Translational development of novel CRISPR approach to treat genomic duplications

治疗基因组重复的新型 CRISPR 方法的转化开发

• 批准号: 10698879
• 负责人: Monkol Lek
• 金额: $ 27.57万
• 依托单位: MYOS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10698879
• 关键词: Amino Acid Sequence; Becker Muscular Dystrophy; Biotechnology; CRISPR therapeutics; CRISPR/Cas technology; Cardiomyopathies; Cell Line; Cell Nucleus; Cessation of life; Characteristics; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; DNA; DNA Sequence Alteration; Development; Disease Progression; Doctor of Philosophy; Dose; Duchenne muscular dystrophy; Dystrophin; Endonuclease I; Equipment; Excision; Exons; Fiber; Future; Genes; Genetic; Genomics; Goals; Health; Human; Human Cell Line; In Vitro; Injections; Intention; Introns; Laboratories; Lead; Length; Life; Measures; Molecular Analysis; Morbidity - disease rate; Mouse Cell Line; Mus; Muscle; Muscular Atrophy; Mutation; Myopathy; Pathogenicity; Pathology; Patient-Focused Outcomes; Patients; Pharmacology; Phase; Production; Proteins; Public Health; RNA; RNA Splicing; Reading Frames; Recombinant adeno-associated virus (rAAV); Reporting; Site; Skeletal Muscle; Small Business Technology Transfer Research; Structural Protein; System; Testing; Therapeutic; Toxicology; Universities; Validation; Work; assay development; base editing; clinical translation; commercialization; dystrophinopathy; first-in-human; gene replacement therapy; gene therapy clinical trial; genotoxicity; improved; improved outcome; in vivo; insertion/deletion mutation; lead candidate; manufacture; micro-dystrophin; mouse model; muscular structure; novel; nuclease; restoration; skills; targeted nucleases; therapeutic development; therapy development; tool; translatable strategy; translational study

PROJECT ABSTRACT The goal of this Phase I STTR project is to accomplish key milestones in commercializing a novel SaCas9 nickase-based system (CRD-002) to treat dystrophinopathies in order to restore the production of healthy, endogenous full-length dystrophin protein otherwise compromised by duplication mutations. Duchenne muscular dystrophy (DMD) is characterized by progressive muscle loss and cardiomyopathy ultimately leading to death in the third decade of life. There is thus a strong rationale for mutation-specific therapies that can restore endogenous full-length dystrophin in order to restore the entire function of the dystrophin protein that cannot be achieved by current microdystrophin gene replacement therapy. Duplication mutations are ideal candidates for mutation-specific correction as they are amenable to full-length gene restoration by removal of duplicated exons to restore the normal reading frame. Pathogenic duplications underlie 10% to 15% of DMD mutations, but there is no specific therapy for duplication mutations downstream of exon 5. The Lek lab developed a novel single nickase approach that efficiently removes DMD duplications at in vitro efficiencies comparable to nuclease approaches. Importantly, nickase produces a scarless edit at the target site, whereas nucleases produce a characteristic insertion/deletion mutation. The novel exon 18-30 tandem duplication mouse model for DMD (Dup18-30), provides a valuable tool to test DMD human therapies. Sequences in DMD exons 18-30 that are shared between the human and mouse genes will be targeted with this novel nickase strategy to induce a scarless edit that restores the gene sequence without introducing frameshifts. The objective of this Phase I STTR project is to characterize the efficacy and pharmacology of the novel single- guide nickase strategy to remove duplications using human-mouse compatible targets for direct clinical translation. Myos, Inc., develops and commercializes novel biomedical therapeutics to improve outcomes for patients. This STTR project is a collaboration with Monkol Lek, Ph.D. of Yale University to optimize the CRD- 002 CRISPR nickase approach. Aim 1 is to characterize human-mouse compatible editing targets to identify lead candidate for in vivo validation. This work will assess the in vitro editing efficiency of three candidate CRISPR guides in human and mouse cell lines, and will validate the predicted target sites and characterize off- target effects. Aim 2 is to perform a functional assessment of CRD-002 in the Dup18-30 mouse model. CRD- 002 packaged in rAAV9 will be injected systemically into Dup18-30 mice followed by molecular analysis of the splice site, analysis of DMD protein sequence, and functional analysis of muscle structure. Successful completion of this project will demonstrate the feasibility of using CRD-002 to restore full-length dystrophin protein. A future Phase II project will support the manufacturing development and the pivotal GLP toxicology study to support the submission of an IND for future clinical trials.

项目摘要 这个第一阶段STTR项目的目标是完成将新型SaCas9商业化的关键里程碑 以镍酶为基础的系统(CRD-002)用于治疗营养不良症，以恢复生产 健康的内源性全长营养不良蛋白，否则会受到复制突变的影响。 Duchenne肌营养不良症(DMD)以进行性肌肉萎缩和心肌病为特征 最终导致在生命的第三个十年死亡。因此，突变特异性有很强的理由 可以恢复内源性全长dystrophin的治疗方法，以恢复患者的整体功能 目前的微营养不良蛋白基因替代疗法无法获得的营养不良蛋白。 重复突变是突变特异性纠正的理想候选者，因为它们服从于全长 通过去除重复的外显子来恢复正常阅读框架的基因修复。病原复制 10%到15%的DMD突变是基础，但下游的重复突变没有特效治疗方法 外显子5。Lek实验室开发了一种新的单镍酶方法，它有效地消除了DMD在 体外效率可与核酸酶方法相媲美。重要的是，ickase在 靶点，而核酸酶产生特有的插入/缺失突变。小说外显子18-30 DMD串联复制小鼠模型(Dup18-30)，为测试DMD人类疗法提供了一个有价值的工具。 人类和小鼠基因共享的DMD外显子18-30中的序列将被作为靶点 新的镍酶策略，诱导无疤痕的编辑，恢复基因序列，而不引入移码。 这项第一阶段STTR项目的目标是表征新型单抗的疗效和药理作用。 使用人鼠相容靶点指导镍酶策略消除重复，直接用于临床 翻译。Myos，Inc.开发和商业化新的生物医学疗法，以改善治疗结果 病人。该STTR项目是与耶鲁大学博士Monkol Lek合作，以优化CRD- 002 CRISPR昵称方法。目标1是表征人-鼠标兼容的编辑目标以识别 体内验证的主要候选者。这项工作将评估三个候选者的体外编辑效率 CRISPR在人和小鼠细胞系中进行指导，并将验证预测的靶点并确定其特性。 目标效果。目的2是在Dup18-30小鼠模型上进行CRD-002的功能评估。CRD- 包装在rAAV9中的002将系统地注射到Dup18-30小鼠体内，随后对 剪接位点、DMD蛋白序列分析、肌肉结构功能分析。 该项目的成功完成将证明使用CRD-002进行全长修复的可行性 营养不良蛋白。未来的第二阶段项目将支持制造业发展和关键的GLP 毒理学研究，以支持提交IND用于未来的临床试验。