Recombinant AAV Gene Therapy Vector Recombination, Integration, and Genotoxicity

描述（由申请人提供）： 注意力重新集中在基因治疗过程中的潜在遗传毒性和相关不良事件上，这是在用逆转录病毒载体治疗的患者中发展的白血病的结果。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，尽管细胞和转基因之间的后续相互作用以及二次突变使得结果预测复杂。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还没有很好地理解。在不了解rAAV整合的情况下，不可能估计插入激活或大规模染色体重排的风险。我们使用报告系统的rAAV DNA重组事件，我们以前使用的rAAV附加体的形成的特点，探测导致rAAV整合的分子相互作用。基于自身互补rAAV（scAAV）载体，这些报告基因提供了基因检测，而不是DNA产物的物理检测，以给出载体基因组命运的灵敏和准确的定量。我们将继续我们已经进行的研究，以了解发夹末端重复序列（TR）的作用，以及它们与细胞因子在培养细胞中的环化，连环化和整合中的相互作用（目标1）。我们将应用这些载体和方法的整合在小鼠肝组织的研究，强调优先使用特定的TR结构和TR介导的重组DNA代谢抑制剂的影响（目的2）。我们将采用一种新的测定法来确定rAAV在体内整合的速率，而不诱导细胞分裂。最后，我们将使用具有类似于未修饰的逆转录病毒载体的插入激活特性的scAAV载体来测定小鼠肝细胞中的致瘤性（目的3）。我们希望该项目将为我们对rAAV整合的了解提供新的细节水平，并有助于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难以估计，因为体内整合机制和频率未知。我们将使用专门的报告载体来报告在靶细胞中发生了什么类型的DNA重组事件。

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.