Mapping Adeno-associated Virus Capsid Structural and Dynamic Transitions

绘制腺相关病毒衣壳结构和动态转变图

基本信息

批准号：
7634896
负责人：
BRIAN P BOTHNER
金额：
$ 36.52万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
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：
2009-07-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7634896
关键词：
Affect Animal Model Animals Antibodies Biochemical Biochemical Genetics Biological Assay Biology Brain Calisthenic Capsid Capsid Proteins Cell Nucleus Cell surface Cells Cellular Tropism Clinical Clinical Trials Coupled Cryoelectron Microscopy DNA Packaging Data Dependovirus Development Exhibits Gene Delivery Gene Transfer Generations Genes Genome Goals Hereditary Disease Hot Spot Human Hydrolysis Imagery In Vitro Infection Influenza Kinetics Libraries Liver Lung Maps Mass Spectrum Analysis Measures Mediating Modeling Molecular Molecular Conformation Mutate N-terminal Parvovirus Parvovirus Infections Pathway interactions Pattern Performance Phospholipase Phospholipase A2 Physiological Poliomyelitis Polysaccharides Population Primates Process Protein Dynamics Protein Region Proteolysis Recombinants Resolution Rhinovirus Roentgen Rays Serotyping Sinus Site Site-Directed Mutagenesis Skeletal Muscle Solutions Structural Models Structure Temperature Testing Therapeutic Thermodynamics Tissues Tropism Viral Virion Virus X-Ray Crystallography adeno-associated viral vector base cellular transduction human disease improved in vivo interest knowledge base member mutant next generation particle public health relevance receptor receptor binding targeted delivery tissue tropism trafficking transduction efficiency uptake vector viral gene delivery

项目摘要

DESCRIPTION (provided by applicant): Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the long-term correction of genetic diseases in animal models and efficiently deliver corrective genes for the treatment of human diseases. They are safe and persist in humans following delivery to lung, sinus, skeletal muscle, brain, and liver tissue. The ability to efficiently 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, trafficking, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The long- range goal of this project is to obtain information on the AAV capsid transition dynamics required for efficient cell entry and intracellular trafficking to the nucleus for replication. Physical, biochemical, and genetic approaches will be used to identify sites on the capsid that are involved in structural changes that occur during cell entry and subsequent transport to the nucleus via the endocytic pathway. Four selected AAV serotypes (AAV1, AAV2, AAV5, and AAV8) which represent the spectrum of sequence and capsid structural diversity so far observed for the primate AAVs, will serve as our models for the proposed studies. Specific aim 1 will utilize solution studies, employing limited proteolysis to coupled mass spectrometry to identify and measure dynamic protein regions. Specific aim 2 will focus on crystallographic visualization of capsid transitions, providing a 3D platform onto which the data resulting from aim 1 can be annotated. Specific aim 3 will validate the observations from specific aims 1 and 2 using biochemical and genetic approaches. It is anticipated that a better physical understanding of the AAVs, as is the main goal of this project, could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and transduction efficiencies. PUBLIC HEALTH RELEVANCE Several Adeno-associated viral (rAAV) vectors can mediate safe gene transfer for the correction of genetic diseases and are in clinical trials. However, very little information is available on the physical transitions of the protein capsid necessary for permissive cellular infection and trafficking to the nucleus for replication. The availability of this information will be valuable for the development of next generation recombinant vectors with improved efficacy. This project aims to fill this dearth in our knowledge base of basic AAV biology.

描述（申请人提供）：腺相关病毒（rAAV）载体可以介导安全的基因转移，以长期纠正动物模型中的遗传疾病，并有效地传递纠正基因以治疗人类疾病。它们在进入肺、鼻窦、骨骼肌、大脑和肝脏组织后在人体中是安全的并持续存在。有效转导不同细胞/组织群体以进行校正基因传递的能力引起了人们对了解其基本生物学的浓厚兴趣。这包括它们的衣壳结构、细胞向性以及进入、运输、脱壳、复制、DNA 包装、衣壳组装和抗体中和的相互作用。该项目的长期目标是获得有关有效细胞进入和细胞内运输至细胞核进行复制所需的 AAV 衣壳转变动力学的信息。将使用物理、生化和遗传方法来识别衣壳上参与细胞进入和随后通过内吞途径转运到细胞核期间发生的结构变化的位点。四种选定的 AAV 血清型（AAV1、AAV2、AAV5 和 AAV8）代表了迄今为止观察到的灵长类 AAV 的序列谱和衣壳结构多样性，将作为我们拟议研究的模型。具体目标 1 将利用溶液研究，采用有限的蛋白水解与质谱联用来识别和测量动态蛋白质区域。具体目标 2 将重点关注衣壳转变的晶体学可视化，提供一个 3D 平台，可以在该平台上注释目标 1 产生的数据。具体目标 3 将使用生化和遗传学方法验证具体目标 1 和 2 的观察结果。作为该项目的主要目标，预计对 AAV 的更好的物理理解可能会产生新一代的校正病毒基因传递载体，并在组织向性和转导效率方面具有协同改进。公共卫生相关性几种腺相关病毒 (rAAV) 载体可以介导安全基因转移以纠正遗传疾病，并且正在进行临床试验。然而，关于允许细胞感染和运输到细胞核进行复制所必需的蛋白质衣壳的物理转变的信息非常少。这些信息的可用性对于开发具有更高功效的下一代重组载体非常有价值。该项目旨在填补我们 AAV 基础生物学知识库的这一空白。