Streamlined development of an IND with the silica nanocapsule loaded with Cas9 genome editors to disrupt the dominant BEST1 mutant allele

项目摘要-追随者项目3.最佳卵黄样黄斑营养不良，或最佳疾病(BD)， 是一种相对常见的遗传性黄斑变性疾病，导致视网膜色素上皮(RPE)。 中心视力功能障碍和进行性丧失。BD是由200多种不同的错义突变引起的 Best1基因。目前，常染色体显性遗传性BD尚无治疗方法，部分原因是缺乏 相关动物模型。要克服许多致病BEST1影响的模型化困难 突变，我们利用患者特定的IPSC-RPE模型来开发一种生物系统，该系统可以快速筛查 基因组编辑方面的线索。就像在追随者项目2中一样，我们假设Cas9通过Leads介导Indel的形成 在突变的BEST1等位基因中，会导致框架移位，导致过早停止密码子，并导致无稽之谈- 介导编辑后的转录本的衰退，消除显性-负面效应，恢复野生- 类型等位基因。我们在项目3中的目标是开发一个包含Cas9有效负载的SNC产品SNC-201 特异性靶向BEST1突变等位基因并比较使用核糖核蛋白(RNP)的各种方法 和Cas9mRNA/sgRNAs。为了实现这一目标，我们将追求四个目标。首先，我们将生成双BEST1等位基因 BD iPSC-RPE报告系统，便于突变等位基因靶向的开/离靶点分析。我们已经开发出 一个BD双报告IPSC系，其突变的BEST1等位基因与3‘td番茄报告基因和野生型连锁 Best1等位基因与3‘GFP报告基因连锁。通过荧光成像，我们可以监测这两个基因的表达 靶向突变的BEST1等位基因和野生型等位基因，这是我们战略中最重要的“偏离目标”的位置。另外，我们 可以监控已分类小区的信道功能。我们将改编这条记者iPSC线，打造一款野性和八款 额外的突变品系。其次，我们比较了领先的SNC-201的模块化编辑器组件。使用 将含有RNPs或Cas9 mRNA/sgRNA的SNC制剂导入双报告系统，我们将迅速 筛选SNC配方以干扰双报告基因IPSC-RPE中的每个突变等位基因。两种编辑方式的比较 通过这项工作将实现Cas9 mRNA/sgRNA和RNP的效率。第三，我们放大合成 SNC RNP线索的一部分，并依赖于Lead Project 1的协议来放大mRNA/sgRNA线索。最后，我们 为SNC-201生成针对选定的突变BEST1等位基因的临床前包。在对安全性进行评估后 关于非人类灵长类动物的SNC-201配方，我们将完成与FDA的一次互动会议 该产品旨在治疗具有多种不同突变的BD患者。在获得反馈后，我们将起草 IND前期会议的临床发展计划。成功完成我们的目标将提供一个 开发针对特定突变的IND的严格、循序渐进的方法-这一策略可能 扩展到所有患有BD的个人-并解决与应用 基因异质性显性疾病的基因编辑。它还将促进IPSC的效用 模型作为定制的临床前工具，以快速开发体细胞基因组编辑策略。

PROJECT SUMMARY– FOLLOWER PROJECT 3. Best Vitelliform Macular Dystrophy, or Best disease (BD), is a relatively common inherited macular degenerative disorder that results in retinal pigment epithelium (RPE) dysfunction and progressive loss of central vision. BD is caused by over 200 different missense mutations in the BEST1 gene. Currently, there are no treatments for autosomal dominant BD due in part to the absence of relevant animal models. To overcome the difficulties in modeling the effects of many pathogenic BEST1 mutations, we utilize patient-specific iPSC-RPE models to develop a biological system that can rapidly screen genome editing leads. As in Follower Project 2, we hypothesize that Cas9-mediated indel formation by the leads in the mutant BEST1 allele will cause frameshifts leading to premature stop codons and resulting in nonsense- mediated decay of the edited transcript, removing the dominant-negative effect and restoring function to the wild- type allele. Our objective in Project 3 is to develop an SNC product, SNC-201, containing a Cas9 payload to specifically target BEST1 mutant alleles and compare various approaches using ribonucleoproteins (RNPs) and Cas9 mRNA/sgRNAs. To achieve this, we will pursue four aims. First, we will generate dual BEST1 allele BD iPSC-RPE reporter systems to facilitate on/off-target analysis of mutant allele targeting. We have developed a BD dual reporter iPSC line that has the mutant BEST1 allele linked to a 3’ tdTomato reporter and the wild-type BEST1 allele linked to a 3’ GFP reporter. Via fluorescent imaging, we can monitor the expression of both the targeted mutant BEST1 allele and the wild-type allele, which is the top ‘off-target’ site for our strategy. Plus, we can monitor channel function on sorted cells. We will adapt this reporter iPSC line to create a wild-type and eight additional mutant lines. Second, we compare the modular editor components of the lead SNC-201s. Using the dual reporter systems transfected with SNC formulations containing RNPs or Cas9 mRNA/sgRNA, we will rapidly screen SNC formulations to disrupt each mutant allele in dual reporter iPSC-RPEs. A comparison of the editing efficiency of Cas9 mRNA/sgRNA and RNP will be achieved through this work. Third, we scale up the synthesis of the SNC RNP leads and rely on Lead Project 1 for protocols to scale up mRNA/sgRNA leads. Finally, we generate a preclinical package for SNC-201 targeting selected mutant BEST1 alleles. After evaluating the safety of an SNC-201 formulation in nonhuman primates, we will complete one INTERACT meeting with the FDA for a product that intends to treat BD patients with multiple different mutations. After obtaining feedback, we will draft a clinical development plan for a pre-IND meeting. Successful completion of our aims will provide a rigorous, stepwise approach to developing an IND for targeting specific mutations – a strategy that could be expanded for all individuals with BD – and address central questions pertaining to the application of gene editing for genotypically heterogeneous dominant diseases. It will also advance the utility of iPSC models as custom preclinical tools to rapidly develop somatic cell genome editing strategies.