Mechanism of high-efficiency transduction of hepatocytes by optimized AAV vectors

该多PI提案的主要目的是表征腺相关性免疫应答的潜在机制。 病毒（AAV）-肝细胞相互作用，并开发一些优化的重组（rAAV） 用于以最小有效剂量和最小免疫力高效转导肝细胞的载体 对Capsid的回应 近年来，我们进行了系统的研究，以更好地了解基本的 AAV-肝细胞相互作用的分子机制，并进行了以下重要观察， 这构成了当前提案的基础： ·鉴定和定点诱变关键表面暴露的酪氨酸、丝氨酸、苏氨酸残基， AAV衣壳，以及下一代高效AAV载体的开发。 ·证明NF-kB通路的瞬时抑制使促炎反应最小化 通过AAV介导的转导诱导。 ·观察到糖皮质激素受体（GR）途径参与AAV 2载体的生命周期。 将追求以下三个具体目标： 具体目的1：研究GR和NF-κ B通路在肝细胞转导过程中的相互作用 优化的AAV 8载体和可能的实施以降低免疫应答。 具体目的2：通过研究频率评价优化的AAV 8载体在人肝细胞中的安全性 整合和可能的插入突变。 具体目标3：开发衣壳和基因组优化的AAV 8载体，用于有效转导AAV 8。 肝细胞，具有最小的免疫应答。 从这些研究中获得的知识将直接适用于下一代的发展 rAAV载体在肝脏定向基因治疗中的最佳用途，特别是血友病B。

The main aims of this multi-PI proposal are to characterize the underlying mechanisms the adeno- associated virus (AAV)-hepatocyte interactions in general, and to develop number of optimized recombinant (rAAV) vectors for high-efficiency transduction of hepatocytes with minimal effective dose and with minimal immune response to capsid. In recent years, we have undertaken systematic studies to gain a better understanding of the fundamental molecular mechanisms of AAV-hepatocyte interactions and have made the following significant observations, which form the basis of the current proposal: • Identified and site-directed mutagenesis of critical surface-exposed tyrosine, serine, threonine residues on AAV capsids, and the development of next generation of highly efficient AAV vectors. • Demonstrated that of transient suppression of the NF-kB pathway minimized pro-inflammatory response induced by AAV-mediated transduction. • Observed involvement of the glucocorticoid receptor (GR) pathway in the life cycle of AAV2 vectors. The following three Specific Aims will be pursued: Specific Aim 1: Studying of cross-talk between GR and NF-kB pathway during transduction of hepatocytes by optimized AAV8 vectors and possible implementation to immune response reduction. Specific Aim 2: Evaluation of safety of optimized AAV8 vectors in human hepatocytes by studying frequency of integration and possible insertional mutagenesis. Specific Aim 3: Development of capsid- and genome- optimized AAV8 vectors for efficient transduction of hepatocytes at low dose and with minimal immune response. The knowledge gained from these studies will be directly applicable in the development of the next generation of rAAV vectors for their optimal use in liver-directed gene therapy and particularly for hemophilia B.