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

描述(申请人提供)：这项建议的主要目的是开发高效转导肝细胞的重组腺相关病毒3型(AAV3)血清型载体，并评价优化的AAV3血清型载体在非人类灵长类动物中的安全性和有效性，长期目标是它们在人类肝脏导向基因治疗中的潜在应用。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非常有效。AAV3载体还可以利用人肝细胞生长因子受体(HGFR)作为细胞共受体，有效地转导原代人肝细胞。基于我们最近关于含有表面暴露的酪氨酸残基突变的下一代AAV2载体的研究，该载体可以在低剂量下很好地转导小鼠肝细胞，我们还产生了酪氨酸突变的AAV3血清型载体，并在小鼠体内异种移植模型中鉴定了一种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来探索优化的AAV3载体是否是高效转导人肝细胞的理想血清型。我们建议验证以下假设：a.在小鼠体内异种移植模型中，优化的AAV3血清型载体可以实现对原代人肝细胞的高效转导。系统递送的优化的AAV3血清型载体的安全性和有效性，在它们潜在地用于人类基因治疗之前，可以在体内肝脏定向基因转移的非人灵长类动物模型中进行评估。具体目标1：优化表面暴露酪氨酸、丝氨酸和苏氨酸突变型AAV 3型载体，用于高效转导人肝细胞。具体目标2：系统分娩后优化的AAV3血清型载体在非人类灵长类动物中的生物分布和安全性。在分子水平上更好地了解AAV3与肝细胞的相互作用可能会导致开发安全有效的载体，使其在人类肝病尤其是血友病B的基因治疗中具有潜在的应用价值。

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.