Molecular Virology Core

项目摘要/摘要 为了了解负面影响血友病A基因治疗的关键因素，我们的计划将 研究与AAV载体生产和设计、因子(FVIII)表达、 以及免疫系统的作用。拟议的基因转移研究关键依赖于对载体的严格控制 病媒给药前后病媒性质的组成和彻底表征。 不幸的是，由于缺乏一些关键的知识，对载体的深入描述受到了阻碍 给定载体制备的参数可能是载体性能的关键。这方面的例证是 在分析AAV载体产品中的杂质方面面临的挑战，例如接近 类似于向量本身。提出的分子病毒学核心(MVC)的科学家们开发了一系列 为基因安全性分析提供额外关键参数的AAV载体的新检测方法 心理治疗。这种前所未有的深度分析将为项目1、2和3提供向量的知识 他们需要的成分来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)制造质量向量以支持所有 项目：为了使研究和项目之间的结果具有可比性，病媒生产的标准相同 需要进行涂抹。拟议的生产工艺将进一步消除各种变量，例如，使用质粒 无主干生产，以及其他创新。此外，MVC将在向量中帮助项目1、2和3 开发、整合新发现并产生改进肝脏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将使用一种新开发的基于PacBio测序和 序列数据的生物信息学分析。传统的AAV载体检测方法，例如通过酶联免疫吸附试验进行AAV滴定， 还将进行Southern印迹分析、银染以及数字聚合酶链式反应/定量聚合酶链式反应。3)深入分析 甲型肝炎病毒产后基因组状态。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。AAV转录组分析也将包括在内。

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.