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

• 批准号: 8085833
• 负责人: BRIAN P BOTHNER
• 金额: $ 34.56万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 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/8085833
• 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; Health; 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; receptor; receptor binding; targeted delivery; tissue tropism; trafficking; transduction efficiency; uptake; vector; viral gene delivery

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（由申请人提供）：腺相关病毒（RAAV）向量可以介导安全基因转移，以长期纠正动物模型中的遗传疾病，并有效地赋予治疗人类疾病的纠正基因。他们是安全的，并且在传递到肺，鼻窦，骨骼肌，脑和肝组织后的人类中持续存在。有效地转导不同细胞/组织群以矫正基因递送的能力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，细胞向上主义和用于进入，运输，脱涂，复制，DNA包装，衣壳组件和抗体中和的相互作用。该项目的长距离目标是获取有关有效细胞进入和细胞内运输所需的AAV衣壳过渡动力学的信息，以复制。物理，生化和遗传学方法将用于识别在细胞进入期间发生的结构变化以及随后通过内吞途径转移到细胞核的结构变化的位点。迄今为止，对于灵长类动物AAV所观察到的四个选择的AAV血清型（AAV1，AAV2，AAV5和AAV8），它代表了序列和衣壳结构多样性，将作为我们提出的研究的模型。具体目标1将利用有限的蛋白水解来耦合质谱法以识别和测量动态蛋白质区域。特定的目标2将重点放在衣壳过渡的晶体学可视化上，提供一个3D平台，可以注释AIM 1引起的数据。特定的目标3将使用生化和遗传方法从特定目标1和2验证观察结果。可以预期，对AAV的更好理解，该项目的主要目标也可能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转导效率的协同提高。公共卫生相关性几种与腺相关的病毒（RAAV）向量可以介导安全的基因转移以纠正遗传疾病，并且正在临床试验中。但是，关于允许细胞感染和运输到细胞核进行复制所需的蛋白质衣壳的物理过渡的信息很少。此信息的可用性对于通过提高功效的下一代重组向量的开发将很有价值。该项目旨在填补我们基本AAV生物学知识基础的缺乏。