3 A Novel Immunoprecipitation (IP) Seq Based Method for Anti AAV Capsid Antibody Epitope Mapping Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell The[.]
AAV VECTOR BIOLOGY AAV Vector Biology Genome-Wide RNAi Screening Identifies Host Restriction Factors Critical for In Vivo AAV Transduction Miguel Mano,1,2 Rudy Ippodrino,2 Serena Zacchigna,2 Lorena Zentilin,2 Mauro Giacca.2 High-Throughput Screening Facility, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy Despite the broad preclinical and clinical success of recombinant AAV vectors in different gene therapy scenarios, several aspects of the biology of these vectors remain obscure In particular, limited information is available on the cellular factors that restrict vector transduction The identification of such factors is an essential requisite to improve in vivo transduction, expand the number of permissive tissues and achieve AAV-mediated gene correction at a clinically applicable level To systematically identify host cell factors that positively or negatively modulate the different steps of AAV transduction, we performed a high-throughput RNAi screening using a genome-wide siRNA library (18,175 human gene targets) Luciferase activity was used to assess AAV2 transduction and cell viability was monitored in parallel This unbiased screening approach identified 1,528 genes affecting AAV2 transduction by more than 4-fold (184 genes by more than 8-fold) Of these genes, 993 are inhibitors of AAV transduction, whereas 535 are required for efficient transduction by AAV vectors; most of these genes have not been previously associated with AAV2 transduction To compare the cellular factors required for transduction by ssAAV and scAAV vectors, we performed an additional imagebased secondary screening of cells transduced with a recombinant ssAAV2 vector expressing DsRed and a scAAV2 vector expressing EGFP Interestingly, the vast majority of the host factors identified in the initial screening affected ssAAV and scAAV transduction to a similar extent Given the interest in improving AAV2 transduction, we chose to follow-up the 10 genes that, once silenced, induced AAV2 transduction more efficiently Unexpectedly, we observed that silencing of out of these 10 genes induced extensive DNA damage, confirmed by a massive accumulation of γ-H2AX foci, phosphorylation of NBS1 and phosphorylation of proteins involved in cell cycle checkpoint control, in particular CHK1/2 Of note, silencing these 10 genes significantly increased AAV transduction by both AAV2 and other serotypes (AAV1, AAV5, AAV6, AAV9) and in various cell types, suggesting that the regulation of AAV permissivity mainly occurs at a step subsequent to receptor binding and virion internalization Finally, delivery of the siRNAs targeting each of these 10 inhibitory genes to the liver of one-month old mice concomitantly with AAV8 vectors expressing EGFP led to a dramatic increase in hepatocyte transduction in vivo Overall, our work highlights the value of using genome-wide unbiased approaches to identify novel molecular players and pathways relevant to AAV transduction Furthermore, our results show that RNAi or pharmacological modulation of the identified restriction factors can be successfully exploited to improve AAV efficiency in vivo Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy Mapping the Antigenic Structure of AdenoAssociated Virus Serotypes and Yu-Shan Tseng,1 Kim Van Vliet,1 Matias Kaplan,1 J Kennon Smith,1 Lavanya Rao,2 Robert McKenna,1 Barry J Byrne,3 Sergei Zolotukhin,3 Aravind Asokan,2 Mavis Agbandje-McKenna.1 Department of Biochemistry and Molecular Biology, Center for Structural Biology, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL; 2Department of Genetics and The Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Department of Pediatrics and Powell Gene Therapy Center, Division of Cell and Molecular Therapy, College of Medicine, University of Florida, Gainesville, FL Adeno-associated virus (AAV) is one of the most promising viral vector systems for therapeutic gene delivery Recently, regulatory approval of an AAV1 gene delivery vector for the treatment of lipoprotein lipase deficiency in the clinic heralded a new and exciting era for this gene delivery system, which has already been successfully utilized in several human clinical trials This includes clinical trials for the treatment of hemophilia B with an rAAV8 vector expressing therapeutic levels of Factor IX protein and the treatment of diseases of the CNS and muscle with rAAV9 vectors, which have been reported to cross the blood brain barrier Although it has been shown that AAV only induces a limited inflammatory response, neutralization from pre-existing human antibodies is detrimental to transduction efficiency and is one of the remaining hurdles to be overcome before full clinical realization of this vector system Hence, characterizing the antigenic structure of the AAVs and engineering neutralization-escaping