Development of CRISPR/Cas9-based exon-skipping strategies for the treatment of USH-associated deafness

开发基于 CRISPR/Cas9 的外显子跳跃策略来治疗 USH 相关耳聋

• 批准号: 10445638
• 负责人: Zheng-Yi Chen
• 金额: $ 72.75万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-22 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445638
• 关键词: Affect; Animal Model; Auditory; Biological Assay; Blindness; Budgets; CRISPR/Cas technology; Cell Line; Cell Therapy; Cell model; Cells; Clinical; Clinical Trials; Cochlear Implants; Code; Communication; Coping Skills; Data; Development; Electrophysiology (science); Engineering; Excision; Exons; Foundations; Gene Expression Profiling; Gene Transduction Agent; Generations; Genes; Genetic Diseases; Genomic DNA; Genomics; Goals; Guide RNA; Hair; Hair Cells; Hearing; Hereditary Disease; Human; Inherited; Inner Hair Cells; Knockout Mice; Labyrinth; Masks; Mediating; Messenger RNA; Modeling; Mus; Mutation; National Institute on Deafness and Other Communication Disorders; Nature; Organ of Corti; Organoids; Patients; Persons; Phenotype; Prevalence; Production; Proteins; Protocols documentation; Recovery; Research; Retina; Severities; Social Distance; Structure; Symptoms; Technology; Testing; Therapeutic; Time; Transgenic Organisms; USH1D gene; USH2A gene; United States; Usher Syndrome; Wild Type Mouse; Work; Zebrafish; autosomal recessive trait; base; clinical application; clinical subtypes; deaf; deafness; design; early childhood; exon skipping; frontier; genetic deafness; genome editing; hearing restoration; hereditary hearing loss; improved; in vivo; induced pluripotent stem cell; mouse model; mutant; novel; novel therapeutic intervention; pandemic disease; pre-clinical; protein structure; therapeutic genome editing; therapeutically effective; therapy development; tool; treatment strategy; whole genome

Abstract: Usher syndrome (USH) is the most common form of inherited deaf-blindness, with a prevalence of 1/6.000. Inherited as an autosomal recessive trait, it affects about 15,000 people in the United States and is responsible for 6% of early childhood deafness. Usher syndrome is classified under three clinical subtypes (USH-1, -2 and -3) according to the severity of the symptoms. Approximately 2/3 of the patients with USH suffer from USH2 and USH1, of whom 75% have mutations in the USH2A, USH1D and 1F genes. Because USH affects both major senses, it is a severely debilitating condition, and intense research is crucial to improve coping strategies and develop therapies for the patients. It is particularly devastating during the current pandemic, with social distancing and the wearing of masks making communication nearly impossible. Treatment for USH is limited to cochlear implants, and there is no treatment for the blindness. Development of an effective therapeutic approach for USH has been challenging due to the large size of USH genes. Therefore, there is an unmet need to develop alternative therapeutic strategies. The goal of this project is to develop and test novel therapy approaches for treating recessive deafness in human hair cells of inner ear organoids derived from human induced pluripotent stem cells (hiPSCs) and in USH mouse models by establishing genome editing-based therapeutic strategies for USH and to lay the foundation for moving genome editing approaches closer clinical trials. We have chosen to focus on the most common mutations in the three major USH genes with the following reasons: 1) The USH2A, 1D, and 1F genes are the most common and important USH genes which are responsible for more than 70% human USH cases with significant clinical application; 2) Due to their large size, traditional gene augmentation or addition therapy is hampered as its coding sequence far exceeds the packaging capacity of standard gene therapy vectors; 3) All three USH2A, 1D, and 1F genes contain similar multiple repetitive domains with in frame common mutations in their protein structures, making them potential targets for exon-skipping-based therapies (see Preliminary data); 4) We have obtained exciting data demonstrating restoration of hearing in an Ush2a mouse model using exon skipping strategy with an available mouse model of USH2A and have successfully generated hiPSCs from Usher patients carrying the most common mutations of USH, and established optimized protocols for generation of large numbers of human inner ear organoids with the production of human hair cells derived from these hiPSCs. This proposal leverages the exceptional deafness genomics information and genome editing expertise of collaborators for the development of novel treatments for HL. In this proposal, we will build on our accomplishments and preliminary data by proposing to complete the following specific aims: 1) to develop CRISPR/Cas9- based exon-skipping strategies to rescue hearing in transgenic USH mouse models and 2) to develop exon-skipping by CRISPR/Cas9 editing strategies to rescue USH mutations using inner ear organoids derived from patient hiPSCs. This proposal develops a new strategy utilizing CRISPR/Cas9 editing to skip exons most afflicted by common recessive mutations in USH2A, USH1D and USH1F as treatment to rescue hearing in animal models and restore phenotypes in human patient iPS cells-derived inner ear organoids with the hope of moving these technologies closer to their clinical application.

