Molecular Virology Core

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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.