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

基本信息

批准号：
10688070
负责人：
Zheng-Yi Chen
金额：
$ 69.57万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
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/10688070
关键词：
Affect Animal Model Auditory Biological Assay Blindness Budgets CRISPR/Cas technology Cell Line Cell Therapy Cell model Cells Classification 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 clinical application clinical subtypes current pandemic 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 mutation correction novel novel therapeutic intervention pre-clinical protein structure therapeutic genome editing therapeutically effective therapy development tool treatment strategy whole genome

项目摘要

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.

摘要：Usher综合征（USH）是遗传性聋哑人的最常见形式，患病率为1/6.000。作为常染色体隐性特征，它影响了美国约15,000人，并负责6％幼儿耳聋。根据三种临床亚型（USH -1，-2和-3），USHER综合征分类为症状的严重程度。大约2/3的USH患者患有USH2和USH1，其中75％ USH2A，USH1D和1F基因中的突变。因为USH会影响这两种主要感官，所以这是一个严重的衰弱病情和激烈的研究对于改善应对策略和为患者制定疗法至关重要。特别是在当前大流行期间造成毁灭性的，社会疏远和戴口罩几乎使交流不可能的。 USH的治疗仅限于耳蜗植入物，并且没有治疗失明的治疗方法。发展由于USH基因的大量，对USH的有效治疗方法一直在挑战。因此，有一个未满足的需要制定替代性治疗策略。该项目的目的是开发和测试新疗法治疗源自人诱导的内耳类器官的人毛细胞中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并为移动基因组编辑方法奠定基础。我们选择专注于三个主要USH基因中最常见的突变，其原因是：1）USH2A，1D和1F基因是最常见和重要的USH基因，负责超过70％的人类案例临床应用； 2）由于其尺寸较大，传统的基因增强或添加疗法被阻碍了其编码序列远远超过了标准基因治疗载体的包装能力； 3）所有三个USH2A，1D和1F基因在其蛋白质结构中包含与框架共同突变中的类似的多个重复域，使其基于外显子的疗法的潜在靶标（请参阅初步数据）； 4）我们获得了令人兴奋的数据证明使用外显子跳过策略在USH2A鼠标模型中恢复听力，并使用USH2A的可用鼠标模型并成功地产生了来自Usher患者的HIPSC，该患者携带了最常见的USH突变，并确定了HIPSC 通过生产人毛细胞生成大量人内耳类器官的优化方案源自这些HIPSC。该建议利用了非凡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。在此提案中，我们将建立在我们的基础上成就和初步数据通过建议完成以下特定目的：1）开发CRISPR/CAS9- 基于转基因的策略，以在转基因USH鼠标模型中营救听力，2）通过 CRISPR/CAS9编辑策略，使用源自患者HIPSC的内耳类器官挽救USH突变。这提案制定了一种利用CRISPR/CAS9编辑的新策略，以跳过最常见的隐性外显子 USH2A，USH1D和USH1F中的突变作为挽救动物模型听力并恢复人类表型的治疗方法患者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。