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.

抽象的 腺相关病毒 (AAV) 载体正处于临床开发阶段，用于传递基因来治疗多种遗传性疾病 疾病包括血友病。虽然在优化基因传递方面已经取得了进展，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。这些发现 强调需要新的方法来减少 AAV 载体剂量，以尽量减少肝毒性、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已经确定了一个 AAV 载体对有效基因校正的限制未被充分认识。 AAV基因的长期研究 在A型血友病狗体内传递FVIII时，我们发现大多数AAV载体基因组都高度重排 在转导的肝组织中。这些重排通常会破坏转基因，因此会损害 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 转导后不产生功能性蛋白质。这些重新排列的 AAV 基因组存在于 整合形式，但也存在可能是游离形式的 AAV 多联体中。目前尚不清楚这些是否 重排发生在载体生产期间或靶细胞转导之后，尽管数据是 累积至少一些重排起源于载体生产细胞。我们的血友病A狗 研究还发现了犬基因组中与细胞生长和癌症相关的基因中的整合事件 与克隆扩增有关。通过以下方法验证这些扩展克隆中整合的 AAV DNA 序列分析表明，在所有情况下，整合载体都被高度重新排列，只有五分之一 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 尽管 AAV DNA 中宿主因素的影响，载体生产者和靶细胞中的途径 重排大多未被研究。我们假设宿主细胞通路的调节可以抑制 AAV DNA 重排，从而改善每个载体 DNA 拷贝的转基因表达。在这个提案中， 我们将 1) 实施深度测序方法，以统计严格的方式量化重排频率 时尚，2) 识别可以用小分子、siRNA 或 microRNA 调节的细胞通路， 抑制载体重排，3) 设计支持有效途径的新颖递送策略 调制、抑制载体重排并提高每个载体拷贝的转基因输出。这些方法将 在 AAV 载体生产期间（具体目标 1）和 AAV 递送至转导的靶细胞后进行评估 （具体目标 2）。项目结束时我们的交付成果将大大加深对 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 以较低的载体剂量安全有效的基因传递。