In vivo precision genome editing to correct genetic disease

项目摘要 遗传性疾病影响全世界每50个新生儿中就有一个，但目前还没有批准的治疗方法。 能够纠正潜在的遗传缺陷。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 通过单次注射到相关组织中来纠正潜在疾病突变的治疗剂， 病人碱基编辑器可以有效地纠正转换点突变，这是最常见的疾病形式- 导致基因突变，而没有不希望的编辑结果，如插入缺失。一次注射， 在随后的富集中，组织培养物中超过95%的细胞可以被编辑，并且在超过60%的非细胞中编辑。 已经在早期的体内工作中证明了靶向成年哺乳动物组织中的分裂细胞。主要编辑 可以纠正长度至少为~50 nt的任何遗传干扰（包括~89%的人类疾病 突变）。我将开发和评估两种精确的基因组编辑技术（基础和主要编辑器） 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剂。 扩张型心肌病（DCM）是一种常见的遗传性心脏病， 在美国有30万人，并可能导致心力衰竭。DCM的常见原因是 心肌细胞中重要基因的单倍不足，包括TTN和LMNA。我会用屏幕 鉴定新的编辑策略以通过增强健康等位基因的转录来治疗单倍性缺失。我会 描述已识别编辑的机制，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 提供基本编辑器和主要编辑器工具，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病毒递送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。然后我将联合收割机 使用最佳载体递送至心肌细胞以治疗TTN单倍不足的小鼠模型的策略。 我将测量对靶编辑和脱靶编辑以及定义这一点的收缩性缺陷的任何改善 模型基础编辑器和主要编辑器可以很容易地重新编程，以纠正一个或甚至多个 通过改变共同递送的指导RNA来同时突变。未来的工作将扩大这种筛选 方法，并将确定的递送方法应用于新模型 遗传疾病的。这项工作的最终目标是开发体内基因组编辑疗法， 很容易适应于治疗甚至罕见的或独一无二的疾病变体。

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.