Safety and efficacy of optimized AAV3 vectors in murine and NHP livers

描述（由申请人提供）：本提案的主要目的是开发用于高效转导肝细胞的重组腺相关病毒3（AAV 3）血清型载体，并评价优化的AAV 3血清型载体在非人灵长类动物中的安全性和有效性，其长期目标是其在人类肝脏定向基因治疗中的潜在用途。我们已经观察到，在10种最常用的AAV血清型中，AAV 3载体非常有效地转染人肝癌细胞系。AAV 3载体还有效地转导原代人肝细胞，因为它们利用人肝细胞生长因子受体（HGFR）作为细胞辅助受体。基于我们最近对开发在表面暴露的酪氨酸残基中含有突变的下一代AAV 2载体的研究，其在低剂量下非常好地抑制鼠肝细胞，我们还产生了酪氨酸突变的AAV 3血清型载体，并鉴定了在鼠异种移植模型中体内有效转导人肝肿瘤的优化载体。我们现在希望利用这些信息中的一些来探索优化的AAV 3载体是否是用于高效转导人肝细胞的理想血清型。我们提出以下假设：a。在小鼠异种移植模型中，通过优化的AAV 3血清型载体可以实现原代人肝细胞的高效转导。B.在其潜在用于人基因治疗之前，可以在用于体内肝脏定向基因转移的非人灵长类动物模型中评估全身递送的优化的AAV 3血清型载体的安全性和功效。具体目标1：开发优化的表面暴露酪氨酸、丝氨酸和苏氨酸突变型AAV血清型3载体，用于高效转导人肝细胞。具体目标2：优化的AAV 3血清型载体在全身递送后在非人灵长类动物中的生物分布和安全性。 在分子水平上更好地理解AAV 3-肝细胞相互作用可能导致开发安全有效的载体，用于其在一般人类肝脏疾病，特别是血友病B的基因治疗中的潜在用途。 公共卫生相关性：该提案的主要目的是开发基于人类病毒（腺相关病毒（AAV））的安全有效的载体，该病毒不会引起已知的疾病。重组腺相关病毒载体目前正用于许多I/II期临床试验，用于多种人类疾病的潜在基因治疗，我们提出的研究有望为进一步开发这些载体提供新的有用信息，这与公共卫生有关。

DESCRIPTION (provided by applicant): 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.