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

这个多PI方案的主要目的是特征腺相关的潜在机制 病毒(AAV)与肝细胞的相互作用一般，并开发出多个优化的重组(RAAV) 以最小有效剂量和最低免疫水平高效转导肝细胞的载体 对卡西德的回应。 近年来，我们进行了系统的研究，以更好地了解 AAV与肝细胞相互作用的分子机制，并作了以下重要观察。 它们构成了当前提案的基础： ·对关键表面暴露的酪氨酸、丝氨酸、苏氨酸残基进行鉴定和定点突变 AAV衣壳，以及下一代高效AAV载体的开发。 ·证明瞬时抑制核因子-kB通路可最大限度地减少促炎反应 由AAV介导的转导诱导。 ·观察到糖皮质激素受体(GR)途径参与AAV2载体的生命周期。 我们将致力于实现以下三个具体目标： 特异性目的1：研究肝细胞转导过程中GR和NF-kB信号通路的相互作用 优化了AAV8载体，并可能实现免疫应答降低。 具体目标2：通过研究频率评价优化的AAV8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AAV8载体，用于有效转导 低剂量，免疫反应最小的肝细胞。 从这些研究中获得的知识将直接应用于下一代的发展 重组AAV载体在肝脏导向基因治疗中的最佳使用，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.