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）影响约150万 世界各地的人们。Bardet-Biedl综合征（BBS）是综合征型RP的第二大常见原因， 其特征为伴有严重光感受器变性的常染色体隐性纤毛病 发生在生命的第一个或第二个十年。BBS与21个基因的变异有关， BBS 7占所有BBS案例的约2%。该提案的总体目标是克服两个 视觉研究的主要障碍：1）准确概括疾病机制的能力， BBS 7在可翻译模型系统中的进展，以及2）永久纠正 致病变异体，并以高效率和特异性恢复感光细胞功能。在 目的1，将通过产生视网膜病变的视网膜病变来检查负责BBS 7发作的疾病机制。 来自携带致病突变的人诱导多能干细胞的类器官模型系统 BBS 7基因在目的2和3中，将使用引物编辑工具和脂质体校正BBS 7变体。 在人类视网膜类器官模型中基于纳米颗粒（LNP）的递送策略，以研究时机和 疾病救助的效率。此外，在体外发现最有效的疗法将在 使用非人灵长类动物来确定Cas9递送到非人灵长类动物中的剂量、免疫原性和效率。 体内感光细胞。成功地完成这些目标将有助于我们的基本 理解BBS中光感受器功能障碍的病理生理机制7，2） 为该领域提供了对靶向基因编辑策略的彻底评估，以治疗视网膜中的BBS 7， 和3）建立LNP作为理想的递送系统以限制细胞毒性和免疫学副作用 这在基于AAV的递送方法中常见。总而言之，这项工作将建立一个管道 用于测试和优化治疗BBS 7和其他遗传性视网膜疾病的疗法，包括 BBS和RP的其他形式。

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.