Prime editing for Crumbs homologue 1 (CRB1) Inherited Retinal Dystrophies

项目总结 CRB1基因突变可导致严重的遗传性视网膜营养不良(IRD)。世界范围 大约80,000名CRB1患者受到影响，美国的患病率为每86,500人中就有1人。没有治疗方法 可用。CRB小鼠模型中的基因增强显示出混合的结果，并成功地进行了概念验证 (POC)使用家族成员CRB2，但除了不利之外，仅具有有限的形态和功能优势 使用CRB1-A的效果。CRB1蛋白定位于粘着连接附近，在维持 它们在光感受器(PRCs)和Müller神经胶质细胞(MGCs)中的稳定性。CRB1在视网膜发育中的作用 疾病一直集中在CRB1-A上。然而，存在三种人类视网膜CRB1亚型：CRB1-A、 人类特有的CRB1-C和新鉴定的CRB1-B。在小鼠中，CRB1-A和CRB1-B的作用方式不同 细胞类型(分别为mGCS和PRCs)。我们的长期目标是阻止进行性视网膜变性 在CRB1 IRD患者中发现。我们的初步数据证实了主要的细胞类型不同的定位 CRB1-A和CRB1-B以及CRB1-C在人身体视网膜中的定位和诱导多能性 干细胞(IPSC)来源的视网膜有机体。此外，大多数CRB1突变会影响不止一个CRB1 异构体。因此，这项资助的目标是确定一种不依赖于异构体的治疗方法。 CRB1IRDS。Prime编辑是一种双链断裂独立的基因编辑系统，可以纠正所有 突变类型。我们的中心假设是，初级编辑服从于CRB1突变的纠正， 使我们能够开发必要的工具来确定其对有丝分裂后视网膜细胞的治疗效果。这 假设将通过追求以下三个具体目标来检验。目标1(C.2843和>A)和目标2(C.3307G和>A) 将评估Prime编辑是否适用于CRB1突变的安装和纠正，并确定其安全性 通过评估目标外最有效的策略来建立个人资料。进一步的目标1和2将描述 衍生的视网膜器质的表型、组织病理学和分子变化。最后，在Aim3中，我们将 确定慢病毒All-in-in-One或AAV Split-Intein Prime编辑策略是否最适合执行有丝分裂后编辑 在视网膜器官中进行编辑。影响：这一新项目的结果将产生新的CRB1视网膜器质性疾病 模型，确定CRB1IRDS的治疗结果指标，并定义 用于改进CRB1IRD的主要编辑工具。这一建议是创新的，正如我们的方法所做的那样 纠正所有受给定CRB1突变影响的CRB1亚型。令人兴奋的是，这项工程的成功完成 该项目将建立一条临床前途径，展示CRB1主要编辑疗法的POC。

PROJECT SUMMARY Mutations in Crumbs homologue-1 (CRB1) gene cause severe inherited retinal dystrophies (IRDs). Worldwide ~80,000 CRB1 patients are affected, with a prevalence in the United States of 1 in 86,500. There is no treatment available. Gene augmentation in Crb mouse models has shown mixed results, with successful proof-of-concept (POC) using family member CRB2 but only limited morphological and no functional benefits in addition to adverse effects using CRB1-A. CRB1 proteins localize adjacent to adherens junctions and are essential in maintaining their stability in photoreceptors (PRCs) and Müller glial cells (MGCs). The role of CRB1 in retinal development and disease has been focused on CRB1-A. However, three human retinal CRB1 isoforms exist: CRB1-A, the human specific CRB1-C, and the newly identified CRB1-B. In mice, CRB1-A and CRB1-B operate in different cell types (MGCs and PRCs, respectively). Our long-term goal is to halt the progressive retinal degeneration found in CRB1 IRD patients. Our preliminary data confirm the predominate cell-type-distinct localizations of CRB1-A and CRB1-B in addition to the localization of CRB1-C in human cadaveric retina and induced pluripotent stem cell (iPSC)-derived retinal organoids. Further, the majority of CRB1 mutations affect more than one CRB1 isoform. Consequently, the objective of this grant is to determine an isoform-independent approach to treat CRB1 IRDs. Prime editing is a double-strand break-independent gene editing system that can correct all mutation types. Our central hypothesis is that prime editing is amenable to the correction of CRB1 mutations, allowing us to develop the tools necessary to ascertain its therapeutic efficacy in post-mitotic retinal cells. This hypothesis will be tested by pursuing the following three specific aims. Aim 1 (c.2843>A) and Aim 2 (c.3307G>A) will assess if prime editing is amenable for the installation and correction of CRB1 mutations and define its safety profile by evaluating off-targeting of the most efficient strategies. Further Aim 1 and 2 will characterize phenotypic, histopathological, and molecular changes in the derived retinal organoids. Lastly, in Aim3 we will define if a lentiviral all-in-one or AAV split-intein prime editing strategy is most amenable to perform post-mitotic editing in retinal organoids. Impact: Results of this novel project would create new CRB1 retinal organoid disease models, identify therapeutic outcome measures for CRB1 IRDs, and define the efficiency and safety profile for prime editing tools for the amelioration of CRB1 IRDs. This proposal is innovative, as our approach would correct all CRB1 isoforms affected by a given CRB1 mutation. Excitingly, the successful completion of this project will establish a preclinical pathway for showing POC for CRB1 prime editing therapeutics.