Establishing a novel gene editing strategy for BBS7 using human retinal organoids

项目摘要/摘要 作为遗传性视网膜变性的主要原因，视网膜色素变性(RP)影响着大约150万人 全世界。Bardet-Biedl综合征(BBS)是综合征性RP的第二大常见原因， 以常染色体隐性遗传性睫状体病为特征的，第一个出现严重的光感受器变性 或者是生命的第二个十年。BBS与21个基因的变异有关，其中BBS7基因的变异约占 占所有BBS案例的2%。这项建议的总体目标是克服视觉研究中的两大障碍：(1) 在可翻译模型中准确概括BBS7疾病机制和进展的能力 系统，以及(2)永久纠正致病变异和恢复感光细胞的能力 功能高效、特异性强。在目标1中，导致BBS7发病的疾病机制 将通过从人类诱导的多能干细胞产生视网膜器官模型系统来检验 在BBS7基因中存在致病突变。在AIMS 2和3中，BBS7变体将使用 主要的编辑工具和基于脂质纳米颗粒(LNP)的人视网膜器官内的递送策略 模型来研究疾病救援的时机和效率。此外，发现最有效的治疗方法是 体外试验将在非人类灵长类动物身上进行，以确定Cas9的剂量、免疫原性和效率 进入体内的感光细胞。成功地完成这些目标将有助于我们对 BBS7中光感受器功能障碍的病理生理机制，2)为该领域提供了一个 彻底评估治疗视网膜中BBS7的有针对性的基因编辑策略，以及3)将LNPs建立为 理想的递送系统，可限制基于AAV的常见细胞毒性和免疫副作用 投递方式。综上所述，这项工作将建立一条测试和优化疗法的管道，以 治疗BBS7和其他遗传性视网膜疾病，包括其他形式的BBS以及RP。

PROJECT SUMMARY/ABSTRACT As the leading cause of inherited retinal degeneration, retinitis pigmentosa (RP) affects about 1.5 million people worldwide. Bardet-Biedl syndrome (BBS) is the second most common causes of syndromic RP, and is characterized as an autosomal recessive ciliopathy with severe photoreceptor degeneration occurring by the first or second decade of life. BBS has been linked to variants in 21 genes, with those in BBS7 accounting for roughly 2% of all BBS cases. The overall goal of this proposal is to overcome two major hurdles in vision research: (1) the ability to accurately recapitulate disease mechanisms and progression for BBS7 in a translatable model system, and (2) the ability to permanently correct disease-causing variants and restore photoreceptor cell function with high efficiency and specificity. In Aim 1, disease mechanisms responsible for the onset of BBS7 will be examined by generating a retinal organoid model system from human induced pluripotent stem cells harboring disease-causing mutations in the BBS7 gene. In Aims 2 and 3, BBS7 variants will be corrected using prime editing tools and a lipid nanoparticle (LNP)-based delivery strategy within the human retinal organoid model to study timing and efficiency of disease rescue. Furthermore, the therapies found to be most effective in vitro will be tested in nonhuman primates to determine dose, immunogenicity, and efficiency of Cas9 delivery into photoreceptor cells in vivo. Successful completion of these aim will 1) contribute to our basic understanding of the pathophysiological mechanisms underlying photoreceptor dysfunction in BBS7, 2) provide the field with a thorough evaluation of a targeted gene editing strategy to treat BBS7 in the retina, and 3) establish LNPs as an ideal delivery system to limit cytotoxic and immunologic side effects commonly observed with AAV-based delivery methods. Taken together, this work will establish a pipeline for testing and optimization of therapies to treat BBS7 and other inherited retinal diseases, including additional forms of BBS as well as RP.