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

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 世界各地的人们。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，伴有严重的光感受器变性 在生命的第一或第二个十年前发生的。BBS与21个基因的变异有关，其中 BBS7约占所有BBS案例的2%。这项建议的总体目标是克服两个 视力研究中的主要障碍：1)准确概括疾病机制和 BBS7在可翻译模型系统中的进展，以及2)永久更正的能力 高效率、高特异性的致病变异体和恢复感光细胞功能。在……里面 目的1、BBS7发病的疾病机制将通过产生视网膜来检查 含有致病突变的人诱导多能干细胞的类器官模型系统 在BBS7基因中。在AIMS 2和3中，BBS7变体将使用主要的编辑工具和LID进行更正 人视网膜器官模型中基于纳米颗粒(LNP)的给药策略以研究时机和 疾病救援的效率。此外，被发现在体外最有效的疗法将在 非人灵长类动物用于确定Cas9在体内的剂量、免疫原性和效率 体内的感光细胞。这些目标的成功实现将有助于我们的基本工作 BBS7，2)光感受器功能障碍的病理生理机制 为该领域提供对治疗视网膜中的BBS7的靶向基因编辑策略的彻底评估， 3)建立LNPs作为一种理想的递送系统，以限制细胞毒性和免疫副作用 通常与基于AAV的交付方法一起观察。总而言之，这项工作将建立一条管道 用于测试和优化治疗BBS7和其他遗传性视网膜疾病的治疗方法，包括 其他形式的BBS以及RP。

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.