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(申请人提供)：腺相关病毒(RAAV)载体可以介导安全的基因转移，用于长期纠正动物模型中的遗传性疾病，并有效地传递用于治疗人类疾病的纠正基因。它们是安全的，并在人类体内持续存在，并在肺、鼻窦、骨骼肌、脑和肝脏组织中传播。能够有效地转导不同的细胞/组织群体进行纠正基因传递的能力已经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、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AAV衣壳转变动力学的信息，这是有效进入细胞和细胞内转运到细胞核以供复制所需的。物理、生化和遗传方法将被用来确定衣壳上与结构变化有关的位置，这些变化发生在细胞进入细胞并随后通过内吞途径运输到细胞核中。4个AAV血清型(AAV1、AAV2、AAV5和AAV8)代表了迄今观察到的灵长类AAVs的序列和衣壳结构多样性，将作为我们拟议研究的模型。具体目标1将利用溶液研究，使用有限的蛋白质分解耦合质谱学来识别和测量动态蛋白质区域。特定的AIM 2将专注于衣壳转变的晶体可视化，提供一个3D平台，可以在该平台上对AIM 1产生的数据进行注释。具体目标3将使用生化和遗传方法验证从具体目标1和2观察到的结果。预计对AAVs有更好的物理了解，这是该项目的主要目标，可以产生新一代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作用。公共卫生相关性几种腺相关病毒(RAAV)载体可以介导安全的基因转移以纠正遗传病，并正在进行临床试验。然而，关于蛋白质衣壳的物理转变的信息很少，这是允许细胞感染和运输到细胞核进行复制所必需的。这些信息的可获得性将对开发效率更高的下一代重组载体具有价值。本项目旨在填补我国基础AAV生物学知识库中的这一空白。

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