In vivo precision genome editing to correct genetic disease

项目总结 全球每50名新生儿中就有1人患有遗传病，但目前尚无已获批准的治疗方法。 能够纠正潜在的基因缺陷。因此，大多数患者在整个过程中继续遭受痛苦。 他们的生活和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 通过一次注射到相关组织中纠正潜在疾病突变的治疗学 有耐心的。碱基编辑可以有效地纠正过渡点突变，这是最常见的疾病形式- 导致基因突变，不会产生不希望看到的编辑结果，如indels。只用一剂，不用 后续的浓缩，95%以上的细胞在组织培养中可以编辑，60%以上的细胞在非 在早期的活体工作中，已经证明了在靶向成年哺乳动物组织中进行细胞分裂。主要编辑 可以纠正长度至少为~50个核苷酸(涵盖人类疾病的~89%)的任何遗传扰动 突变)。我将开发和评估两种精确的基因组编辑技术(基础编辑和主要编辑) 在小鼠模型中使用合适的体内给药工具来开发遗传病的治疗方法。 扩张型心肌病(DCM)是一种常见的遗传性心脏病，影响 在美国有30万人，并可能导致心力衰竭。DCM的常见原因有 心肌细胞中TTN和LMNA等重要基因的单倍性不足。我会用屏幕来 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。这就做 描述已识别编辑的机制，以了解转录因子的相关变化 占有率和染色质状态。同时，我将使用荧光报告鼠来表征体内的 提供基本编辑工具和主要编辑工具，以找到编辑心肌细胞的最佳方法。这 工作将包括通过腺相关递送后组织和细胞特异性编辑的特征 病毒、脂质纳米颗粒或聚合物纳米颗粒。然后我将结合识别的治疗性编辑 以最佳载体向心肌细胞输送治疗TTN单倍体功能不全小鼠模型的策略。 我将测量目标上和目标外的编辑，以及定义这一点的收缩缺陷的任何改进 模特。基本编辑程序和主要编辑程序可以很容易地重新编程以更正一个甚至多个 通过改变共同传递的引导RNA而同时发生突变。未来的工作将扩大这一筛选 增加单倍体功能不全的方法学，并将已确定的分娩方法应用于新模型 遗传病。这项工作的最终目标是开发体内基因组编辑疗法，可以 容易适应于治疗甚至罕见或独一无二的疾病变种。

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.