Preserving Genome Integrity In AAV-Mediated Gene Therapy

摘要 腺相关病毒(AAV)载体正处于临床开发阶段，用于运送基因治疗多发性遗传病 包括血友病在内的疾病。虽然在优化基因传递方面取得了进展，但在一些研究中 在一项研究中，所需的AAV载体剂量很高，导致毒性甚至致命后果。这些发现 强调需要新的方法来减少AAV载体剂量，以最大限度地减少肝脏毒性，抗AAV 免疫反应和遗传毒性。我们最近的研究和其他人的工作发现了一种 AAV载体对有效基因校正的局限性未得到充分认识。AAV基因的长期研究 在血友病A犬中传递FVIII，我们发现大多数AAV载体基因组高度重排 在转导的肝脏组织中。这些重排通常会扰乱转基因，因此会造成损害。 转基因产物的表达-出乎意料的是，我们的数据表明，大多数AAV载体基因组 转导后不能产生功能蛋白。这些重排的甲型肝炎病毒基因组存在于 整合的形式，但也在AAV串联体中，可能是异体形式。目前还不清楚这些是否 重排发生在载体生产期间或靶细胞转导之后，尽管数据是 累积至少部分重排起源于载体产生细胞。我们的血友病A狗 研究还确定了犬类基因组中与细胞生长和癌症相关的基因内的整合事件 与克隆性扩张有关。对这些扩增克隆中整合的AAV DNA的验证 序列分析表明，在所有情况下，整合的载体都高度重排，只有五个中的一个 对完整的转基因进行编码。大量文献报道AAV与宿主DNA修复的相互作用 载体产生细胞和靶细胞中的通路，尽管宿主因素对AAV DNA的影响 重排大多未被研究过。我们假设宿主细胞通路的调节可以抑制AAV DNA重排，从而提高了每个载体DNA拷贝的转基因表达。在这份提案中， 我们将1)实现深度排序方法来量化重排频率，在统计上严格 时尚，2)识别可由小分子、siRNA或microRNA调节的细胞通路， 抑制载体重排，以及3)设计支持有效途径的新递送策略 调节，抑制载体重排，并提高每个载体拷贝的转基因产量。这些方法将 在AAV载体生产期间(特定目标1)和在转导的靶细胞中传递AAV之后进行评估 (具体目标2)。我们在项目结束时的可交付成果将大大增强对 AAV与宿主细胞DNA处理途径的相互作用，以及调节这些途径以允许 以较低的媒介剂量安全有效地传递基因。

ABSTRACT Adeno-associated virus (AAV) vectors are in clinical development for delivery of genes to treat multiple genetic diseases including hemophilia. While progress has been made to optimize gene delivery, in some studies the required AAV vector doses were high, leading to toxicity and even fatal outcomes in one study. These findings highlight the need for novel approaches to reduce the AAV vector dose to minimize liver toxicity, anti-AAV immune responses, and genotoxicity. Our recent studies and work from others have identified an underappreciated limitation to efficient gene correction with AAV vectors. In a long term study of AAV gene delivery of FVIII in hemophilia A dogs, we found that most of the AAV vector genomes were highly rearranged in transduced liver tissues. These rearrangements typically disrupted the transgene, and so would compromise expression of the transgene product—unexpectedly, our data indicated that most of the AAV vector genomes present did not produce functional protein after transduction. These rearranged AAV genomes were present in integrated forms but also in AAV concatemers that may be episomal forms. It is unclear whether these rearrangements occurred during vector production or after transduction of the target cells, though data is accumulating that at least some of the rearrangements originate in vector producer cells. Our hemophilia A dog study also identified integration events in the canine genome within genes linked to cell growth and cancer that were associated with clonal expansions. Validation of integrated AAV DNA in these expanded clones by sequence analysis showed that in all cases integrated vectors were highly rearranged, with only one of five encoding an intact transgene. An extensive literature documents interactions of AAV with host DNA repair pathways in both vector producer and target cells, though the influence of host factors in AAV DNA rearrangements is mostly unstudied. We hypothesize that modulation of host cell pathways can suppress AAV DNA rearrangements, thereby allowing improved transgene expression per vector DNA copy. In this proposal, we will 1) implement a deep sequencing method to quantify rearrangement frequency in a statistically rigorous fashion, 2) identify cellular pathways that can be modulated with small molecules, siRNAs, or microRNAs that suppress vector rearrangements, and 3) devise novel delivery strategies that support efficient pathway modulation, suppress vector rearrangement, and boost transgene output per vector copy. These methods will be assessed during AAV vector production (Specific Aim 1) and after AAV delivery in the transduced target cells (Specific Aim 2). Our deliverables at the end of the project will be a greatly enhanced understanding of the interaction of AAV with host cell DNA handling pathways, and methods for modulating these pathways to allow safe and effective gene delivery at lower vector doses.