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

项目概要/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）影响约150万人 国际吧Bardet-Biedl综合征（BBS）是综合征型RP的第二大常见原因， 其特征为常染色体隐性纤毛病，第一次出现严重的感光细胞变性， 或人生的第二个十年。BBS与21个基因的变异有关，其中BBS7中的变异约占20%。 2%的BBS案例。本提案的总体目标是克服视觉研究中的两个主要障碍：（1） 在可翻译模型中准确概括疾病机制和BBS7进展的能力 系统，和（2）永久纠正致病变异和恢复感光细胞的能力 高效特异的功能。在目标1中，负责BBS发作的疾病机制7 将通过从人诱导多能干细胞产生视网膜类器官模型系统来检查 在BBS7基因中携带致病突变。在目标2和3中，将使用以下方法纠正BBS7变体： 主要编辑工具和基于脂质纳米颗粒（LNP）的人类视网膜类器官内的递送策略 模型来研究疾病救助的时机和效率。此外，发现最有效的治疗方法是 将在非人灵长类动物中进行体外测试，以确定Cas9递送的剂量、免疫原性和效率。 转化为体内的感光细胞。成功地完成这些目标将有助于1）我们的基本理解 的病理生理机制的基础上感光细胞功能障碍的BBS 7，2）提供了一个领域， 彻底评估靶向基因编辑策略以治疗视网膜中的BBS7，以及3）将LNP建立为 限制基于AAV通常观察到细胞毒性和免疫副作用的理想递送系统 交付方法。总之，这项工作将建立一个测试和优化治疗的管道， 治疗BBS 7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.