The main aims of this proposal are to develop recombinant adeno-associated virus 3 (AAV3) serotype vectors for high-efficiency transduction of hepatocytes, and to evaluate the safety and efficacy of the optimized AAV3 serotype vectors in non-human primates, with the long-term goal of their potential use in liver-directed gene therapy in humans. We have observed that of the 10 most commonly used AAV serotypes, AAV3 vectors transduce human liver cancer cell lines extremely efficiently. AAV3 vectors also transduce primary human hepatocytes efficiently, as they utilize human hepatocyte growth factor receptor (HGFR) as a cellular co-receptor. Based on our recent studies on the development of the next generation of AAV2 vectors containing mutations in the surface-exposed tyrosine residues, which transduce murine hepatocytes exceedingly well at low doses, we have also generated tyrosine-mutant AAV3 serotype vectors, and identified an optimized vector that efficiently transduces human liver tumors in a murine xenograft model in vivo. We now wish to parlay some of this information to explore whether optimized AAV3 vectors are the ideal serotype for high-efficiency transduction of human hepatocytes. We propose to test the following hypotheses: a. High-efficiency transduction of primary human hepatocytes can be achieved by optimized AAV3 serotype vectors in a mouse xenograft model in vivo. b. The safety and efficacy of the systemically delivered optimized AAV3 serotype vectors, prior to their potential use in human gene therapy, can be evaluated in a non-human primate model for liver-directed gene transfer in vivo. The following two Specific Aims will be pursued:
Specific Aim 1 : Development of optimized surface-exposed tyrosine-, serine and threonine-mutant AAV serotype 3 vectors for high-efficiency transduction of human hepatocytes.
Specific Aim 2 : Biodistribution and safety of optimized AAV3 serotype vectors in non-human primates following systemic delivery. A better understanding of the AAV3-hepatocyte interactions at the molecular level is likely to lead to development of safe and effective vectors for their potential use in gen therapy of human liver diseases in general, and hemophilia B in particular.

Public Health Relevance

The main aim of this proposal is to develop safe and effective vectors based on a human virus, the adeno- associated virus (AAV), which causes no known disease. Recombinant AAV vectors are currently being used in a number of Phase I/II clinical trials for the potential gene therapy of a wide variety of human diseases, and our proposed studies are expected to yield new and useful information towards further development of these vectors, which has relevance to public health.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Exploratory/Developmental Grants (R21)
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Gene and Drug Delivery Systems Study Section (GDD)
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Tucker, Jessica
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University of Florida
Schools of Medicine
United States
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Srivastava, Arun; Carter, Barrie J (2017) AAV Infection: Protection from Cancer. Hum Gene Ther 28:323-327
Ling, Chen; Li, Baozheng; Ma, Wenqin et al. (2016) Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses. Hum Gene Ther Methods 27:143-9
Ling, Chen; Yin, Zifei; Li, Jun et al. (2016) Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors. Mol Ther Methods Clin Dev 3:16029
Srivastava, Arun (2016) In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol 21:75-80
Ling, Chen; Bhukhai, Kanit; Yin, Zifei et al. (2016) High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing. Sci Rep 6:35495
Ling, Chen; Wang, Yuan; Lu, Yuan et al. (2015) Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo. J Virol 89:952-61
Li, Baozheng; Ma, Wenqin; Ling, Chen et al. (2015) Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo. Hum Gene Ther Methods 26:211-20
Ling, Chen; Wang, Yuan; Lu, Yuan et al. (2015) The Adeno-Associated Virus Genome Packaging Puzzle. J Mol Genet Med 9:
Li, Shaoyong; Ling, Chen; Zhong, Li et al. (2015) Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors. Mol Ther 23:1867-76
Wang, Lina; Yin, Zifei; Wang, Yuan et al. (2015) Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal. J Gen Virol 96:2780-7

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