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.

项目摘要 - 追随者项目3。最佳卵形黄斑营养不良或最佳疾病（BD）， 是一种相对常见的Macualar变性疾病，导致视网膜色素上皮（RPE） 功能障碍和中央视力的逐渐丧失。 最佳基因。 相关的动物模型。 Muterize，我们利用患者特异性的IPSC-RPE模型来开发可以快速筛选的生物系统 基因组编辑铅。 在突变体1等位基因Wille frameshifts中，导致了过早的停止密码子，并导致胡说八道 - 介导的编辑转录本的衰减，消除了巨大的阴性效应和恢复功能 键入等位基因。 专门针对Best1突变等位基因并使用核糖核蛋白（RNP）比较各种方法 和Cas9 mRNA/sgrnas。 BD IPSC-RPE记者系统促进突变等位基因靶向靶向的靶向。 BD双报告基因IPSC线最佳突变体1 allele与3'TDTOMATO RETORTER相关 Best1等位基因通过荧光成像链接到3'GFP记者。 有针对性的突变体1等位基因和野生型等位基因，这是策略的顶级“脱离目标”。 可以监视分类的单元格上的通道函数。 Aditional突变线 用SNC公式贡献RNP或Cas9 mRNA/SGRNA转染的双报告基因系统，我们将迅速 屏幕snc公式，以双重报告中的每个突变等位基因的编辑比较。 CAS9 mRNA/sgrNA和RNP的效率将获得第三名。 SNC RNP的引导和依赖于铅项目1来扩展mRNA/sgrna的铅 在评估安全性后，生成用于SNC-201的临床前包装。 在非人类灵长类动物中的SNC-2012公式中，我们将与FDA启用OnEterAct会议 获得多个不同突变的乳头BD患者的产品。 临床开发计划，以实现我们的目标。 严格，逐步的方法来开发针对特定突变的IND - 一种可能的策略 为所有BD的人扩展 - 与应用有关的中心问题 基因编辑，用于基因型异质主要疾病。 模型作为定制的准直式工具，以快速开发体细胞基因组编辑策略。