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）， 是一种相对常见的遗传性黄斑变性障碍，导致残留色素上皮（RPE） 功能障碍和中央视力的逐渐丧失。 BD是由200多个不同的错义突变引起的 最好的1基因。目前，没有针对常染色体显性bd的治疗方法，部分原因是 相关动物模型。为了克服建模许多致病性最佳作用的困难1 突变，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 基因组编辑线索。与追随者项目2一样，我们假设CAS9介导的indel形成。 在突变体中，最好的1等位基因会导致移架，导致过早的停止密码子，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 输入等位基因。我们在项目3中的目标是开发SNC产品SNC-2012，其中包含CAS9有效载荷 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。为了实现这一目标，我们将追求四个目标。首先，我们将生成Dual Best1等位基因 BD IPSC-RPE报告基因系统促进/非目标分析突变等位基因靶向。我们已经发展了 BD双报告基因IPSC系列，其最佳1等位基因与3'TDTOMATO RETORTER相关联和野生型 Best1等位基因链接到3'GFP记者。通过荧光成像，我们可以监视两者的表达 有针对性的突变体1等位基因和野生型等位基因，这是我们策略的顶级“脱离目标”站点。另外，我们 可以监视分类单元格上的通道功能。我们将适应此记者IPSC系列以创建野生型和八个 其他突变线。其次，我们比较铅SNC-201S的模块化编辑器组件。使用 双重记者系统翻译的SNC公式包含RNP或CAS9 mRNA/SGRNA，我们将迅速 屏幕SNC公式破坏双重记者IPSC-RPE中的每个突变等位基因。编辑的比较 通过这项工作将实现Cas9 mRNA/sgrNA和RNP的效率。第三，我们扩展合成 SNC RNP的铅并依靠铅项目1来规定缩放mRNA/sgrna铅。最后，我们 生成用于SNC-201的临床前包装，以靶向选定的突变体BEST1等位基因。评估安全性后 在非人类素数中的SNC-2012制定中，我们将完成与FDA的一次互动会议 旨在治疗多个不同突变的BD患者的产品。获得反馈后，我们将起草 临床开发计划的预定会议。成功完成我们的目标将提供 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 - 解决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。它也将推动IPSC的实用程序 作为自定义临床前工具的模型，可以快速开发体细胞基因组编辑策略。