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

• 批准号: 8299123
• 负责人: BRIAN P BOTHNER
• 金额: $ 34.45万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299123
• 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（Adeno-associated viral, rAAV）载体可以介导安全的基因转移，在动物模型中长期纠正遗传疾病，并有效地传递纠正基因，用于治疗人类疾病。它们在人体中是安全的，并且在肺、窦、骨骼肌、脑和肝组织中持续存在。有效地转导不同的细胞/组织群体进行纠正性基因传递的能力引起了人们对了解其基本生物学的极大兴趣。这包括它们的衣壳结构、细胞趋向性和进入、运输、剥离、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效进入细胞和细胞内转运到细胞核进行复制所需的AAV衣壳转移动力学信息。物理、生化和遗传方法将用于鉴定衣壳上参与细胞进入和随后通过内吞途径转运到细胞核过程中发生的结构变化的位点。选取的四种AAV血清型（AAV1, AAV2， AAV5和AAV8）代表了迄今为止观察到的灵长类AAV序列和衣壳结构多样性的频谱，将作为我们提出的研究的模型。具体目标1将利用溶液研究，利用有限的蛋白质水解耦合质谱法来识别和测量动态蛋白质区域。具体目标2将侧重于衣壳转变的晶体可视化，提供一个3D平台，目标1产生的数据可以在其上进行注释。具体目标3将使用生化和遗传方法验证具体目标1和2的观察结果。预计对aav的更好的物理理解，作为本项目的主要目标，可能会产生新一代的纠正性病毒基因传递载体，在组织趋向性和转导效率方面具有协同改善。几种腺相关病毒（rAAV）载体可以介导基因转移以纠正遗传性疾病，目前正处于临床试验阶段。然而，关于允许细胞感染和转运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很少。这些信息的可用性将对开发具有更高功效的下一代重组载体有价值。本项目旨在填补我们基础AAV生物学知识库的这一空白。

项目成果

Fluorometric Estimation of Viral Thermal Stability.

病毒热稳定性的荧光估计。

• DOI: 10.21769/bioprotoc.1199
• 发表时间: 2014
• 期刊: Bio-protocol
• 影响因子: 0.8
• 作者: Rayaprolu,Vamseedhar;Kruse,Shannon;Kant,Ravi;Movahed,Navid;Brooke,Dewey;Bothner,Brian
• 通讯作者: Bothner,Brian

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex.

• DOI: 10.1021/acschembio.7b00649
• 发表时间: 2018-02-16
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: van Erp PBG;Patterson A;Kant R;Berry L;Golden SM;Forsman BL;Carter J;Jackson RN;Bothner B;Wiedenheft B
• 通讯作者: Wiedenheft B