Structural studies of AAV capsids and their glycan receptor interactions

AAV 衣壳及其聚糖受体相互作用的结构研究

• 批准号: 7666688
• 负责人: Mavis Agbandje-Mckenna
• 金额: $ 27.52万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-17 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7666688
• 关键词: Address; Affect; Affinity; Antibodies; Baculovirus Expression System; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Biology; Capsid; Carbohydrates; Cell Adhesion; Cell Surface Receptors; Cell surface; Cells; Cellular Tropism; Characteristics; Communities; Complex; DNA; DNA Packaging; Data; Dependovirus; Development; Disease; Engineering; Funding; Gene Delivery; Generations; Genetic; Genomics; Goals; Heparin; Heparin Binding; Human; Immune response; Infection; Inorganic Sulfates; Maps; Measurement; Mediating; Mutagenesis; Nature; Nucleic Acids; Organ; Pathway interactions; Phenotype; Polysaccharides; Population; Primates; Property; Proteins; Receptor Cell; Recombinants; Research Personnel; Resources; Roentgen Rays; Role; Safety; Serotyping; Sialic Acids; Specificity; Structure; Surface; Tissues; Tropism; Unspecified or Sulfate Ion Sulfates; Variant; Viral; Virus; X-Ray Crystallography; adeno-associated viral vector; carbohydrate receptor; cell type; gene therapy; improved; inhibitor/antagonist; insight; interest; member; mutant; non-genomic; nonhuman primate; programs; receptor; three dimensional structure; tissue tropism; trafficking; transduction efficiency; vector; viral gene delivery

DESCRIPTION (provided by applicant): The Adeno-associated viruses (AAVs) are not associated with any diseases and their ability to package non-genomic DNA and to transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The goal is to improve their specificity and efficacy as vectors. However, while the majority of the characterization of the AAVs has been directed at serotype 2 (AAV2), studies on some of the more recently identified antigenically distinct human and primate viruses show enhanced transduction properties for particular cell types compared to AAV2. This property is mediated by their capsid sequence. Thus while providing the gene therapy community with a more expansive choice of potential AAV vectors for development, specific tissue/organ targeting for improving safety profiles and efficacy as well as engineering a faster onset of transduction would be greatly aided by identifying the capsid features of the other AAVs that correlates with their distinct tissue tropism and transduction phenotypes as well as their antigenic reactivities. The overall objective of this proposal is a structure-function analysis of the AAV capsid to identify features that (I) determine differential cell tropism; (II) affect transduction efficiency, (III) and are utilized for cell receptor recognition. The analysis will also provide information on capsid features that are conserved and thus could be important for the fidelity of viral capsid assembly interactions, and on capsid regions that dictate the distinct antigenicity of the AAV clade groups. Genetically manipulating these features could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and specificity, transduction efficiencies, and the ability to evade existing host immune responses. To achieve our objectives, we will determine the capsid structures for representative members of the AAV clade groups, alone and in complex with identified carbohydrate receptors, by X-ray crystallography, and functionally annotate AAV capsid regions involved in the receptor interactions using mutagenesis, cell binding and transduction assays, and biophysical measurements of binding affinity. We have developed a scaleable baculovirus system for the expression of wild type and mutant virus capsids for these studies.

描述（由申请人提供）：腺相关病毒（AAV）与任何疾病及其包装非基因组DNA的能力无关，并使不同的细胞/组织种群转导不同的细胞/组织种群进行纠正基因递送引起了人们对理解其基本生物学的重大兴趣。这包括它们的衣壳结构，细胞向潮流和用于进入，脱落，复制，DNA包装，衣壳组件和抗体中和的相互作用。目的是提高其作为向量的特异性和功效。但是，尽管AAV的大多数表征已针对血清型2（AAV2），但与AAV2相比，对一些最近发现的抗原差异的人类和灵长类动物病毒的研究表明，特定细胞类型的转导性能增强。该特性由它们的衣壳序列介导。因此，在为基因疗法社区提供开发的潜在AAV向量的更广泛选择，而特定的组织/器官靶向提高安全概况和功效以及工程更快的转导开始，可以通过确定其他AAV的capsID特征，从而极大地有助于其与其独特的组织动力抗体和转移态度相关的capsID特征。该提案的总体目的是对AAV CAPSID的结构 - 功能分析，以识别（i）确定差异细胞对流的特征； （ii）影响转导效率，（iii），用于细胞受体识别。该分析还将提供有关保守的衣壳特征的信息，因此对于病毒capsid组装相互作用的保真度以及决定AAV进化枝组的独特抗原性的衣壳区域可能很重要。从遗传上操纵这些特征可能会导致新一代的纠正病毒基因递送载体，并具有协同的组织对流和特异性，转导效率以及逃避现有宿主免疫反应的能力。为了实现我们的目标，我们将通过X射线晶体学单独和与已识别的碳水化合物受体的代表成员确定Capsid结构，并通过X射线晶体学和功能上注释AAV AAV CAPSID，使用诱变，细胞结合和转导测定和生物生物物理测量值的诱变，细胞结合和转导的结合测定和生物物理测量值。我们已经开发了一种可伸缩的杆状病毒系统，用于这些研究的野生型和突变病毒衣壳的表达。