Develop an engineered Cas effector for in vivo cell-targeted delivery in the eye to treat autosomal dominant BEST disease

项目总结/摘要-后续项目2 贝斯特病（BD）是一种流行的、主要是常染色体显性的黄斑变性疾病，其导致 视网膜色素上皮（RPE）功能障碍和进行性中心视力丧失。BD结果来自>200个已知 BEST 1基因的错义突变。然而，目前还没有可行的治疗方案， 在占主导地位的BD中，基因组编辑可以提供一种有前途的治疗策略。这个追随者项目的目标 2是使用Spotlight Therapeutics开发一种安全有效的BD基因组编辑治疗方法。 靶向活性基因编辑器（TAGE）平台。TAGE是一种新型的、非病毒的、非纳米颗粒的 在一个实施方案中，提供了一种递送系统，其中功能性CRISPR-Cas效应子与抗体（Ab）融合并且细胞穿透CRISPR-Cas效应子与抗体（Ab）融合。 肽（CPP）部分，能够实现体内细胞靶向递送，细胞内运输至细胞核， 随后的基因编辑TAGE-101将通过识别和优化这些 模块化组分和与结合突变Best 1的人单向导RNA（sgRNA）的复合。一 将生成临床前数据包，证明R218 C突变体BEST 1的>10%编辑/敲除 等位基因、恢复的功能和可接受的离体脱靶和遗传毒性特征，使用患者iPSC来源的 RPE和非GLP非人灵长类动物（NHP）毒性研究。INTERACT和IND前会议， FDA计划提交临床前和上游工艺开发数据包， 开发计划，并探索是否可以用患者特异性指南解决其他BD突变体 在TAGE-101的主IND下。在五年资助期结束时，TAGE-101开发项目 候选人将准备启动IND使能研究，并最终在BEST 1患者中进行临床试验。这 项目和项目3旨在开发针对BEST 1突变等位基因的基于CRISPR-Cas的基因编辑疗法 使用不同的交付平台。协同作用将存在于诸如sgRNA开发、离体 与人类细胞检测核心合作进行临床前表征研究，以及监管 与监管核心合作的BD战略。该项目还将与项目1产生协同作用， NHP试点研究，通过视网膜下注射解决RPE通道病，与大型动物合作 核心这个项目的成功完成将提供一个严格的，逐步的方法来提名一个发展 候选人准备启动IND使能研究的所有个人与BD造成的特定突变。它 还将推进iPSC模型作为定制临床前工具的实用性，以快速开发体细胞基因组 编辑策略与新型细胞靶向CRISPR基因编辑平台相结合。

PROJECT SUMMARY/ABSTRACT – FOLLOWER PROJECT 2 Best Disease (BD) is a prevalent, primarily autosomal dominant, macular degenerative disorder that results in retinal pigment epithelium (RPE) dysfunction and progressive loss of central vision. BD results from >200 known missense mutations in the BEST1 gene. Whereas there are currently no viable treatment options for autosomal dominant BD, genome editing could offer a promising therapeutic strategy. The goal of this Follower Project 2 is to develop a safe and effective genome editing treatment for BD using Spotlight Therapeutics’ proprietary Targeted Active Gene Editors (TAGE) platform. TAGE is a novel, nonviral, non-nanoparticle delivery system wherein a functional CRISPR-Cas effector is fused to antibody (Ab) and cell-penetrating peptide (CPP) moieties, enabling in vivo cell-targeted delivery, intracellular trafficking to the nucleus and subsequent gene editing. A TAGE-101 lead will be developed through identification and optimization of these modular components and complexation with a human single guide RNA (sgRNA) that binds mutant Best1. A preclinical data package will be generated demonstrating >10% editing/knockout of the R218C mutant BEST1 allele, restored function, and acceptable off-target and genotoxicity profiles ex vivo, using patient iPSC-derived RPEs, and in non-GLP nonhuman primate (NHP) toxicity studies. INTERACT and pre-IND meetings with the FDA are planned to present a preclinical and upstream process development data package and a future development plan and to explore whether additional BD mutants can be addressed with patient-specific guides under a master IND for TAGE-101. At the end of the five-year grant horizon, the TAGE-101 development candidate will be poised to initiate IND-enabling studies and, ultimately, clinically tested in BEST1 patients. This Project and Project 3 aim to develop CRISPR-Cas-based gene editing therapies targeting a BEST1 mutant allele employing different delivery platforms. Synergy will exist in areas such as sgRNA development, ex vivo preclinical characterization studies in partnership with the Human Cell Assay Core, as well as a regulatory strategy for BD in partnership with the Regulatory Core. This project will also have synergy with Project 1 around pilot NHP studies, addressing RPE channelopathies via subretinal injection, in partnership with the Large Animal Core. Successful completion of this project will provide a rigorous, stepwise approach to nominate a development candidate poised to initiate IND-enabling studies for all individuals with BD resulting from a specific mutation. It will also advance the utility of iPSC models as custom preclinical tools to rapidly develop somatic cell genome editing strategies in conjunction with a novel cell-targeted CRISPR gene editing platform.