Molecular Virology Core

分子病毒学核心

• 批准号: 10560536
• 负责人: WEIDONG XIAO
• 金额: $ 38.67万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-05 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10560536
• 关键词: Baculoviruses; Bioinformatics; Biological Assay; Cells; Clinical; Clinical Trials; Data; Data Analyses; Data Collection; Development; Dose; Enzyme-Linked Immunosorbent Assay; Factor VIII; Future; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genome; Goals; Hemophilia A; Hepatic; Human; Immune system; In Vitro; Insecta; Knowledge; Methods; Molecular; Molecular Virology; Outcome; Patients; Performance; Pharmaceutical Preparations; Plasmids; Population Analysis; Preparation; Production; Property; Quality Control; Role; Safety; Scientist; Sequence Analysis; Series; Silver Staining; Southern Blotting; System; Transfection; Vertebral column; Viral; adeno-associated viral vector; data integration; design; digital; experimental study; gene therapy; genome analysis; improved; in vivo; innovation; manufacturing process; manufacturing quality; manufacturing systems; manufacturing technology; next generation sequencing; novel; particle; programs; success; tool; transcriptome; vector; vector control

PROJECT SUMMARY/ABSTRACT To understand the critical factors that negatively impact gene therapy for hemophilia A, our program will investigate the key problems associated with AAV vector production and design, factor VIII (FVIII) expression, and role of the immune system. Proposed gene transfer studies critically rely on strict control of vector composition and thorough characterization of vector properties before and after vector administration. Unfortunately, in-depth characterization of vectors is stymied by the lack of knowledge of some of the critical parameters of a given vector preparation that may be key to vector performance. This is exemplified by challenges in analyzing the impurities within AAV vector products such as defective viral particles that closely resemble the vector itself. The scientists of the proposed Molecular Virology Core (MVC) developed a series of new assays on AAV vectors which provide additional critical parameters to be analyzed for safety of gene therapy. This unprecedented depth of analysis will provide Projects 1, 2, and 3 with the knowledge of vector composition that they require to interpret the outcomes of gene transfer experiments and help develop safer vectors. The MVC will pursue the following Specific Aims: 1) Manufacturing of quality vectors to support all projects: For results between studies and projects to be comparable, identical standards of vector production need to be applied. The proposed manufacturing process will further eliminate variables, e.g., use of plasmid backbone-free production, among other innovations. In addition, MVC will aid Projects 1, 2 and 3 in vector development, incorporating new discoveries and generating novel AAV vectors that improve hepatic FVIII gene delivery. 2) Providing in-depth molecular characterization of AAV vectors for in vitro and in vivo studies: MVC will provide single AAV genome analysis using a newly developed assay based on PacBio sequencing and bioinformatic analysis of the sequence data. Traditional AAV vector assays such as AAV titering by ELISA, Southern blot analysis, silver staining as well as digital PCR/qPCR will also be performed. 3) In-depth analysis for AAV genome status post-delivery. AAV genomes status in vivo will be analyzed by next generation sequencing as well as traditional Southern blot and qPCR. AAV transcriptome analysis will also be included.

项目摘要/摘要 要了解对基因治疗对血友病A的负面影响的关键因素，我们的计划将 研究与AAV矢量产生和设计相关的关键问题，VIII因子（FVIII）表达， 和免疫系统的作用。拟议的基因转移研究严重依赖于对载体的严格控制 载体给药之前和之后的矢量特性的组成和彻底表征。 不幸的是，对矢量的深入表征由于缺乏对某些关键的知识而受到阻碍 给定向量准备的参数可能是向量性能的关键。这是由 分析AAV矢量产物中的杂质（例如有缺陷的病毒颗粒）的挑战 类似于向量本身。拟议的分子病毒核心（MVC）的科学家开发了一系列 在AAV矢量上的新测定，该测定法提供了供基因安全性分析的其他关键参数 治疗。这个空前的分析深度将为项目1、2和3提供矢量的知识 他们需要解释基因转移实验的结果并帮助发展更安全的组成 向量。 MVC将追求以下特定目的：1）制造质量向量以支持所有人 项目：对于研究和项目之间的结果，矢量生产的相同标准相同 需要应用。拟议的制造过程将进一步消除变量，例如使用质粒 无骨干场的生产以及其他创新。此外，MVC还将在矢量中帮助项目1、2和3 开发，结合​​新发现并产生新颖的AAV矢量，以改善肝FVIII基因 送货。 2）为体外和体内研究提供AAV载体的深入分子表征： MVC将使用基于PACBIO测序和 序列数据的生物信息学分析。传统的AAV矢量测定法，例如Elisa的AAV滴度， 还将进行Southern印迹分析，银色染色以及数字PCR/QPCR。 3）深入分析 用于AAV基因组状态后。下一代将对体内的AAV基因组状态进行分析 测序以及传统的Southern印迹和QPCR。还将包括AAV转录组分析。