Molecular interactions between soluable host factors and a gene delivery vector

可溶性宿主因子与基因传递载体之间的分子相互作用

基本信息

批准号：
8731791
负责人：
VIJAY S REDDY
金额：
$ 23.69万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-10 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8731791
关键词：
Acute Adenovirus Infections Adenoviruses Affinity Antiviral Agents Binding Blood Blood Circulation Blood Coagulation Factor Body Fluids CAR receptor Capsid Cells Clinic Complex Cryoelectron Microscopy Data Defect Development Double Stranded DNA Virus Epithelial Cells Factor X Fiber Gene Delivery Gene Transduction Agent Glutamic Acid Heparan Sulfate Proteoglycan Hepatocyte Human Adenovirus Infections Human Adenoviruses Immune response Infection Integration Host Factors Intervention Intravenous Investigation Knowledge Ligand Binding Domain Light Liver Location Maps Mediating Methods Modeling Molecular Mutation Pathway interactions Peptides Physiological Point Mutation Protease Domain Proteins Resolution Respiratory Mucosa Saliva Serine Protease Structure Surface Tropism Vaccines Virus X ray diffraction analysis X-Ray Diffraction base carboxylate cell type gene therapy improved in vivo mutant public health relevance research study respiratory tissue tropism vector vector vaccine

项目摘要

DESCRIPTION (provided by applicant): Human adenoviruses (HAdV) are nonenveloped dsDNA viruses that infect a variety of cell types. Species C HAdV5 is the most commonly used vector for gene therapy and vaccine applications in the clinic. Recently, it has been shown that association of species A and C HAdVs with several blood coagulation factors (e.g., Factors X, IX) following intravenous delivery allow these viruses to gain access to epithelial cells and liver hepatocytes in vivo, respectively. This clotting factor-mediated pathway results in the unintended retargeting of C-type HAdVs to hepatocytes. Remarkably, this retargeting overrides the selectivity of natural receptor (CAR) for the virus fiber protein. Blocking of the hexon-FX interaction, either by pharmacological intervention or mutation of HAd5 hexon protein, abolishes liver transduction in vivo. Although low-resolution structural information on HAdV-FX association is available from cryoEM studies, detailed knowledge on the molecular interactions between the GLA-domain of FX with the hexon on the HAd capsid is still lacking. We recently determined the crystal structure for a HAdV5 based vector, designated Ad35F, at near atomic resolution by X- ray diffraction. Despite this new structural information, we still lack important knowledge of how the virus capsid influences tissue tropism in vivo. The absence of an accurate model of HAdV-FX interactions hampers the development of antivirals and gene therapy vectors. Preliminary diffraction experiments on Ad35F crystals soaked with chemically synthesized GLA (cs-GLA) peptide have been quite positive. Initial Fo-Fc maps at 6¿ resolution indicate that footprint of th GLA domain binding site on the hexon subunits is different from the location previously suggested. This proposal seeks to greatly improve our understanding of adenovirus host cell tropism in vivo by 1) determining the structure of HAdV in complex with the FX-GLA domain at near atomic resolution by employing X-ray diffraction methods. These analyses will build on the extensive knowledge and expertise that we gained in solving the crystal structure of Ad35F at 3.5 ¿ resolution and 2) analyzing the structure of single point mutant (E451Q) in the hexon subunit that drastically reduces FX binding to HAdV suggesting that the region of the hexon containing this mutation is crucial for clotting factor association. We anticipate that these investigations will provide greater understanding of mechanism and underlying molecular interactions involved in adenovirus transduction of liver hepatocytes.

描述（由申请人提供）：人类腺病毒（HAdV）是感染多种细胞类型的无包膜双链 DNA 病毒，HAdV5 是临床上最常用的基因治疗和疫苗应用载体。静脉注射后，A 种和 C 种 HAdV 与多种凝血因子（例如因子 X、IX）的关联使这些病毒能够获得上皮细胞和肝脏这种凝血因子介导的途径导致 C 型 HAdV 意外重新定位至肝细胞，值得注意的是，这种重新定位超越了天然受体 (CAR) 对病毒纤维蛋白的选择性。尽管可以从冷冻电镜中获得有关 HAdV-FX 关联的低分辨率结构信息，但通过药物干预或 HAd5 六邻体蛋白突变，相互作用会消除体内肝脏转导。研究表明，关于 FX 的 GLA 结构域与 HAd 衣壳上的六邻体之间分子相互作用的详细知识仍然缺乏，我们最近通过 X 射线衍射确定了基于 HAdV5 的载体（指定为 Ad35F）的晶体结构。尽管有了这些新的结构信息，我们仍然缺乏关于病毒衣壳如何影响体内组织向性的重要知识，缺乏 HAdV-FX 相互作用的准确模型阻碍了抗病毒药物和基因治疗的发展。对浸有化学合成 GLA (cs-GLA) 肽的 Ad35F 晶体进行的初步衍射实验在 6¿ 处的初始 Fo-Fc 图谱非常积极。分辨率表明六邻体亚基上的 GLA 结构域结合位点的足迹与之前建议的位置不同。该提议旨在通过 1) 与 FX 复合物中的 HAdV 的决定结构来极大地提高我们对体内腺病毒宿主细胞向性的理解。 -GLA 域通过采用 X 射线衍射方法进行近原子分辨率分析，这些分析将建立在我们在解析 3.5 ¿ 的 Ad35F 晶体结构过程中获得的广泛知识和专业知识的基础上。分辨率和2）分析六邻体亚基中的单点突变体（E451Q）的结构，该突变体显着减少FX与HAdV的结合，这表明包含该突变的六邻体区域对于凝血因子关联至关重要，我们预计这些研究将提供更大的结果。了解肝细胞腺病毒转导的机制和潜在分子相互作用。