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: 7922351
• 负责人: Mavis Agbandje-Mckenna
• 金额: $ 15.63万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922351
• 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(申请人提供)：腺相关病毒(AAV)与任何疾病无关，它们包装非基因组DNA和转导不同细胞/组织群体以进行纠正基因传递的能力引起了人们对了解它们的基本生物学的浓厚兴趣。这包括它们的衣壳结构、细胞取向和进入、脱壳、复制、DNA包装、衣壳组装和抗体中和的相互作用。目的是提高它们作为载体的特异性和有效性。然而，虽然大多数AAVs的特征是针对2型(AAV2)的，但对一些最近发现的抗原性不同的人类和灵长类病毒的研究表明，与AAV2相比，特定细胞类型的转导特性更强。这一特性是由它们的衣壳序列介导的。因此，在为基因治疗界提供更广泛的潜在AAV载体选择的同时，通过识别其他AAVs的衣壳特征将大大有助于提高安全性和有效性的特定组织/器官靶向以及设计更快的转导开始，这些特征与它们不同的组织趋向性和转导表型以及它们的抗原反应性相关。本提案的总体目标是对AAV衣壳进行结构-功能分析，以确定以下特征：(1)确定不同的细胞嗜性；(2)影响转导效率；(3)并用于细胞受体识别。该分析还将提供关于衣壳特征的信息，这些特征是保守的，因此对于病毒衣壳组装相互作用的保真度可能是重要的，以及关于决定AAV分支群独特抗原性的衣壳区域。对这些特征进行基因操作可以产生新一代纠正性病毒基因递送载体，在组织趋向性和特异性、转导效率以及逃避现有宿主免疫反应方面具有协同改善作用。为了实现我们的目标，我们将通过X射线结晶学确定AAV分支成员中具有代表性的成员的衣壳结构，并通过诱变、细胞结合和转导试验以及结合亲和力的生物物理测量从功能上注释参与受体相互作用的AAV衣壳区域。我们已经开发了一种可扩展的杆状病毒系统，用于表达野生型和突变型病毒衣壳，用于这些研究。