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

• 批准号: 7131316
• 负责人: Douglas M McCarty
• 金额: $ 31.95万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 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/7131316
• 关键词: DNA; DNA damage; Retroviridae; SCID mouse; cell cycle; emotions; gene therapy; genome; hepatectomy; liver; liver cells; model; quality of life; reading; role; tissue /cell culture; tissues; transfection

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）： 人们的注意力重新集中在基因治疗过程中由于逆转录病毒载体治疗的患者出现白血病而可能产生的基因毒性和相关不良事件。逆转录病毒载体的潜在遗传毒性可以根据已知的整合率来理解，尽管随后细胞和转基因之间的相互作用以及二次突变使结果的预测变得复杂。相比之下，重组腺相关病毒（rAAV）基因治疗载体整合的频率、机制或对相邻染色体序列的可能影响尚不清楚。如果不了解 rAAV 整合，就不可能估计插入激活或大规模染色体重排的风险。我们使用 rAAV DNA 重组事件的报告系统（我们之前曾用它来表征 rAAV 附加体的形成）来探测导致 rAAV 整合的分子相互作用。基于自互补 rAAV (scAAV) 载体，这些报告基因提供基因检测，而不是 DNA 产物的物理检测，从而对载体基因组的命运进行灵敏而准确的定量。我们将继续我们已经开展的研究，了解发夹末端重复 (TR) 序列的作用，以及它们在培养细胞中环化、多联化和整合中与细胞因子的相互作用（目标 1）。我们将应用这些载体和方法来研究小鼠肝组织中的整合，强调优先使用特定的TR结构以及DNA代谢抑制剂对TR介导的重组的影响（目标2）。我们将采用一种新的测定法来确定 rAAV 在体内的整合率，而不诱导细胞分裂。最后，我们将使用具有与未修饰的逆转录病毒载体类似的插入激活特性的 scAAV 载体来测定小鼠肝细胞的致瘤性（目标 3）。我们期望这个项目将为我们对 rAAV 整合的了解提供一个新的细节水平，并帮助为使用这些载体铺平道路，同时清楚地了解任何潜在风险。公众：rAAV 基因治疗载体的遗传毒性风险很难估计，因为整合机制和体内频率尚不清楚。我们将使用专门的报告载体来报告靶细胞中发生的 DNA 重组事件类型。