Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry

腺相关病毒基因治疗载体：进入细胞时的分子相互作用

基本信息

批准号：
9277018
负责人：
MICHAEL S. CHAPMAN
金额：
$ 71.34万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9277018
关键词：
Adverse effects Affinity Arginine Back Binding Biological Models Calibration Cell Line Cell surface Cells Complex Cryoelectron Microscopy Crystallization DNA delivery Dependovirus Development Disease Electron Microscopy Feeds Foundations Gene Transduction Agent Genes Genetic Goals Haploid Cells Hemophilia A Hereditary Disease Hybrids Intervention Ion Channel Laboratories Ligand Binding Ligands Malignant Neoplasms Maps Mediating Methodology Methods Modeling Molecular Analysis Molecular Conformation Motion Multienzyme Complexes Mutagenesis Names Nuclear Pharmacologic Substance Phenotype Phospholipase A2 Phosphotransferases Polysaccharides Predisposition Protein Dynamics Proteins Publishing Relaxation Research Residual state Resistance Resolution Ribosomes Roentgen Rays Serotyping Small Interfering RNA Specificity Structure TNFSF5 gene Testing Uncertainty Validation Viral Work base cell type crosslink data exchange extracellular feeding flexibility improved insight method development molecular pathology mutant public health relevance receptor restraint structural biology targeted treatment trafficking trans-Golgi Network virus host interaction

项目摘要

Project Description Adeno-associated virus (AAV) is a leading gene therapy vector for delivery of DNA to correct genetic errors or predispositions to disease. Our objective is an understanding of the virus-host interactions that mediate cell entry. This foundation will support the widely sought goal of manipulating cell specificity, efficiently transducing desired cells and reducing off-target side effects. This goal has motivated extensive genetic and phenotypic characterization, which, with structure, provide an exceptional paradigm for general insights into viral entry. Our binding and siRNA studies cast doubt on published co-receptors, motivating a gene trap screen in a haploid cell line. The gene most frequently hit in AAV-resistant mutant cells, encoded a protein that we named AAVR which binds with nM affinity, inhibits transduction, and is essential for all AAV serotypes and cell types tested. We have expressed the AAV-binding domains, and will determine a structure of the complex by cryo- electron microscopy (EM) before further characterizing the interactions through mutagenesis. Other genes were implicated by the screen. We will determine which encoded proteins interact with AAV or AAVR through photo-induced cross-linking, pull-down, and mass spectrometric identification of interacting domains. These will be expressed for binding analysis, and hybrid x-ray/EM structure. The gene trap indicates potential partners, not just at the cell surface, but during AAV's trafficking to the peri-nuclear trans Golgi network, and which will illuminate how AAV's phospholipase A2 domain is released for endosomal escape. EM methods, applicable to AAV, will be developed for general use. For refinement of hybrid structures, our map-fitting optimization will incorporate parsimony restraints on model flexibility to avoid the common problem of over-fitting at intermediate resolution. Difference map analysis will be improved, through model-based calibration, to enhance the sensitivity with which small ligands and subtle conformational changes can be analyzed by high resolution EM. This will be applicable to studies of AAV's attachment to extracellular glycans, or to the binding of ligands to ion channels, ribosomes or enzyme complexes in other laboratories. Within the MIRA framework, contributions to collaborative studies of protein dynamics will continue. These are integrating crystal structure with NMR relaxation dispersion and residual dipolar couplings to characterize rate- limiting milli-/micro-second protein motions in arginine kinase as it turns over. Our model system is illuminating poorly understood general principles of intrinsic motions and conformational selection. These fundamental questions are the primary goal, but the research also feeds back into methods development. The NMR relaxation exchange data allows validation tests that our parsimonious model parameterization captures real conformational changes. In summary, we will continue to work on challenging structural biology with both fundamental and applied goals that drive the development of widely applicable methodology.

项目描述腺相关病毒 (AAV) 是一种领先的基因治疗载体，用于传递 DNA 以纠正遗传错误或疾病倾向。我们的目标是了解介导细胞的病毒与宿主的相互作用入口。该基金会将支持广泛寻求的操纵细胞特异性、有效转导的目标所需的细胞并减少脱靶副作用。这一目标激发了广泛的遗传和表型研究表征，其结构为病毒进入的一般见解提供了一个特殊的范例。我们的结合和 siRNA 研究对已发表的共受体提出了质疑，激发了基因陷阱筛选单倍体细胞系。 AAV 抗性突变细胞中最常受到攻击的基因编码了一种我们命名为的蛋白质 AAVR 以 nM 亲和力结合，抑制转导，对所有 AAV 血清型和细胞类型至关重要已测试。我们已经表达了 AAV 结合域，并将通过冷冻确定复合物的结构电子显微镜（EM），然后通过诱变进一步表征相互作用。其他基因也受到屏幕的影响。我们将确定哪些编码蛋白与 AAV 或 AAVR 通过光诱导交联、下拉和相互作用的质谱鉴定域。这些将被表达用于结合分析和混合 X 射线/EM 结构。基因陷阱表明潜在的伙伴，不仅在细胞表面，而且在 AAV 运输到核周反高尔基体的过程中网络，这将阐明 AAV 的磷脂酶 A2 结构域如何释放以进行内体逃逸。适用于 AAV 的 EM 方法将被开发用于通用用途。为了细化混合结构，我们的地图拟合优化将结合模型灵活性的简约限制，以避免常见问题中间分辨率下的过度拟合。通过基于模型的方法将改进差异图分析校准，以提高小配体和细微构象变化的灵敏度通过高分辨率 EM 进行分析。这将适用于 AAV 与细胞外聚糖附着的研究，或其他实验室中配体与离子通道、核糖体或酶复合物的结合。在 MIRA 框架内，对蛋白质动力学合作研究的贡献将继续。这些都是将晶体结构与 NMR 弛豫色散和残余偶极耦合相结合，以表征速率限制精氨酸激酶翻转时的毫秒/微秒蛋白质运动。我们的模型系统很有启发性对内在运动和构象选择的一般原理知之甚少。这些基本的问题是主要目标，但研究也反馈到方法的开发中。核磁共振松弛交换数据允许验证测试，我们的简约模型参数化捕获真实的构象变化。总之，我们将继续致力于具有挑战性的结构生物学推动广泛适用的方法论发展的基本目标和应用目标。