Rational and combinatorial engineering of AAV vectors

AAV载体的合理组合工程

基本信息

批准号：
7846494
负责人：
RICHARD J SAMULSKI
金额：
$ 0.72万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-05 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7846494
关键词：
Adenoviruses Amino Acids Antibodies Binding Biochemical Biological Assay Capsid Cell Line Cell Surface Receptors Cells Common Cold Communities Complex Custom DNA Shuffling Dependovirus Endothelial Cells Engineering Epithelial Eye Factor IX Family Firefly Luciferases Gene Delivery Generations Genetic Recombination Genome Goals Hepatocyte Hot Spot Human Image In Vitro Intercellular adhesion molecule 1 Intestines Libraries Life Low Density Lipoprotein Receptor Lung Lymphoid Tissue Minor Molecular Models Mus Mutagenesis Natural Selections Organ Parvovirus Pathway interactions Patients Phenotype Process Receptor Cell Research Design Respiratory Tract Infections Rhinovirus Route Sequence Analysis Serotyping Serum Site-Directed Mutagenesis Structure Testing Tissues Transformed Cell Line Transgenes Translations Triage Tropism Variant Viral X-Ray Crystallography adeno-associated viral vector base cell type cellular transduction combinatorial design directed evolution gene therapy in vitro Assay in vivo molecular modeling mutant neutralizing antibody novel preference receptor receptor binding tissue tropism transcytosis transduction efficiency transgene expression vector

项目摘要

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.

描述（由申请人提供）：病毒血清型通过自然选择以及遗传重组和诱变的迭代过程发展了多种组织向对流。在此框架内，最近鉴定了与腺相关病毒（AAV）具有广泛的组织偏爱的血清型，为基因治疗群落提供了AAV载体的多功能工具包。我们的目标是对AAV血清型的各种组织向对性的结构功能相关性建立透彻的理解。为了实现这一目标，我们设计了一种全面的两管齐下的方法来解开AAV1-9的结构属性，同时将这些血清型作为“蓝图”作为新颖的AAV矢量设计。该方法利用了DNA改组迅速发展的新表型的能力，这些表型从亲本血清型中得出，然后是对新型AAV突变体的合理操纵以在氨基酸水平上建立结构属性的能力。第一种策略涉及通过AAV血清型衣壳序列的DNA随后将Compinatorial AAV库生成，然后将细胞类型/受体特异性突变体的定向演变。这种方法的目的是消除在鉴定赋予特定表型的AAV CAPSID上所谓的“热点”中的偏见。第二种方法与使用位置定向的诱变有关对AAV血清型衣壳上这种特定区域的合理操纵。后一种策略的目的是在氨基酸水平上建立AAV衣壳的结构功能相关。预计这两种互补策略将产生关键的结构信息，这将为靶向组织的AAV矢量定制设计奠定基础。研究设计涉及（1）通过上述策略（1）使用分子建模，冷冻em，在体外生物化学分析的电池中对此类新型变体进行表征的细胞类型/受体特异性AAV突变体的定向演化，（2）在体外的生物化学分析以及（3）将其转化为vectors viv viv viv viv，（3）。