Preserving Genome Integrity In AAV-Mediated Gene Therapy

在 AAV 介导的基因治疗中保持基因组完整性

• 批准号: 10558679
• 负责人: Frederic D Bushman
• 金额: $ 64.52万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10558679
• 关键词: Animal Model; Animals; Attention; Base Sequence; Biology; Blood Coagulation Disorders; CD8-Positive T-Lymphocytes; Canis familiaris; Capsid; Cells; Clonal Expansion; DNA; DNA Integration; DNA Repair; DNA Repair Pathway; DNA Structure; Data; Dependovirus; Dose; Effectiveness; Event; F8 gene; Factor VIII; Fatal Outcome; Frequencies; Gene Delivery; Gene Modified; Gene Rearrangement; Gene therapy trial; Genes; Genetic Diseases; Genome; Genomics; Goals; Hemophilia A; Hepatotoxicity; Holliday Junction Resolvases; Human; Immune response; Insertional Mutagenesis; Integration Host Factors; Inverted Repeat Sequences; Inverted Terminal Repeat; Link; Literature; Liver; Longitudinal Studies; Malignant Neoplasms; Mediating; Methods; MicroRNAs; Modeling; Nature; Nonhomologous DNA End Joining; Outcome; Output; Pathway interactions; Production; Proteins; Reaction; Reporting; Risk; Risk Reduction; Safety; Sampling; Sequence Analysis; Small Interfering RNA; Structure; Therapeutic; Time; Tissues; Toxic effect; Transgenes; Treatment Efficacy; Validation; Viral; Viral Genome; Work; adeno-associated viral vector; cell growth; clinical development; deep sequencing; design; dog genome; experience; gene correction; gene product; gene therapy; genome integrity; genotoxicity; homologous recombination; improved; next generation sequencing; novel; novel strategies; nuclease; particle; preservation; prevent; repaired; small hairpin RNA; small molecule; small molecule inhibitor; therapeutic gene; transduction efficiency; transgene expression; vector; vector genome; viral DNA

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.

抽象的 腺相关病毒（AAV）载体正在临床发育中用于递送基因以治疗多个基因 包括血友病在内的疾病。尽管已经取得了进展来优化基因递送，但在某些研究中 一项研究中所需的AAV载体剂量很高，导致毒性甚至致命的结果。这些发现 强调需要减少AAV载体剂量以最大程度减少肝脏毒性的新方法的需求 免疫反应和遗传毒性。我们最近的研究和其他工作已经确定了 用AAV向量校正有效基因校正的限制不足。在对AAV基因的长期研究中 在血友病A狗中的FVIII传递，我们发现大多数AAV矢量基因组都被高度重新排列 在转导的肝组织中。这些重排通常破坏了转换，因此会妥协 转换产物的表达 - 不预计，我们的数据表明大多数AAV矢量基因组 转导后没有产生功能性蛋白质。这些重新排列的AAV基因组存在 集成形式，但也以aav confemers的形式为偶发形式。目前尚不清楚这些是否 重排发生在矢量产生期间或目标细胞翻译后，尽管数据是 累积至少一些重排起源于矢量生产细胞。我们的血友病是狗 研究还确定了与细胞生长和癌症相关的基因中的犬类基因组中的整合事件 与克隆扩张有关。通过这些扩展的克隆中集成AAV DNA的验证 序列分析表明，在所有情况下，整合的向量都是高度重新排列的，只有五个 编码完整的转换。广泛的文献记录了AAV与宿主DNA修复的相互作用 尽管AAV DNA中宿主因子的影响 重排大多未被研究。我们假设宿主细胞途径的调制可以抑制AAV DNA重排，从而可以改善每个向量DNA拷贝的转化表达。在此提案中， 我们将1）实施一种深层测序方法，以量化统计严格的重排频率 时尚，2）识别可以通过小分子，siRNA或microRNA调节的细胞途径 抑制向量重排，3）制定支持有效途径的新型交付策略 调制，抑制矢量重排和每个向量副本增强变换输出。这些方法将 在AAV矢量产生（特定目标1）和在翻译的目标细胞中递送后进行评估 （特定目标2）。我们在项目结束时的可交付成果将是对 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的方法 较低矢量剂量的安全有效基因输送。