Molecular Virology Core

项目总结/摘要 为了了解对血友病A基因治疗产生负面影响的关键因素，我们的项目将 研究与AAV载体生产和设计，因子VIII（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严格控制载体 在载体施用之前和之后，对载体的组成和载体性质进行彻底表征。 不幸的是，由于缺乏对一些关键因素的了解， 给定载体制剂的参数可能是载体性能的关键。这方面的例子有： 在分析AAV载体产物中的杂质，例如与AAV载体紧密结合的缺陷病毒颗粒， 类似于向量本身。提出分子病毒学核心（MVC）的科学家开发了一系列 AAV载体的新测定法，其提供了用于分析基因安全性的额外关键参数 疗法这种前所未有的分析深度将为项目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 向量。MVC将追求以下具体目标：1）制造质量矢量，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，采用相同的病媒生产标准 需要应用。拟定的制造工艺将进一步消除变量，例如，使用质粒 无骨架生产，以及其他创新。此外，MVC将以矢量方式帮助项目1、2和3 开发，纳入新发现并产生改善肝FVIII基因的新型AAV载体 交付. 2)为体外和体内研究提供AAV载体的深入分子表征： MVC将使用新开发的基于PacBio测序的测定法提供单一AAV基因组分析， 序列数据的生物信息学分析。传统的AAV载体测定如通过ELISA的AAV滴定， 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AAV基因组递送后的状态。下一代将分析体内AAV基因组状态 测序以及传统的Southern印迹和qPCR。还将包括AAV转录组分析。

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.