Rational and combinatorial engineering of AAV vectors

基因治疗社区， AAV载体的工具包。我们的目标是建立一个彻底的理解的结构功能相关的 AAV血清型的不同组织嗜性。为了实现这一目标，我们制定了一个全面的，两个- 一种解决AAV 1 -9结构属性的方法，同时利用这些血清型 作为新型AAV载体设计的“蓝图”。这种方法利用了DNA改组的能力， 衍生自亲本血清型的新表型，然后合理操作新的AAV突变体， 在氨基酸水平上建立结构属性。第一种策略涉及生成一个组合 通过AAV血清型衣壳序列的DNA改组随后细胞定向进化的AAV文库 型/受体特异性突变体。这一方法的目的是消除在确定如此- 称为AAV衣壳上的“热点”，其赋予特定表型。第二种方法涉及 使用以下方法合理操作AAV血清型衣壳上的此类特异性区域：

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