525 Absence of Integration Hotspots in Non Human Primates after Intravenous Injection of AAV2/5 AAT coPBGD Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene[.]
AAV VECTOR DESIGN & APPLICATION purified form using immuno-magnetic beads Where isolated Purkinje lysate used to induce neuronal stem cells to learnable Purkinje cells that can be implanted in mice brain after induction of focal epilepsy Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and reimplanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells Immunohistochemical study revealed that the cells are positive of callbindin which is the marker for Purkinje cells AAV Vector Design & Application 523 Vector Genome Length and SelfComplementarity Affect AAV Capsid Uncoating Eric D Horowitz,1 Aravind Asokan.1 Gene Therapy Center, The University of North Carolina at Chapel Hill, NC The 4.7kb single-stranded DNA genome of adeno-associated virus (AAV) is packaged into a highly confined space (diameter ∼22nm) As demonstrated in the case of bacteriophages, the viral genome exerts a substantial force on the inner walls of the capsid, resulting in high internal pressure In the current study, we investigated whether AAV vectors packaging genomes of different length or self-complementary genomes display similar uncoating profiles Using a novel biophysical uncoating assay, high resolution electron microscopy and cellular imaging techniques, we determined that subgenomic and selfcomplementary vector genomes dramatically impact AAV capsid uncoating We observed a linear correlation between genome size and uncoating efficiency for ssAAV vectors in the 3.4kb to 4.7kb range, with capsids packaging smaller genomes displaying decreased uncoating Capsids packaging oversized ssDNA genomes (5.8kb) displayed uncoating efficiency similar to those packaging subgenomic (3.4kb) DNA, most likely due to genome fragmentation as demonstrated earlier Surprisingly, self-complementary AAV vectors were in general more stable and displayed decreased uncoating efficiency compared to their single-stranded counterparts Further, subgenomic ssAAV and scAAV vectors were less efficient at nuclear entry than wild type AAV capsids in vitro Studies evaluating the infectious titers/transduction efficiencies of AAV vectors packaging different genome constructs will also be presented Our results support the notion that smaller or self-complementary genomes are likely to exert lesser pressure on the AAV capsid, thereby decreasing uncoating efficiency and markedly affecting the performance of AAV vectors Careful design of AAV vector genomes might help reduce vector dose required for clinical applications 524 Patterns of scAAV Vector Genome Insertion Associated with Oncogenic Events in a Mouse Model for Genotoxicity Lucia E Rosas,1 Jessica L Grieves,3 Kimberly Zaraspe,1 Krista M.D La Perle,3 Haiyan Fu,1 Douglas M McCarty.1,2 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Dept of Pediatrics, The Ohio State University, Columbus, OH; 3Dept of Veterinary Biosciences, The Ohio State Unviversity, Columbus, OH Recombinant adeno-associated virus (rAAV) gene delivery vectors have gained an extensive record of safety and efficacy in numerous animal models of human disease Infrequent reports of rAAV-associated hepatocellular carcinomas (HCC) in mice have been limited to specific vectors In this study, we set out to understand the potential for AAV genotoxicity and identify patterns of insertion or features of the vector genome that could promote tumor formation To maximize the chances of generating rAAV-related tumors, we included a self-complementry AAV (scAAV) vector designed to S202 promote insertional activation, with a strong promoter and no protein coding sequences or transcriptional termination signal (scAAVCBA-null), and evaluated the incidence of HCC in a liver tumor prone mouse model (C3H/HeJ) Vector-chromosome junctions were recovered from tumor tissue in CBA-null vector-infected mice by inverse PCR or LM-PCR and analyzed by conventional sequencing Consistent with its high propensity for read-through transcription, 15 out of 18 junctions recovered from CBA-null vector tumors were associated with known proto-oncogenes or tumor suppressors, and two were clearly intergenic One was within a repetitive sequence that could not be mapped Three separate common insertion sites were identified, with four insertions in the 5’UTR of Hras1, three within intron of Sos1, and two in the promoter region of Fgf3 Most vector-associated oncogenic events were likely due to readthrough transcription of downstream sequences, as the CBA-null vector was designed to However, insertions in the Fgf3 gene were in an inverse orientation relative to transcription of the oncogene, suggesting enhancer effects Two vector insertions were associated with putative tumor suppressor genes, suggesting a gene disruption mechanism Increased transcription of the associated oncogene was demonstrated in three tumors with insertions in Fgf3, Sos1, and Fgf10 The C3H/HeJ mouse model offers a useful tool for evaluating features of rAAV vector genomes that may promote genotoxicity 525 Absence of Integration Hotspots in NonHuman Primates after Intravenous Injection of AAV2/5-AAT-coPBGD Christine Kaeppel,1 María Astrid Peda Rodríguez,2 Esperanza López-Franco,3 Antonio Fontanellas,3 