4. Characterizing Essential Receptor Binding Residues for AAV1

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we have found that this region also functions as an epitope for mouse antiAAV7 NtAbs using the same in vivo approach (unpublished data).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, 8 and 9 capsids. Using this library and anti-AAV1, 2, 7, 8, 9 and naïve mouse sera collected from 4 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, 8 and 9-derived peptides spanning 8 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.

4. 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 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

5. 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 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 do 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