Rational and combinatorial engineering of AAV vectors

描述（由申请人提供）：病毒血清型通过自然选择和基因重组和突变的迭代过程进化出不同的组织趋向性。在这个框架内，最近发现的具有广泛组织趋向性的腺相关病毒（AAV）血清型为基因治疗界提供了一个多功能的AAV载体工具包。我们的目标是建立对AAV血清型不同组织倾向的结构-功能相关性的透彻理解。为了实现这一目标，我们设计了一种全面的、双管齐下的方法来揭示AAV1-9的结构属性，同时利用这些血清型作为新型AAV载体设计的“蓝图”。该方法利用DNA洗牌的能力，从亲代血清型中快速进化出新的表型，然后对新的AAV突变体进行合理的操作，在氨基酸水平上建立结构属性。第一种策略包括通过AAV血清型衣壳序列的DNA洗牌生成组合AAV文库，然后进行细胞类型/受体特异性突变体的定向进化。这种方法的目的是消除在鉴定AAV衣壳上赋予特定表型的所谓“热点”时的偏见。第二种方法是利用位点定向诱变对AAV血清型衣壳上的这些特定区域进行合理操纵。后一种策略的目标是在氨基酸水平上建立AAV衣壳的结构-功能相关性。这两种互补策略有望产生关键的结构信息，为组织靶向AAV载体的定制设计奠定基础。研究设计包括：(1)通过上述策略合成AAV文库，定向进化细胞类型/受体特异性AAV突变体，(2)使用分子建模，低温电镜，体外生化分析等方法表征这些新变体，以及(3)将其转化为载体，用于体内独立的基因传递应用。

DESCRIPTION (provided by applicant): Viral serotypes have evolved diverse tissue tropisms through natural selection and an iterative process of genetic recombination and mutagenesis. Within this framework, the recent identification of Adeno-Associated Virus (AAV) serotypes with broad tissue tropisms has provided the gene therapy community with a versatile toolkit of AAV vectors. Our goal is to establish a thorough understanding of the structure-function correlates of the diverse tissue tropisms of AAV serotypes. To achieve such, we have devised a comprehensive, two-pronged approach to unravel structural attributes of AAV1-9, while simultaneously exploiting these serotypes as "blueprints" for novel AAV vector design. The approach exploits the ability of DNA shuffling to rapidly evolve novel phenotypes derived from parental serotypes followed by rational manipulation of novel AAV mutants to establish structural attributes at the amino acid level. The first strategy involves generation of a combinatorial AAV library through DNA shuffling of AAV serotype capsid sequences followed by directed evolution of cell type/receptor-specific mutants. The objective of this approach is to eliminate bias in the identification of so-called "hot spots" on the AAV capsid that impart a specific phenotype. The second approach is concerned with rational manipulation of such specific regions on AAV serotype capsids using site-directed mutagenesis. The goal of the latter strategy is to establish structure-function correlates of the AAV capsid at the amino acid level. The two complementary strategies are expected to generate critical structural information that will lay the groundwork for custom design of tissue-targeted AAV vectors. Research design involves the (1) synthesis of an AAV library through aforementioned strategies, directed evolution of cell type/receptor-specific AAV mutants, (2) characterization of such novel variants using molecular modeling, cryo-EM, a battery of biochemical assays in vitro, and (3) their translation into vectors for independent gene delivery applications in vivo.