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: 10338480
• 负责人: Frederic D Bushman
• 金额: $ 63.15万
• 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/10338480
• 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 Rearrangement; Gene therapy trial; Gene-Modified; Genes; Genetic Diseases; Genome; Genomics; Goals; Hemophilia A; Hepatotoxicity; Holidays; Human; Immune; Immune response; Insertional Mutagenesis; Integration Host Factors; Inverted Repeat Sequences; Inverted Terminal Repeat; Lead; Link; Literature; Liver; Longitudinal Studies; Malignant Neoplasms; Mediating; Methods; MicroRNAs; Nature; Nonhomologous DNA End Joining; Outcome; Output; Pathway interactions; Production; Proteins; Reaction; Reporting; Resolvase; Risk; 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; repair model; 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.

摘要 腺相关病毒（AAV）载体处于临床开发中，用于递送基因以治疗多种遗传病。 包括血友病在内的疾病。虽然已经在优化基因递送方面取得了进展，但在一些研究中， 所需的AAV载体剂量很高，在一项研究中导致毒性甚至致命的结果。这些发现 强调需要新的方法来减少AAV载体剂量以使肝毒性最小化，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已经确定了一个 未被充分认识的限制是用AAV载体进行有效的基因校正。在对AAV基因的长期研究中， 在血友病A狗中递送FVIII时，我们发现大多数AAV载体基因组高度重排， 在转导的肝组织中。这些重排通常会破坏转基因， 出乎意料的是，我们的数据表明，大多数AAV载体基因组 本发明的细胞在转导后不产生功能蛋白。这些重排的AAV基因组存在于 整合的形式，但也可以是附加型形式的AAV多联体。目前尚不清楚这些 重组发生在载体生产过程中或转导靶细胞后，尽管数据是 积累至少一些重排起源于载体生产细胞。我们的A型血友病狗 这项研究还确定了犬基因组中与细胞生长和癌症相关的基因的整合事件， 与克隆扩张有关。通过以下方法验证这些扩增克隆中整合的AAV DNA： 序列分析表明，在所有情况下，整合载体高度重排，只有一个五 编码完整的转基因。大量文献记录了AAV与宿主DNA修复的相互作用 载体生产者和靶细胞中的途径，尽管宿主因子在AAV DNA中的影响， 重排大多未被研究。我们假设调节宿主细胞通路可以抑制AAV DNA重排，从而允许每个载体DNA拷贝的转基因表达提高。在这一提议中， 我们将1）实施深度测序方法，以统计上严格的方式量化重排频率， 2）鉴定可以用小分子、siRNA或microRNA调节的细胞途径， 抑制载体重排，和3）设计支持有效途径新的递送策略 在一些实施方案中，转基因载体可以通过调节、抑制载体重排和提高每个载体拷贝的转基因输出来实现。这些方法将 在AAV载体生产（特异性目标1）期间和在转导的靶细胞中的AAV递送之后进行评估 （具体目标2）。我们在项目结束时的交付成果将是对 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 在较低的载体剂量下安全和有效的基因递送。