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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要 Crumbs 同源基因 1 (CRB1) 基因突变会导致全球严重遗传性视网膜营养不良 (IRD)。约 80,000 名 CRB1 患者受到影响，在美国患病率为 86,500 分之一。目前尚无治疗方法。 Crb 小鼠模型中的基因增强显示出好坏参半的结果，并成功进行了概念验证。（POC）使用家族成员 CRB2，但除了不利的影响外，仅在形态上有限，没有功能上的好处使用 CRB1-A 的效应定位于粘附连接附近，对于维持粘附连接至关重要。 CRB1 在光感受器 (PRC) 和 Müller 胶质细胞 (MGC) 中的稳定性在视网膜发育中的作用。然而，人类视网膜 CRB1 亚型存在三种：CRB1-A，即 CRB1-A。人类特有的CRB1-C和新发现的CRB1-B 在小鼠中，CRB1-A和CRB1-B以不同的方式运作。我们的长期目标是阻止进行性视网膜变性。我们的初步数据证实了 CRB1 IRD 患者的主要细胞类型不同定位。 CRB1-A和CRB1-B以及CRB1-C在人尸体视网膜中的定位和诱导多能性此外，大多数 CRB1 突变影响不止一种 CRB1。经过检查，这笔赠款的目的是确定一种独立于异构体的治疗方法。 CRB1 IRDs.Prime编辑是一种双链断裂无关的基因编辑系统，可以纠正所有的缺陷。我们的中心假设是prime编辑可以修正CRB1突变，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功效。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编辑是否适合安装和纠正CRB1突变并确定其安全性通过评估最有效策略的脱靶来描述目标 1 和 2 将表征。最后，在 Aim3 中，我们将研究衍生的视网膜类器官的表型、组织病理学和分子变化。定义慢病毒一体化或 AAV 分裂内含肽编辑策略是否最适合执行有丝分裂后影响：这个新项目的结果将创造新的 CRB1 视网膜类器官疾病。模型，确定 CRB1 IRD 的治疗结果测量，并定义 CRB1 IRD 的效率和安全性概况用于改善 CRB1 IRD 的主要编辑工具该提案具有创新性，正如我们的方法一样。纠正受给定 CRB1 突变影响的所有 CRB1 同种型令人兴奋的是，此任务成功完成。该项目将建立一条临床前途径，用于展示 CRB1 初等编辑疗法的 POC。