vectors is a mandatory vector development step Towards this goal and to recapitulate the polyclonal response that exists in humans, we have generated a panel of novel mouse monoclonal antibodies (MAbs) against the capsids of AAV8 and AAV9 Biochemical and molecular biology approaches were employed to characterize the in vitro and in vivo neutralization properties of these antibodies Structural studies using cryo-electron microscopy and image reconstruction, combined with pseudo-atomic model building, was employed to characterize and map the antibody binding sites on the AAV capsids The structural data maps the binding site for these MAbs to capsid regions known to play functional roles in infection The visualized epitopes will inform development of the next generation of rAAV vectors which will be capable of evading antibody neutralization while retaining parental tropism A Novel Immunoprecipitation (IP)-SeqBased Method for Anti-AAV Capsid Antibody Epitope Mapping Hiroyuki Ido,2 Kei Adachi,1 Wade H Holman,1 Hiroyuki Nakai.1 Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR; 2Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga, Japan High prevalence of pre-existing neutralizing antibodies (NtAbs) against adeno-associated virus (AAV) capsids in humans poses a significant barrier to successful AAV vector-mediated gene therapy There has been strong enthusiasm about developing “stealth” AAV vectors that can evade NtAbs; however, creation of such AAVs requires more comprehensive information about NtAb epitopes, which currently remains very limited In this regard, we have recently developed a next generation sequencing-based high-throughput approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains and can be applied to anti-AAV NtAb epitope mapping In our previous study, we produced DNA-barcoded AAV2R585E hexapeptide (HP) scanning capsid mutant libraries in which AAV2-derived HPs were replaced with those derived from other serotypes These libraries were injected S1 AAV VECTOR BIOLOGY intravenously into mice harboring anti-AAV1 or AAV9 capsid antibodies, which led to the identification of 452-QSGSAQ-457 in the AAV1 capsid and 453-GSGQN-457 in the AAV9 capsid as epitopes for anti-AAV NtAbs in mouse sera (Nat Commun 5:3075, 2014) These epitopes correspond to the highest peak of the threefold symmetry axis protrusion on the capsid In addition, we have found that this region also functions as an epitope for mouse antiAAV7 NtAbs using the same in vivo approach (unpublished data) Here, we develop a novel in vitro immunoprecipitation (IP)-based AAV Barcode-Seq method to identify anti-AAV antibody epitopes as opposed to the previously reported in vivo approach, and address a hypothesis that the highest peak around the amino acid positions 452-457 in the capsid is a potential dominant epitope for antibodies against various AAV serotypes To this end, we first optimized the IP conditions using a recombinant AAV9 vector, anti-AAV9 mouse sera and protein A/G agarose beads Then we produced a DNA-barcoded AAV HP scanning capsid mutant library comprising 78 AAV clones, which included nineteen AAV2R585E-derived HP mutants and five AAV9-derived HP mutants that spanned a 14-amino-acid region in and flanking the vicinity of the highest peak of the AAV1, 2, 7, and capsids Using this library and anti-AAV1, 2, 7, 8, and naïve mouse sera collected from mice per serotype/native, we performed IP and recovered AAV library genomes from both immunoprecipitates and supernatants, which were then subjected to the AAV Barcode-Seq analysis As a result, we found that mutants harboring AAV1, 2, 7, and 9-derived peptides spanning amino acids within the vicinity of the highest peak, not other mutants or the control AAV2R585E or AAV9, were clearly captured by the corresponding anti-AAV serotype sera of some, if not all, of the immunized mice, indicating that the identified peptides constitute a dominant epitope Thus, these results demonstrate that a combination of AAV Barcode-Seq with IP is an easy and effective approach to map anti-AAV antibody epitopes in a high-throughput manner Characterizing Essential Receptor Binding Residues for AAV1 Lin-Ya Huang,1 Robert Ng,1 Edward B Miller,1 Robert McKenna,1 Aravind Asokan,2 Mavis Agbandje-McKenna.1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville; 2Department of Genetics and Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill AAVs have drawn attention as promising gene delivery vectors due to their non pathogenicity, wide host range, ability to package foreign DNA, and ability to establish long-term transgene expression To date over 100 genetic variants been isolated and thirteen serotypes have been characterized These exhibit differences in tissue tropism and transduction efficiency that is dictated by their capsid amino acid compositions and structure, which also determines differences in cell surface receptor/co-receptor usage The glycan receptor for AAV1 has