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（由应用提供）：人类腺病毒（HADV）是未开发的DSDNA病毒，感染了多种细胞类型。物种C HADV5是诊所中最常用的基因疗法和疫苗应用的载体。最近，已经表明，静脉输送后，物种A和C HADV与几个血液凝血因子（例如，X，IX）的关联，使这些病毒可以进入上皮细胞和肝脏。体内肝细胞分别。这种服装因子介导的途径导致C型HADV对肝细胞的意外重新定位。值得注意的是，这种重新定位超出了天然受体（CAR）对病毒纤维蛋白的选择性。通过药理学干预或HAD5 HEXON蛋白的突变阻断己酮-FX相互作用，可以废除体内的肝转移。尽管从冷冻研究中获得了有关HADV-FX关联的低分辨率结构信息，但仍然缺乏FX GLA域与HEXON上的Hexon capsid上的分子相互作用的详细知识。我们最近通过X射线衍射在接近原子分辨率下确定了基于HADV5的载体（指定为AD35F）的晶体结构。尽管有了新的结构信息，但我们仍然缺乏有关病毒capsid如何影响体内组织偏见的重要知识。没有精确的HADV-FX相互作用模型阻碍了抗病毒药和基因治疗载体的发展。在用化学合成的GLA（CS-GLA）浸泡的AD35F晶体上进行的初步衍射实验非常阳性。在6“分辨率的初始FO-FC地图”指标表明，己酮亚基上Th Gla结构域结合位点的足迹与先前建议的位置不同。该提案旨在通过1）通过采用X射线衍射方法来大大提高我们对体内腺病毒宿主细胞对流的理解。这些分析将基于我们在解决AD35F时获得的广泛知识和专业知识的基础，该AD35F的晶体结构为3.5€和2）分析单点突变体（E451Q）的结构（E451Q）的结构，该结构大大降低了FX与HADV结合的FX构成该突变因素与hADV的结合，以使其与HADV的结合进行了封闭。我们预计，这些研究将对肝脏肝细胞腺病毒翻译涉及的机制和潜在的分子相互作用提供更多的了解。