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

描述（由申请人提供）：本申请的主要目的是开发重组腺相关病毒3 （AAV3）血清型载体，用于肝细胞的高效转导，并评估优化后的AAV3血清型载体在非人灵长类动物中的安全性和有效性，长期目标是将其用于人类肝脏定向基因治疗。我们观察到，在10种最常用的AAV血清型中，AAV3载体对人肝癌细胞系的转导效率极高。AAV3载体利用人肝细胞生长因子受体（HGFR）作为细胞共受体，也能有效地转导原代人肝细胞。基于我们最近对含有表面暴露酪氨酸残基突变的下一代AAV2载体的研究，这些突变在低剂量下可以很好地转导小鼠肝细胞，我们也产生了酪氨酸突变的AAV3血清型载体，并确定了一种优化的载体，可以在小鼠异种移植模型中有效地转导人肝脏肿瘤。我们现在希望利用这些信息来探索优化的AAV3载体是否是人类肝细胞高效转导的理想血清型。我们提出验证以下假设：a.优化的AAV3血清型载体可在小鼠异种移植模型中实现人肝细胞的高效转导。b.系统递送优化的AAV3血清型载体在用于人类基因治疗之前的安全性和有效性，可以在非人灵长类动物模型中进行体内肝脏定向基因转移评估。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3型载体，用于人肝细胞的高效转导。特异性目的2：优化的AAV3血清型载体在非人灵长类动物全身递送后的生物分布和安全性。在分子水平上更好地了解aav3 -肝细胞相互作用可能会导致开发安全有效的载体，用于一般人类肝脏疾病的基因治疗，特别是B型血友病。

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.