been identified as terminal N-linked sialic acid (SIA) Using structural biology (X-ray crystallography) combined with molecular biology and biochemistry; we have mapped the SIA glycan receptor binding residues on AAV1 and verified the role of these residues in cellular transduction in CHO cells with different terminal glycans Significantly, this site is analogous to that identified as the galactose (GAL) binding site on AAV9 Thus these two serotypes, which are ~83% identical, utilize the same binding pocket to engage different glycan receptors However, the amino acid composition of this pocket differs between these serotypes and suggests the need for specific interactions with their receptors for successful cellular infection Efforts to dissect these specificities with respect to glycan binding and transduction ability of the amino acids in this pocket and to identify the critical residues for SIA engagement will be presented S2 Transduction of Primary Human Hepatocytes In Vitro and in Humanized Murine Livers In Vivo by Recombinant AAV3 Vectors Chen Ling,1 Yuan Wang,1,2 Yuanhui Zhang,1 Koen Vercauteren,3 Lieven Verhoye,3 Yuan Lu,4 George Aslanidi,1 Li Zhong,5 Guangping Gao,5 Changquan Ling,2 Philip Meuleman,3 Arun Srivastava.1 Pediatrics, University of Florida, Gainesville, FL; 2Traditional Chinese Medicine, Second Military Medical University, Shangahi, China; 3Center for Vaccinology, Ghent University, Ghent, Belgium; 4Orthopedics and Rehabilitation, University of Florida, Gainesville, FL; 5Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Recombinant AAV vectors have shown remarkable efficacy in gene therapy of several human diseases For example, AAV2 vectors have restored vision in adults and children with Leber’s Congenital Amaurosis; an AAV1 vector (“Glybera”) for the treatment of lipoprotein lipase deficiency is the first gene therapy drug approved in the Western world; and AAV8 vectors have yielded sustained expression of therapeutic levels of factor IX in patients with hemophilia B Despite these successes, significant hurdles remain, especially for liver-directed gene therapy These are in part related to immune responses, including the presence of pre-existing neutralizing antibodies (nAbs) Furthermore, although there is a 1- to 2-log10 dose advantage of the AAV8 vector in transducing naïve murine hepatocytes in vivo, this does not appear to be the case for human hepatocytes Thus, the search for an alternative AAV serotype vector continues AAV3 has largely been ignored because of lack of in vivo transduction in mouse models We have previously reported that AAV3 vectors transduce human liver cancer cell lines and primary human hepatocytes more efficiently than other serotypes, and that an optimized tyrosine-mutant AAV3 vector efficiently transduces human liver tumors in a murine xenograft model in vivo Here, we compared the transduction efficiency of wild-type (wt) scAAV2, scAAV3, and scAAV8 vectors in primary human hepatocytes in vitro Our data document that AAV3 vectors transduced primary human hepatocytes ~10-fold more efficiently than AAV2, whereas AAV8 vectors failed to transduce these cells The transduction efficiency of a capsid-optimized ssAAV3 (S663V+T492V) vector was ~2-fold higher than the wt However, since primary human hepatocyte cultures in vitro not truly reflect liver functions in vivo, we next evaluated the transduction efficiencies of wt scAAV3 and scAAV8 vectors in urokinase-type plasminogen activator-severe combined immunodeficiency [uPA(+/+)-SCID] mice whose liver was reconstituted with primary human hepatocytes Our results show that, whereas little transgene expression occurred in untransplanted mice, IV-injection of 4x1010 vgs of scAAV3 vectors in humanized mice resulted in ~5.6-fold increase 3-weeks post-vector administration In contrast, the extent of transgene expression mediated by AAV8 vectors in mice transplanted with human hepatocytes was ~2.3fold lower than that in untransplanted mice, presumably due to less-efficient transduction of human hepatocytes Taken together, our studies suggest that optimized AAV3 vectors may prove to be a useful alternative to AAV8 vectors for the potential gene therapy of liver diseases in humans especially since AAV3 has lower incidence of nAbs in humans than other serotypes Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy .. .AAV VECTOR BIOLOGY intravenously into mice harboring anti- AAV1 or AAV9 capsid antibodies, which led to the identification of 452-QSGSAQ-457 in the AAV1 capsid and 4 53- GSGQN-457 in the AAV9 capsid. .. of AAV Barcode -Seq with IP is an easy and effective approach to map anti- AAV antibody epitopes in a high-throughput manner Characterizing Essential Receptor Binding Residues for AAV1 Lin-Ya Huang,1... that this region also functions as an epitope for mouse antiAAV7 NtAbs using the same in vivo approach (unpublished data) Here, we develop a novel in vitro immunoprecipitation (IP)- based AAV Barcode-Seq