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

Crumbs 同源物 1 (CRB1) 遗传性视网膜营养不良的 Prime 编辑

• 批准号: 10636325
• 负责人: Peter Martin John Quinn
• 金额: $ 40.97万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636325
• 关键词: Adherens Junction; Adverse effects; Affect; Birth; Blindness; Cadaver; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependovirus; Development; Disease; Disease model; Extracellular Domain; Family member; Genes; Goals; Grant; Homologous Gene; Human; Inherited; Knockout Mice; Lead; Leber' s amaurosis; Mediating; Modification; Molecular; Morphology; Mus; Mutation; Neuroglia; Organoids; Outcome Measure; Pathogenicity; Pathway interactions; Patients; Phenotype; Photoreceptors; Prevalence; Protein Isoforms; Proteins; Rattus; Reporter; Research; Retina; Retinal Degeneration; Retinal Diseases; Retinal Dystrophy; Retinitis Pigmentosa; Role; Safety; System; Techniques; Testing; Treatment Efficacy; United States; Viral; Visual Fields; Work; base editing; cell type; conditional knockout; early childhood; gene augmentation therapy; induced pluripotent stem cell; innovation; insertion/deletion mutation; intein; mouse model; mutant; novel; phase I/IIa trial; postmitotic; pre-clinical; prime editing; prime editor; therapeutic genome editing; therapy outcome; tool; transition mutation; transversion mutation; vector

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.

项目摘要 Crumbs同源1（CRB1）基因的突变引起严重的遗传性视网膜营养不良（IRD）。全世界 〜80,000名CRB1患者受到影响，在美国有86,500例患病率。没有治疗 可用的。 CRB小鼠模型中的基因增强表现出了混合的结果，并获得了成功的概念证明 （POC）使用家族成员CRB2，但除逆境外，只有有限的形态和功能益处 使用CRB1-A的效果。 CRB1蛋白质本地化与粘附连接相邻，对于维持 CRB1在剩余发展中的作用 疾病一直集中在CRB1-A上。但是，存在三种人类残留CRB1同工型：CRB1-A， 人类特异性CRB1-C和新确定的CRB1-B。在小鼠中，CRB1-A和CRB1-B不同 细胞类型（分别为MGC和PRC）。我们的长期目标是停止渐进的永久性变性 在CRB1 IRD患者中发现。我们的初步数据证实了细胞类型的主要位置的位置 除了在人尸体视网膜中CRB1-C的定位外，CRB1-A和CRB1-B除了诱导多能 干细胞（IPSC）衍生的视网膜器官。此外，大多数CRB1突变会影响一个以上的CRB1 同工型。因此，这笔赠款的目的是确定与同工型的治疗方法 CRB1 IRDS。 Prime编辑是双链独立的基因编辑系统，可以纠正所有 突变类型。我们的核心假设是，主要的编辑可以校正CRB1突变， 允许我们开发确定其在丝裂后残留细胞中的治疗效率所需的工具。这 假设将通过追求以下三个特定目标来检验。目标1（c.2843> a）和目标2（c.3307g> a） 将评估主要编辑是否适合安装和校正CRB1突变并定义其安全性 通过评估最有效策略的目标来评估概况。进一步的目标1和2将表征 衍生的视网膜器官的表型，组织病理学和分子变化。最后，在AIM3中，我们将 定义慢病毒是多合一或AAV分裂素数素数编辑策略最适合执行有丝分裂的 在视网膜器官中进行编辑。影响：这个新型项目的结果将产生新的CRB1视网膜器官疾病 模型，确定CRB1 IRD的治疗结果指标，并定义 用于改善CRB1 IRD的主要编辑工具。该建议是创新的，就像我们的方法一样 纠正受给定CRB1突变影响的所有CRB1同工型。令人兴奋的是，成功完成 项目将建立临床前途径，用于显示CRB1 Prime编辑疗法的POC。