翻译后摘要：Usher综合征（USH）是最常见的遗传性眼盲症，患病率为1/6. 000。 作为一种常染色体隐性遗传性状，它影响了美国约15，000人，并导致6%的 儿童早期耳聋Usher综合征根据其临床表现分为三种临床亚型（USH-1、USH-2和USH-3）。 症状的严重程度。大约2/3的USH患者患有USH 2和USH 1，其中75%患有USH 2和USH 1。 USH 2A、USH 1D和1F基因突变。因为USH影响两个主要的感官，这是一个严重的衰弱 条件，深入研究是至关重要的，以改善应对策略和开发治疗的病人。显得尤为 在当前的大流行期间，社交距离和戴口罩使沟通几乎 不可能的USH的治疗仅限于人工耳蜗植入，并且没有治疗失明的方法。发展 由于USH基因的大尺寸，USH的有效治疗方法具有挑战性。因此，有一个 开发替代治疗策略的需求尚未得到满足。该项目的目标是开发和测试新的治疗方法 用于治疗源自人诱导的内耳类器官的人毛细胞中的隐性耳聋的方法 多能干细胞（hiPSC）和USH小鼠模型，通过建立基于基因组编辑的治疗策略， USH并为将基因组编辑方法更接近临床试验奠定基础。我们选择专注于 最常见的突变在三个主要USH基因中，原因如下：1）USH 2A，1D和1F基因 是最常见和最重要的USH基因，其负责超过70%的人类USH病例， 2）由于其体积大，传统的基因扩增或添加治疗受到阻碍，因为其编码 序列远远超过标准基因治疗载体的包装能力; 3）所有三种USH 2A、1D和1F基因 含有类似的多个重复结构域，在其蛋白质结构中具有框内共同突变， 基于外显子跳跃疗法的潜在靶点（见初步数据）; 4）我们已经获得了令人兴奋的数据，表明 使用可用的USH 2A小鼠模型，使用外显子跳跃策略在USH 2A小鼠模型中恢复听力 并成功地从携带最常见USH突变的Usher患者中产生了hiPSC，并建立了 优化的方案，用于产生大量的人内耳类器官，同时产生人毛细胞 来源于这些hiPSC。该提案利用了特殊的耳聋基因组学信息和基因组编辑 合作者的专业知识，用于开发HL的新型治疗方法。在本建议中，我们将建立在我们的 通过提出完成以下具体目标的成就和初步数据：1）开发CRISPR/Cas9- 基于外显子跳跃策略，以拯救转基因USH小鼠模型的听力; 2）通过 CRISPR/Cas9编辑策略使用源自患者hiPSC的内耳类器官拯救USH突变。这 一项提案开发了一种新的策略，利用CRISPR/Cas9编辑来跳过最受常见隐性遗传影响的外显子。 USH 2A、USH 1D和USH 1F突变作为治疗以挽救动物模型中的听力并恢复人类的表型 患者iPS细胞衍生的内耳类器官，希望将这些技术更接近其临床应用。