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 homologue-1（CRB 1）基因突变可导致严重的遗传性视网膜营养不良（IRD）。全球 约有80，000名CRB 1患者受到影响，在美国的患病率为1/86，500。没有治疗 available. Crb小鼠模型中的基因扩增显示出混合结果，并成功验证了概念 (POC)使用家族成员CRB 2，但除了不利影响外，仅具有有限的形态学益处且无功能益处 使用CRB 1-A的效果。CRB 1蛋白定位于粘附连接附近，并且在维持粘附连接中至关重要。 它们在光感受器（PRCs）和Müller神经胶质细胞（MGCs）中的稳定性。CRB 1在视网膜发育中的作用 而疾病则集中在CRB 1-A上。然而，存在三种人视网膜CRB 1同种型：CRB 1-A， 人特异性CRB 1-C和新鉴定的CRB 1-B。在小鼠中，CRB 1-A和CRB 1-B以不同的方式运作。 细胞类型（分别为MGCs和PRC）。我们的长期目标是阻止视网膜退化 在CRB 1 IRD患者中发现。我们的初步数据证实了占主导地位的细胞类型不同的定位， CRB 1-A和CRB 1-B以及CRB 1-C在人尸体视网膜中的定位和诱导的多能性视网膜病变的研究 干细胞（iPSC）衍生的视网膜类器官。此外，大多数CRB 1突变影响一种以上CRB 1 同种型因此，该补助金的目的是确定一种异构体独立的治疗方法， CRB1 IRD。Prime editing是一种不依赖于双链断裂的基因编辑系统， 突变类型我们的中心假设是，主要编辑可以纠正CRB 1突变， 使我们能够开发必要的工具，以确定其在有丝分裂后视网膜细胞中的治疗效果。这 将通过追求以下三个具体目标来检验假设。目标1（c.2843>A）和目标2（c.3307G>A） 将评估prime editing是否适用于CRB 1突变的安装和校正，并确定其安全性 通过评估最有效策略的非靶向来分析概况。进一步的目标1和2将描述 衍生的视网膜类器官中的表型、组织病理学和分子变化。在AIM 3中，我们将 定义慢病毒全合一或AAV分裂内含肽引发编辑策略是否最适合于进行有丝分裂后编辑， 在视网膜类器官中进行编辑。影响：这个新项目的结果将产生新的CRB 1视网膜类器官疾病 模型，确定CRB 1 IRD的治疗结局指标，并定义CRB 1 IRD的有效性和安全性特征。 CRB 1 IRD改进的主要编辑工具。这一建议是创新的，因为我们的做法将 纠正受给定CRB 1突变影响的所有CRB 1亚型。令人兴奋的是，成功完成这一 该项目将建立一个临床前途径，以显示CRB 1主要编辑疗法的POC。