Molecular Virology Core

分子病毒学核心

基本信息

批准号：
10333188
负责人：
WEIDONG XIAO
金额：
$ 39.08万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
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/10333188
关键词：
Baculoviruses Bioinformatics Biological Assay Cells Clinical Clinical Trials Data Data Analyses Data Collection Development Dose Enzyme-Linked Immunosorbent Assay F8 gene 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 Technology Transfection Vertebral column Viral adeno-associated viral vector base design digital experimental study gene therapy genome analysis improved in vivo innovation manufacturing process next generation sequencing novel particle programs success tool transcriptome vector vector control

项目摘要

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.

项目概要/摘要为了了解对 A 型血友病基因治疗产生负面影响的关键因素，我们的计划将研究与 AAV 载体生产和设计、因子 VIII (FVIII) 表达相关的关键问题，和免疫系统的作用。拟议的基因转移研究严重依赖于载体的严格控制载体施用前后载体特性的组成和彻底表征。不幸的是，由于缺乏一些关键知识，载体的深入表征受到阻碍。给定载体制备的参数可能是载体性能的关键。举例来说分析 AAV 载体产品中的杂质（例如有缺陷的病毒颗粒）面临的挑战类似于向量本身。拟议的分子病毒学核心（MVC）的科学家们开发了一系列 AAV 载体的新检测提供了用于分析基因安全性的额外关键参数治疗。这种前所未有的深度分析将为项目 1、2 和 3 提供矢量知识他们需要解释基因转移实验的结果并帮助开发更安全的成分向量。 MVC 将追求以下具体目标： 1) 制造优质载体以支持所有项目：为了使研究和项目之间的结果具有可比性，载体生产的标准相同需要应用。拟议的制造工艺将进一步消除变量，例如使用质粒无骨干生产等创新。此外，MVC 将在矢量方面为项目 1、2 和 3 提供帮助开发，结合新发现并生成改善肝脏 FVIII 基因的新型 AAV 载体送货。 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 测序和新开发的检测方法提供单一 AAV 基因组分析序列数据的生物信息分析。传统的 AAV 载体检测，例如通过 ELISA 测定 AAV 滴度，还将进行 Southern 印迹分析、银染色以及数字 PCR/qPCR。 3）深入分析用于了解分娩后 AAV 基因组状态。下一代将分析 AAV 体内基因组状态测序以及传统的 Southern blot 和 qPCR。 AAV 转录组分析也将包括在内。