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

摘要：Usher综合征是最常见的遗传性聋盲，患病率为1/6.000。 作为一种常染色体隐性遗传，它在美国影响着大约15,000人，并导致6%的 儿童早期耳聋。Usher综合征分为三个临床亚型(USH-1、-2和-3)。 症状的严重程度。大约三分之二的USH患者患有USH2和USH1，其中75% USH2A、USH1D和1F基因突变。由于USH影响两种主要感官，它是一种严重衰弱的 因此，加强研究对于改善应对策略和为患者开发治疗方法至关重要。它特别是 在当前的大流行期间造成了毁灭性的影响，社交距离和戴口罩几乎使交流 不可能。USH的治疗仅限于人工耳蜗植入，目前还没有治疗失明的方法。发展中的 由于USH基因的大小，治疗USH的有效方法一直具有挑战性。因此，有一个 未得到满足的需求，以开发替代治疗策略。该项目的目标是开发和测试新的治疗方法。 人诱导内耳有机化合物治疗人毛细胞隐性耳聋的研究进展 通过建立基于基因组编辑的治疗策略，在USH小鼠模型中建立多能干细胞(HiPSCs)和USH小鼠模型 并为推动基因组编辑方法更接近临床试验奠定了基础。我们选择将重点放在 最常见的三种USH基因突变的原因如下：1)USH2A、1D和1F基因 是最常见和最重要的USH基因，70%以上的人类USH病例与显著的 临床应用；2)由于它们的大小，传统的基因扩增或添加治疗因其编码而受到阻碍。 序列远远超过标准基因治疗载体的包装能力；3)所有三个USH2A、1D和1F基因 含有相似的多个重复结构域，在它们的蛋白质结构中具有框内共同的突变，使它们 基于外显子的治疗的潜在靶点(见初步数据)；4)我们已经获得了令人兴奋的数据，证明了 Ush2a小鼠模型利用外显子跳跃策略恢复听力 并成功地从携带USH最常见突变的Usher患者中培养出HiPSCs，并建立了 利用人毛细胞生产大量人内耳有机物质的优化方案 来源于这些HiPSCs。这项建议利用了特殊的耳聋基因组学信息和基因组编辑 合作者在开发HL新疗法方面的专业知识。在这项提案中，我们将在我们的基础上 取得的成就和初步数据，建议完成以下具体目标：1)制定CRISPR/CAS9-- 基于外显子跳跃策略挽救转基因USH小鼠模型的听力2)建立外显子跳跃 CRISPR/Cas9编辑策略使用来自患者HiPSCs的内耳有机体来挽救USH突变。这 Proposal开发了一种新的策略，利用CRISPR/Cas9编辑来跳过最受普通隐性遗传影响的外显子 USH2A、USH1D和USH1F突变作为挽救动物模型听力和恢复人类表型的治疗方法 患者iPS细胞衍生的内耳有机体，希望将这些技术推向临床应用。

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.