Genetic Approach to Therapy for DFNA9

DFNA9 的基因治疗方法

• 批准号: 10681990
• 负责人: Cynthia Casson Morton
• 金额: $ 77.41万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-20 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681990
• 关键词: Adult; Age; Alleles; Auditory system; Biological; Biology; Biopsy; Boston; CRISPR/Cas technology; Categories; Characteristics; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Dermal; Development; Disease; Disease model; Early Intervention; Engineering; Enzymes; Equilibrium; Evaluation; Fibroblasts; Functional disorder; GJB2 gene; Gene Delivery; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Guide RNA; Health; Hearing; Heterozygote; Histologic; Human; In Vitro; Individual; Injections; Knock-in; Knock-in Mouse; Laboratories; Labyrinth; Mediating; Mesenchymal; Messenger RNA; Methodology; Methods; Modeling; Mus; Mutation; Nonsense-Mediated Decay; Organism; Organoids; Pathogenicity; Pathology; Patients; Pediatric Hospitals; Phenotype; Plasmids; Pluripotent Stem Cells; Population; Procedures; Proteins; Protocols documentation; Reagent; Research; Resources; Safety; Sensorineural Hearing Loss; Severities; Site; Skin; Specificity; System; Technology; Temporal bone structure; Testing; Therapeutic; Therapeutic Intervention; Time; Tissue Sample; Tissues; Transcript; Transfection; Treatment Efficacy; Variant; cell type; deafness; design; design and construction; design verification; early onset; equilibration disorder; experimental study; gain of function; gain of function mutation; gene correction; gene discovery; gene therapy; genetic approach; hearing impairment; hereditary hearing loss; in vivo; induced pluripotent stem cell; inner ear development; innovation; insertion/deletion mutation; interest; mRNA Expression; mouse model; mutant; next generation sequencing; novel strategies; postnatal; preservation; prevent; progressive hearing loss; round window; safety assessment; success; tool development; transduction efficiency; translational goal; vector

ABSTRACT/PROJECT SUMMARY Impressive advances in gene discovery in the auditory system have occurred in the last decades, making specific targeted approaches for therapeutics realistic goals of great interest in the field of hearing and deafness. Hearing loss (HL) is an increasingly significant health problem in populations worldwide, with a substantial proportion due to genetic causes. Given the health burden and ongoing rapid discoveries, it is now essential to pursue new strategies for specific early interventions that could prevent or mitigate severity and progression of HL. One such disorder is DFNA9, an adult-onset sensorineural HL with balance dysfunction, caused by mutations in COCH, encoding cochlin, the most abundantly detected protein in the inner ear. This disease model is similar to and representative of the majority of genetic HL disorders with a dominant mode of inheritance and with a deleterious gain-of-function of the mutant protein. Aims 1 and 2 of this proposal involve utilization of the powerful and versatile CRISPR-Cas9 gene editing technology for specific targeting and disruption of the dominant missense pathogenic COCH variants p.G88E and p.A449T. We will utilize human fibroblasts from patients with these two variants, and derived pluripotent stem cells and organoids. These biological resources will serve as tools for the development of effective methods for allele-specific gene disruption of the COCH pathogenic variants, while leaving the normal allele intact and functional. The organoids will be assessed as a possible in vitro system for elucidating the biology of COCH aggregates pathognomonic of DFNA9 temporal bones. Furthermore, we will utilize two Coch knock-in (KI) mouse models with these variants for implementation of somatic tissue gene targeting in the inner ear. These approaches will establish methodologies for gene editing not only for DFNA9, but also for a broader category of other HL disorders with a dominant gain-of-function mechanism of pathology, with the ultimate goal of translation to clinical trials and therapeutic intervention.

摘要/项目摘要 过去几十年来，听觉系统基因发现取得了令人印象深刻的进展，使得特定的 听力和耳聋领域非常感兴趣的治疗现实目标的针对性方法。听力 损失（HL）是全世界人口中一个日益严重的健康问题，其中很大一部分是由于 遗传原因。考虑到健康负担和持续的快速发现，现在必须寻求新的 可以预防或减轻 HL 严重程度和进展的特定早期干预策略。这样的一位 疾病是 DFNA9，一种成人发病的感音神经性 HL，具有平衡功能障碍，由 COCH 突变引起， 编码耳蜗蛋白，这是内耳中检测到的最丰富的蛋白质。该疾病模型类似于 代表大多数遗传性 HL 疾病，具有显性遗传模式和有害性 突变蛋白的功能获得。 该提案的目标 1 和 2 涉及利用强大且多功能的 CRISPR-Cas9 基因编辑 用于特异性靶向和破坏显性错义致病性 COCH 变异 p.G88E 的技术 和p.A449T。我们将利用来自具有这两种变异的患者的人类成纤维细胞，并衍生出多能性 干细胞和类器官。这些生物资源将作为开发有效方法的工具 用于 COCH 致病变异的等位基因特异性基因破坏，同时保持正常等位基因完好无损 功能性的。这些类器官将作为一种可能的体外系统进行评估，以阐明 COCH 的生物学特性 DFNA9 颞骨的特征性聚集。此外，我们将利用两只 Coch 敲入 (KI) 小鼠 具有这些变体的模型用于在内耳中实施体细胞组织基因靶向。这些 方法将不仅为 DFNA9 建立基因编辑方法，还为更广泛的类别建立基因编辑方法。 其他具有主要病理功能获得机制的 HL 疾病，其最终目标是转化 临床试验和治疗干预。