Rational and combinatorial engineering of AAV vectors

AAV载体的合理组合工程

• 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
• 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

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基因重组和诱变的迭代过程进化出了多种组织向性。在此框架内，最近鉴定出具有广泛组织向性的腺相关病毒 (AAV) 血清型，为基因治疗界提供了 AAV 载体的多功能工具包。我们的目标是全面了解 AAV 血清型不同组织向性的结构-功能相关性。为了实现这一目标，我们设计了一种全面的、双管齐下的方法来阐明 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 改组的能力​​，快速进化出源自亲代血清型的新表型，然后合理操作新的 AAV 突变体，以在氨基酸水平上建立结构属性。第一个策略涉及通过 AAV 血清型衣壳序列的 DNA 改组生成组合 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识别 AAV 衣壳上赋予特定表型的所谓“热点”时出现的偏差。第二种方法涉及使用定点诱变对 AAV 血清型衣壳上的此类特定区域进行合理操作。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。这两种互补策略预计将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，细胞类型/受体特异性 AAV 突变体的定向进化，(2) 使用分子建模、冷冻电镜、一系列体外生化测定来表征此类新变体，以及 (3) 将其翻译成载体，用于体内独立的基因递送应用。