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.

摘要