In vivo precision genome editing to correct genetic disease

体内精准基因组编辑以纠正遗传疾病

• 批准号: 10771419
• 负责人: Gregory A. Newby
• 金额: $ 24.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10771419
• 关键词: ATAC-seq; Adult; Affect; Alleles; Animal Model; Binding; Binding Sites; Brain; Cardiac Myocytes; Cell division; Cells; ChIP-seq; Chromatin; Clinical; Clinical Trials; DNA Sequence Alteration; Databases; Defect; Dependovirus; Detection; Dilated Cardiomyopathy; Disease; Dose; Echocardiography; Engineering; Flow Cytometry; Frequencies; Future; Genes; Genetic; Genetic Diseases; Genetic Models; Genetic Transcription; Genome; Goals; Guide RNA; Heart; Heart Diseases; Heart failure; Human; Injectable; Injections; Interphase Cell; Intervention; Intravenous; Length; Life; Liver; Measures; Mentors; Methodology; Methods; Microscopy; Modality; Modeling; Mus; Muscle; Mutation; Myocardium; Newborn Infant; Nucleotides; Other Genetics; Outcome; Patients; Persons; Phase; Phenotype; Photography; Point Mutation; Population; Postdoctoral Fellow; Promoter Regions; Rare Diseases; Reporter; Site; Specificity; Technology; Therapeutic; Tissues; United States; Up-Regulation; Variant; Viral; Work; base; base editing; base editor; cell type; delivery vehicle; genome editing; human disease; improved; in vivo; induced pluripotent stem cell; insertion/deletion mutation; interest; lipid nanoparticle; mouse genome; mouse model; mutation correction; nanoparticle; nanoparticle delivery; nanopolymer; precise genome editing; prime editing; prime editor; promoter; reduce symptoms; repair strategy; repaired; screening; success; therapeutic development; therapeutic genome editing; tissue culture; tool; transcription factor

PROJECT SUMMARY Genetic diseases impact over 1 in 50 newborns worldwide and yet there are no approved therapies capable of correcting the underlying genetic defects. As a result, most patients continue to suffer throughout their life and require frequent interventions to ameliorate symptoms. I aim to develop in vivo genome editing therapeutics that correct the underlying disease mutation in relevant tissues by a single injection into the patient. Base editors can efficiently correct transition point mutations, the most common form of disease- causing genetic mutation, without undesired editing outcomes such as indels. With one dose and no subsequent enrichment, over 95% of cells in tissue culture can be edited, and editing in over 60% of non- dividing cells in targeted adult mammalian tissues has been demonstrated in early in vivo work. Prime editors can correct any genetic perturbation of up to at least ~50 nt in length (encompassing ~89% of human disease mutations). I will develop and assess both precision genome editing technologies (base and prime editors) using suitable in vivo delivery tools in mouse models to develop therapeutics for genetic disease. Dilated cardiomyopathy (DCM) is a frequent form of genetic heart disease, affecting an estimated 300,000 people in the United States, and can lead to heart failure. Common causes of DCM are haploinsufficiency of important genes in cardiomyocytes including TTN and LMNA. I will employ screens to identify new editing strategies to treat haploinsufficiencies by enhancing transcription of the healthy allele. I will characterize the mechanism of identified edits to understand the associated changes in transcription factor occupancy and chromatin states. Simultaneously, I will use fluorescent reporter mice to characterize the in vivo delivery of base editor and prime editor tools in order to find the best method for editing cardiomyocytes. This work will include characterizations of tissue- and cell-specific editing following delivery via adeno-associated virus, lipid nanoparticles, or polymer nanoparticles. I will then combine the identified therapeutic editing strategy with the best vehicle for delivery to cardiomyocytes to treat a mouse model of TTN haploinsufficiency. I will measure on- and off-target editing as well as any improvement in the contractility defect that defines this model. Base editors and prime editors can be readily reprogrammed to correct one or even multiple simultaneous mutations by altering the co-delivered guide RNA. Future work will expand this screening methodology to additional haploinsufficiency disorders, and applying identified delivery methods to new models of genetic disease. The ultimate goal of this work is to develop in vivo genome editing therapeutics that can be readily adapted to treat even rare or one-of-a-kind disease variants.

项目概要 遗传病影响着全球超过五分之一的新生儿，但尚无批准的治疗方法 能够纠正潜在的遗传缺陷。结果，大多数患者在整个过程中持续遭受痛苦 他们的生活，需要经常干预以改善症状。我的目标是开发体内基因组编辑 通过单次注射到相关组织中纠正潜在疾病突变的疗法 病人。碱基编辑器可以有效地纠正转变点突变，这是最常见的疾病形式 导致基因突变，而不会产生意外的编辑结果，例如插入缺失。一剂且无 随后的富集，组织培养中超过95%的细胞可以被编辑，并且超过60%的非组织细胞可以被编辑。 早期的体内工作已证明成年哺乳动物目标组织中的分裂细胞。首席编辑 可以纠正长度至少~50 nt的任何遗传扰动（包括~89%的人类疾病） 突变）。我将开发和评估精确基因组编辑技术（基础编辑器和主要编辑器） 在小鼠模型中使用合适的体内递送工具来开发遗传疾病的治疗方法。 扩张型心肌病（DCM）是一种常见的遗传性心脏病，影响估计 在美国有30万人，可导致心力衰竭。 DCM 的常见原因是 心肌细胞中重要基因（包括 TTN 和 LMNA）的单倍体不足。我将使用屏幕来 确定新的编辑策略，通过增强健康等位基因的转录来治疗单倍体不足。我会 表征已识别编辑的机制，以了解转录因子的相关变化 占据和染色质状态。同时，我将使用荧光报告小鼠来表征体内 提供碱基编辑器和初等编辑器工具，以便找到编辑心肌细胞的最佳方法。这 工作将包括通过腺相关传递后组织和细胞特异性编辑的表征 病毒、脂质纳米颗粒或聚合物纳米颗粒。然后我将结合确定的治疗编辑 使用最佳载体递送至心肌细胞来治疗 TTN 单倍体不足的小鼠模型的策略。 我将测量脱靶编辑以及定义这一点的收缩性缺陷的任何改进 模型。碱基编辑器和主要编辑器可以轻松地重新编程以纠正一个甚至多个 通过改变共同传递的指导RNA来同时突变。未来的工作将扩大这种筛选 其他单倍体不足疾病的方法，并将已确定的递送方法应用于新模型 的遗传病。这项工作的最终目标是开发体内基因组编辑疗法 很容易适应治疗罕见或独一无二的疾病变异。