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 在过去的几十年中，听觉系统中基因发现的令人印象深刻的进步，使得特定 在听力和耳聋领域中，有针对性的治疗方法实现现实目标。听力 损失（HL）在全球人口中越来越重大的健康问题，应有的比例很大 遗传原因。鉴于健康负担和正在进行的快速发现，现在必须追求新的 特定的早期干预措施的策略，可以预防或减轻HL的严重程度和进展。一个这样的 疾病是DFNA9，这是一种由Coch突变引起的，具有平衡功能障碍的成人感觉神经性HL， 编码Cochlin，是内耳中最丰富的蛋白质。该疾病模型类似于 代表大多数遗传性HL疾病，具有主导性的继承方式和有害 突变蛋白的功能获得。 该提案的目标1和2涉及使用强大而多功能的CRISPR-CAS9基因编辑 特定靶向和破坏主要错义病原体Coch变种的技术P.G88E 和P.A449T。我们将利用这两种变体的患者的人类成纤维细胞，并得出多能 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 对于科克致病变异的等位基因特异性基因破坏，同时使正常的等位基因完整，并且 功能。器官将被评估为阐明Coch生物学的体外系统 DFNA9颞骨的聚集疗法。此外，我们将利用两只Coch敲入（Ki）小鼠 具有这些变体的模型，用于在内耳内实现靶向体细胞组织基因。这些 方法将不仅为DFNA9建立基因编辑的方法论，还为更广泛的类别建立 其他HL疾病具有主要的病理功能获得机制，其最终目标是翻译的目标 进行临床试验和治疗干预。