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

使用装载有 Cas9 基因组编辑器的二氧化硅纳米胶囊简化 IND 的开发，以破坏占主导地位的 BEST1 突变等位基因

• 批准号: 10668168
• 负责人: Krishanu Saha
• 金额: $ 69.69万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-16 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668168
• 关键词: Acceleration; Address; Alleles; Animal Model; Animals; Calcium; Cell Separation; Cellular Assay; Characteristics; Chloride Channels; Clustered Regularly Interspaced Short Palindromic Repeats; Custom; Degenerative Disorder; Development; Development Plans; Disease; Disease model; Dominant-Negative Mutation; Ensure; Feedback; Formulation; Functional disorder; Future; Genes; Genetic Engineering; Genome; Genotype; Goals; Guide RNA; Human; Individual; Inherited; Investigational Drugs; Lead; Learning; Link; Mediating; Messenger RNA; Missense Mutation; Modeling; Monitor; Mutation; Nonsense Codon; Nonsense-Mediated Decay; Optical Coherence Tomography; Organoids; Pathogenicity; Patients; Photoreceptors; Process; Proteins; Protocols documentation; Publishing; Rapid screening; Reporter; Retina; Ribonucleoproteins; Safety; Silicon Dioxide; Structure of retinal pigment epithelium; System; Testing; Transcript; Transfection; Vision; Vitelliform macular dystrophy; Work; autosome; basolateral membrane; biological systems; cellular targeting; clinical development; experience; fluorescence imaging; functional restoration; genome editing; improved; induced pluripotent stem cell; insertion/deletion mutation; lead candidate; macula; meetings; monomer; mutant; nanocapsule; next generation sequencing; nonhuman primate; novel; off-target site; pre-Investigational New Drug meeting; pre-clinical; programs; scale up; somatic cell gene editing; tool; translational applications

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.

项目概要-后续项目3.最佳卵黄状黄斑营养不良，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） 功能障碍和进行性中心视力丧失。BD是由200多个不同的错义突变引起的， BEST 1基因。目前，没有常染色体显性遗传BD的治疗方法，部分原因是缺乏 相关动物模型。为了克服模拟许多致病性BEST 1效应的困难， 突变，我们利用患者特异性iPSC-RPE模型来开发一种生物系统， 基因组编辑领先。如在后续项目2中，我们假设Cas9介导的indel形成是由前导序列引起的。 在突变BEST 1等位基因将引起移码，导致过早终止密码子，并导致无义- 介导编辑的转录本的衰变，消除显性负效应并恢复野生型的功能， 型等位基因我们在项目3中的目标是开发SNC产品SNC-201，其包含Cas9有效载荷 特异性靶向BEST 1突变等位基因，并比较使用核糖核蛋白（RNP）的各种方法 和Cas9 mRNA/sgRNA。为了实现这一目标，我们将努力实现四个目标。首先，我们将产生双BEST 1等位基因 BD iPSC-RPE报告系统，以促进突变等位基因靶向的靶上/脱靶分析。我们已经开发 BD双报告基因iPSC系，其具有与3' tdTomato报告基因连锁的突变BEST 1等位基因和野生型 BEST 1等位基因与3' GFP报告基因连锁。通过荧光成像，我们可以监测这两种基因的表达。 靶向突变BEST 1等位基因和野生型等位基因，这是我们的策略的顶部“脱靶”位点。另外我们 可以监测分选细胞上的通道功能。我们将改造这一报告基因iPSC系，以创建野生型和八种iPSC。 其他突变株系。其次，我们比较了SNC-201的模块化编辑器组件。使用 用含有RNP或Cas9 mRNA/sgRNA的SNC制剂转染的双报告子系统，我们将快速地 筛选SNC制剂以破坏双报告基因iPSC-RPE中的每个突变等位基因。编辑比较 通过这项工作，将实现Cas9 mRNA/sgRNA和RNP的效率。第三，我们扩大合成 的SNC RNP线索，并依赖于铅项目1的协议，以扩大mRNA/sgRNA线索。最后我们 产生靶向选定突变BEST 1等位基因的SNC-201的临床前包装。在评估了安全性之后， SNC-201制剂在非人灵长类动物中的应用，我们将与FDA完成一次互动会议， 用于治疗具有多种不同突变的BD患者的产品。在获得反馈后，我们将起草 IND前会议的临床开发计划。成功完成我们的目标将提供一个 严格的，逐步的方法来开发针对特定突变的IND-这是一种可以 扩展到所有患有BD的个人-并解决与应用有关的核心问题 用于基因型异质性显性疾病的基因编辑。它还将推进iPSC的实用性 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。