Jesús Prieto,4 Stuart Beattie,5 Stephan Hermening,5 Harald Petry,5 Gloria GonzálezAseguinolaza,3 Christof von Kalle,1 Manfred Schmidt.1 National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany; 2DIGNA Biotech S.L., Madrid-Pamplona, Spain; 3Center for Applied Medical Research, University of Navarra, Pamplona, Spain; 4University Hospital of Navarra, University of Navarra, Pamplona, Spain; 5Amsterdam Molecular Therapeutics, Amsterdam, Netherlands Long-term gene expression can be achieved following recombinant adeno-associated virus (AAV) vector delivery Although AAV integration seems to be a rare event, a previous study has shown that a high dose AAV delivery into neonatal mutant mice may lead to hepatocellular carcinoma formation upon vector integration We hypothesized that AAV gene transfer in non-human primates (NHP, Macaca fascicularis) considered to be a more predictive model for AAV vector safety assessment in ongoing and upcoming clinical trials Acute intermittent porphyria (AIP) is a rare life-threatening autosomal dominant genetic disease in which mutations in the porphobilinogen deaminase (PBGD) gene produce insufficient activity of a protein necessary for heme synthesis The baculovirus produced vector system AAV2/5-AAT-codon optimized PBGD (coPBGD) has been developed for the long-term correction of AIP To test the safety and transduction efficacy of this vector, six adult NHP received either 1*1013 gc/kg or 5*1013 gc/kg of AAV2/5-AAT-coPBGD intravenously via the peripheral saphenous vein DNA from four liver sections of each animal was collected 30 days post-injection Vector integration site (IS) analysis by linear amplification-mediated (LAM-)PCR was performed starting on the 5-prime and 3-prime end of the vector using different restriction endonucleases to reach a comprehensive IS profile In total, >100000 AAV derived LAM-amplicons were characterized after 454 sequencing, revealing 752 NHP-specific IS, 583 of which were exactly mappable to the rhesus genome On average, more IS were detected in the animals which received a higher amount of vector (36.9 vs 25.8 IS, respectively) By comparing the location of AAV2/5-AAT-coPBGD IS and their chromosomal distribution to a random data set of 10000 AAV IS, we demonstrated Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy AAV VECTOR DESIGN & APPLICATION a close to random integration pattern, without preferences of AAV integration within or proximal to genes, CpG islands or DNA palindromic regions Evaluation of integration hotspots by common IS analysis revealed a low level clustering near genes considered to be not worrisome Even if a large number of IS have been detected, the presence of a high frequency of concatemers (up to 99%) was demonstrated in each sample Finally, the feasibility of full inverted terminal repeat (ITR) sequencing was proven, while partially deleted ITR with preferred breakpoints in the first loop of the ITR, starting from the sequencing initiating site, were detected Taken together, these findings with hundreds of IS derived from a preclinical NHP model are unique in their context, showing random AAV integration and thus a presumably safe IS profile These results are promising for our upcoming AIP clinical trial and future AAV gene therapy studies 526 Intravasuclar Delivery of RAAVRH.8 Generates Widespreading Transduction of Neuronal and Glial Cell Types in the Adult Mouse Central Nervous System Bin Yang,1 Chunyan Cao,2,7 Hongwei Zhang,2,3,8 Qin Su,2 Seemin Seher Ahmed,2,3 Li Zhong,2,4 Ran He,2 Miguel Sena-Esteves,2,5 Terrence R Flotte,2,3,6 Robert Brown,5 Zuoshang Xu,1 Guangping Gao.2,3 Biochem & Mol Pharm, UMMS, Worcester; 2Gene Therapy Center, UMMS, Worcester; 3Microbiol & Physiol Syst, UMMS, Worcester; 4Medicine, UMMS, Worcester; 5Neurology, UMMS, Worcester; 6Pediatrics, UMMS, Worcester; 7Physiology, Med Sch of Tongji Univ, Shanghai, China; 8Pharm Sci, SDSU, Brookings The ability of intravenously delivered recombinant adenoassociated virus serotype (rAAV9) to cross the blood-brain barrier (BBB) and efficiently transduce different cell types of the central nervous system (CNS) in neonatal mice was discovered by Kaspar’s group and confirmed by several other groups including ours Our recently published study indicated that such extraordinary gene transfer capability of rAAV to the neonatal CNS was not restricted to rAAV9; several other primate-derived rAAVs, especially rAAVrh.10, are at least as efficient as rAAV9, if not better However, due to the further developed BBB in adult mice, rAAV9 transduction of the adult CNS, particularly the neuronal cell types, is significantly reduced Here, we report the discovery of another primate AAV, AAVrh.8 that can cross the BBB and mediate robust transduction of all major neuronal and glial cell types characterized in the adult mouse CNS In this study, 12 different rAAVs encoding the EGFP reporter gene were introduced to 10-week-old mice via tail vein injections, followed by characterization of EGFP transduction distributions 21 days later As IV injection of rAAVs to adult mice generated much more robust EGFP transduction of the vasculature in the CNS as compared to that of neonates, it was difficult to characterize other transduced cell types by immunofluorescence We took two approaches to address this issue: avidin-biotin-peroxidase