Rational and combinatorial engineering of AAV vectors

描述（由申请方提供）：病毒血清型通过自然选择和遗传重组和诱变的迭代过程进化出不同的组织嗜性。在此框架内，最近鉴定的腺相关病毒（AAV）血清型与广泛的组织嗜性提供了一个通用的工具包的AAV载体的基因治疗社区。我们的目标是建立一个彻底的理解的结构-功能相关的不同组织嗜性的AAV血清型。为了实现这一点，我们设计了一种全面的、双管齐下的方法来解开AAV 1 -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改组的能力来快速进化源自亲本血清型的新型表型，然后合理操作新型AAV突变体以在氨基酸水平上建立结构属性。第一种策略涉及通过AAV血清型衣壳序列的DNA改组，然后通过细胞类型/受体特异性突变体的定向进化来产生组合AAV文库。该方法的目的是消除在鉴定赋予特定表型的AAV衣壳上的所谓“热点”时的偏差。第二种方法涉及使用定点诱变合理操作AAV血清型衣壳上的此类特定区域。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。研究设计涉及（1）通过上述策略合成腺相关病毒库，定向进化细胞类型/受体特异性腺相关病毒突变体，（2）使用分子建模、冷冻EM、一系列体外生化测定来表征此类新型变体，以及（3）将其翻译成载体，用于体内独立基因递送应用。

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.