the 4C-RGD peptide targeted to integrin receptors. Near wild type titers were obtained from all of the peptidemodified vectors. Modified vectors were used to infect four different human endothelial cell (EC) populations (human umbilical vein EC, HUVEC; human coronary artery EC; HCAEC; human saphenous vein EC, HSaVEC; and human coronary microvascular EC, HCMVEC) and human HeLa cells. At least one modified AAV1 vector targeted to each of these receptors significantly increased transduction of all EC lines (average 10 to 60-fold). Specificity was assessed using either soluble peptides or anti-receptor antibody. To determine the mechanism of increased transduction, endothelial or HeLa cell lines were transduced with modified vectors in the presence of inhibitors of endosomal acidification or proteasome activity, adenovirus, or after treatment with neuraminidase to remove cell surface sialic acid. Inhibiting endosomal acidification greatly reduced transduction of both cell types with all vectors tested, suggesting that both targeted and un-modified AAV1 vectors enter cells via endocytosis and require some degree of endosomal maturation for effective gene transfer. Removing sialic acid from the cell surface drastically reduced transduction with all vectors except the RGDmodified vector. This suggested that while the VEGFR2 and Tie2 targeted vectors were significantly more efficient than unmodified vectors at transducing EC, enhancement was due to novel co-receptor usage and/or altered intracellular trafficking rather than redirected cellular attachment. These results further suggest different requirements for receptor/co-receptor usage for different targeted AAV vectors. Co-infection with adenovirus showed variable effects depending on the targeting ligand. When self-complementary vectors were used to infect EC, adenovirus greatly increased transduction of unmodified AAV1 vectors but only moderately increased transduction of targeted vectors. Proteasome inhibitors had no effect on transduction mediated by the RGD-modified vector, and only slightly increased transduction of the VEGFDR2 and Tie2 targeted vectors (1.5 to 3-fold), whereas proteasome inhibitors increased transduction of unmodified AAV1 vectors over 14-fold on HUVEC. Taken together, these studies suggest that intracellular trafficking pathways are defined by initial vector cellular interactions and that transduction of endothelial cells can be increased by either targeting a new receptor/cellular entry pathway, or by altering post-attachment cellular entry/trafficking pathways.

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