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Recombinant AAV Gene Therapy Vector Recombination, Integration, and Genotoxicity

重组 AAV 基因治疗载体重组、整合和基因毒性

基本信息

批准号：
7487331
负责人：
Douglas M McCarty
金额：
$ 30.43万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7487331
关键词：
Adverse event Affect Animals Attention Binding Biological Assay Cell Cycle Cell division Cells Chromosomal Rearrangement Complex Cultured Cells DNA DNA Damage DNA Double Strand Break DNA Repair Detection Episome Evaluation Event Frequencies Gene Transduction Agent Genetic Genetic Recombination Genetic Transcription Green Fluorescent Proteins Hepatocyte In Vitro Insertional Activations Interphase Cell Knowledge Lead Liver Measures Mediating Metabolism Methods Modeling Mus Muscle Mutagens Mutation Neuraxis Numbers Oncogenic Organ Transplantation Outcome Partial Hepatectomy Patients Pharmaceutical Preparations Principal Investigator Process Property Rate Reading Recombinant adeno-associated virus (rAAV)Recombinants Reporter Reporting Research Research Personnel Retroviral Vector Risk Risk Assessment Risk Estimate Role SCID Mice Standards of Weights and Measures Structure System Terminal Repeat Sequences Testing Therapeutic Tissues Transfection Transgenes Tumorigenicity Virus Integration adeno-associated viral vector base chemotherapy gene therapy genotoxicity in vitro Model in vivo inhibitor/antagonist leukemia monomer repaired tumor tumorigenesis vector vector genome viral DNA

项目摘要

DESCRIPTION (provided by applicant): Attention has re-focused on the potential for genotoxicity and related adverse events in the course of gene therapy as a consequence of leukemias that developed in patients treated with retroviral vector. The potential for genotoxicity from retroviral vectors can be understood on the basis of known integration rates, though subsequent interactions between cell and transgene, as well as secondary mutations, make prediction of outcome complex. In contrast, recombinant adeno-associated virus (rAAV) gene therapy vector integration is not well understood in terms of frequency, mechanism, or possible effects on adjacent chromosmal sequnces. Without an understanding of rAAV integration, it is not possible to estimate the risks of either insertiohal activation or large-scale chromosomal rearrangements. We use a reporter system for rAAV DNA recombination events, which we have previously used to characterize the formation of rAAV episomes, to probe the molecular interactions that lead to rAAV integration. Based on the selfcomplementary rAAV (scAAV) vector, these reporters provide genetic detection, rather than physical detection of DNA products, to give a sensitive and accurate quantification of the fate of the vector genomes. We will continue studies that we have already undertaken in understanding the role of the hairpin terminal repeat (TR) sequences, and their interactions with cellular factors in circularization, concatemerization, and integration in cultured cells (Aim 1). We will apply these vectors and methods to the study of integration in mouse liver tissue, emphasizing the preferential use of specific TR structures and the effects of inhibitors of DNA metabolism on TR-mediated recombinations (Aim 2). We will employ a new assay to determine the rate of rAAV integration in vivo without induction of cell division. Finally, we will use an scAAV vector with insertional activation properties similar to unmodified retrovirus vectors to assay for tumorigenicity in mouse hepatocytes (Aim 3). We expect that this project will provide a new level of detail to our knowledge of rAAV integration and help pave the way to the use of these vectors with a clear understanding of any potential risks. Public: The risk of genotoxicity from rAAV gene therapy vectors is difficult to estimate because the mechanism of integration and the frequency in vivo are unknown. We will use specialized reporter vectors to report what types of DNA recombination events have taken place in the target cells.

描述（由申请人提供）：