complex (ABC) staining and 3D deconvolution processing of Z-stack images acquired from immunofluorescently labeled CNS sections Of the 12 rAAVs tested, five (rAAV7, rAAV8, rAAV9, rAAVrh.8, rAAVrh.10, and rAAVrh.39) had transduction levels comparable to, if not better than, rAAV9 Of note, rAAV.rh8 out-performed the rest of the top in most regions of the CNS Importantly, several regions of clinical importance such as the cortex, hippocampus, substantia nigra, caudate-putamen and corpus callosum showed extensive transduction of neurons, astrocytes and oligodendrocytes Even though the transduction levels were weaker, the distribution and the number of cells transduced were quite comparable to those seen in the neonates In addition, we performed double-immunofluorescent staining and confirmed that AAV.rh8 is capable of transducing Darpp-32+, TH+, Calbindin+, ChAT+ neurons, GFAP+, APC+ glial cells, and CD31+ endothelial cells of the blood vessel Our results have demonstrated rAAV.rh8 is a versatile and Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy efficient viral vector in developing promising strategies to treat neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Parkinson’s disease and Huntington’s disease in the adult patient population 527 Altering Tissue Glycan Patterns Improves the Transduction Profile of AAV9 Vectors Shen Shen,1 Junjiang Sun,1 Matthew Hirsch,1 Sai Chavala,2 Paul Monahan,1 Aravind Asokan.1 Gene Therapy Center, The University of North Carolina at Chapel Hill, NC; 2Department of Ophthalmology, The University of North Carolina at Chapel Hill, NC Cross species differences in the transduction profile of AAV vectors in preclinical studies is a significant barrier towards predicting clinical outcomes A well-studied example in this regard is differences in tissue glycosylation patterns, particularly sialic acid linkage in different species Removal of sialic acid (desialylation) exposes the underlying galactose residue, which is an aspect of tissue glycosylation common to all major animal species including human beings Our lab and others recently determined galactose as the primary cell surface attachment receptor for AAV9 infection in vitro In the current study, we exploit the potential of recombinant sialidase to transiently generate galactosylated tissue glycans in vivo and investigate its potential use as an adjuvant to improve the transduction profile of AAV9 vectors Intravenous injection of sialidase prior to AAV9 vector administration dramatically enhanced endothelial sequestration and liver tropism Localized sialidase administration in the joints and eyes of Balb/c mice prior to AAV9 injection resulted in 10-100 fold increase in transgene expression in those tissues Increased galactose density in peripheral tissue also reduced systemic leakage of AAV9 vectors from the site of injection Our studies support the notion that co-administration or pretreatment of different tissue types such as the lung, joints, the central nervous system, or eye with recombinant sialidase might serve as a strategy to expose high avidity glycan receptors that will (a) prevent systemic spread and restrict AAV9 transduction to the site of administration; (b) increase gene transfer efficiency and decrease AAV9 vector dose in clinical applications; and (c) provide a uniform platform to evaluate AAV9 vectors in preclinical studies by eliminating cross-species variation in sialylation patterns 528 rAAV8/9-Mediated Muscle Transduction with Tacrolimus in Non-Human Primate Akiko Ishii,1 Hironori Okada,2 Hiromi Hayashita-Kinou,2 Takashi Okada,2 Shin’ichi Takeda.2 Neurology, University of Tsukuba, Ibaraki, Japan; 2Molecular Therapy, National institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan Background: Recombinant adeno-associated virus (rAAV) is a promising viral vector for gene therapy of muscular dystrophy, proven to improve dystrophic mice and dog phenotype Recently, emergence of the circulating dystrophin-specific T cells was reported in Duchenne muscular dystrophy patients with AAV-mediated gene therapy Therefore, immunomodulation would be necessary requirement for successful gene therapy To establish efficient and safe immunosuppressive regimen of AAV-mediated gene therapy for muscular dystrophy, we tested AAV2, AAV8, and AAV9 vectors expressing LacZ and microdystrophin (M3) gene with tacrolimus to investigate primate skeletal muscle transduction Materials and Methods: We estimated AAV-neutralizing antibody by ELISA in the colony of to 3-year-old Cynomolgus monkey and selected sero-negative animals We utilized AAV2-LacZ, AAV8-LacZ, AAV9-LacZ, AAV2-M3-FLAG, AAV8-M3-FLAG, and AAV9-M3FLAG, each to transduce distinct monkeys Two days before AAV S203 ... a close to random integration pattern, without preferences of AAV integration within or proximal to genes, CpG islands or DNA palindromic regions Evaluation of integration hotspots by common... feasibility of full inverted terminal repeat (ITR) sequencing was proven, while partially deleted ITR with preferred breakpoints in the first loop of the ITR, starting from the sequencing initiating... these findings with hundreds of IS derived from a preclinical NHP model are unique in their context, showing random AAV integration and thus a presumably safe IS profile These results are promising