Allele-specific inactivation for dominant negative NEFL Mutations

抽象的。 导致脊髓运动神经元退化的显性突变是最常见的致残 遗传性疾病，没有有效的治疗方法。基因组编辑正在迅速走向临床应用， 然而，要将这种潜力转化为主要神经疾病的现实，仍然存在许多挑战。特别是， 对显性突变的治疗性编辑需要精确到只针对突变的等位基因，这 通常与野生型等位基因只有一个核苷酸不同。此外，对于许多基因来说，大量的 显性突变可导致病理改变。为每个人设计和测试治疗试剂 突变是令人望而生畏的，但针对顺式病毒杂合变异的常见位置与疾病突变可能 克服这一挑战，治疗很大比例的患者。夏科-玛丽-牙病2E型(CMT2E) 是一个罕见但具有说明性的例子，因为它会导致严重的衰弱疾病，并已知由&gt；30引起 NEFL基因的不同突变，每年都有更多的报道。这项建议的主要目标是 以CMT2E为例，开发并验证显性运动神经元病的治疗性基因编辑平台 一个测试案例。NEFL基因罕见的功能丧失突变是以隐性方式遗传的 证明杂合子携带者是健康的，有力地证明了基因的单一功能拷贝 就足够了。这表明，有针对性地灭活疾病等位基因将是一种有效的治疗方法。 策略。事实上，我们的初步数据显示，专门针对严重的CMT2E突变对 预防体外病理。我们建立了一个基于人类诱导多能干细胞的CMT2E模型 细胞(IPSCs)，观察到由突变型IPSCs分化而来的运动神经元的严重表型。我们的人类 基于IPSC的CMT2E模型为设计治疗性编辑策略提供了理想的平台。第一个目标是， 我们将仔细测试两个不同CMT2E突变的突变特异性编辑，并开发出严格的表型 检测人ipsc来源运动神经元的治疗效果。在第二个目标中，我们将在实验中 确定控制NEFL表达的重要非编码调控序列，其中有很大比例 发现了许多常见的变种。在第三个目标中，我们将系统地筛选常见的杂合子 可通过针对等位基因的编辑来删除蛋白质编码或关键调控区域和 使疾病等位基因失活。这些目标的完成将为临床前研究奠定基础。 CMT2E的基因编辑疗法。这些研究还将为以下战略方法提供概念验证 可推广到其他显性神经遗传性疾病，如其他形式的CMT2和ALS。

Abstract. Dominant mutations causing degeneration of spinal motor neurons are among the most common disabling genetic diseases and have no effective treatment. Genome editing is rapidly moving toward clinical application, yet many challenges remain to translate this potential into reality for dominant neurologic diseases. In particular, therapeutic editing for dominant mutations requires exquisite precision to target only the mutant allele, which often differs from the wild-type allele by only a single nucleotide. Furthermore, for many genes, a multitude of dominant mutations can result in pathology. Designing and testing therapeutic reagents for every individual mutation is daunting, but targeting common sites of heterozygous variation in cis with the disease mutation could overcome this challenge and treat a large proportion of patients. Charcot-Marie-Tooth disease type 2E (CMT2E) is a rare but illustrative example, as it causes severe debilitating disease and is known to be caused by >30 different mutations in the NEFL gene, with more reported yearly. The primary objective of this proposal is to develop and validate a therapeutic gene editing platform for dominant motor neuron diseases, using CMT2E as a test case. Rare, loss-of-function mutations in the NEFL gene are inherited in a recessive manner and demonstrate that the heterozygous carriers are healthy, strong evidence that a single functional copy of the gene is sufficient. This suggests that targeted inactivation of the disease allele would be an effective treatment strategy. Indeed, our preliminary data shows that specifically targeting a severe CMT2E mutation is effective at preventing pathology in vitro. We have developed a model of CMT2E based on human induced pluripotent stem cells (iPSCs), and observed severe phenotypes in motor neurons differentiated from mutant iPSCs. Our human iPSC-based model of CMT2E provides an ideal platform to design therapeutic editing strategies. In the first aim, we will carefully test mutation-specific editing for two different CMT2E mutations and develop rigorous phenotypic assays for therapeutic effect in human iPSC-derived motor neurons. In the second aim, we will experimentally identify the important non-coding regulatory sequences that control NEFL expression, where a large proportion of common variants are found. In the third aim, we will systematically screen for the common heterozygous variants that can be targeted by allele-specific editing to excise protein coding or critical regulatory regions and inactivate the disease allele. Completion of these aims will build the foundation for pre-clinical development of gene editing therapies for CMT2E. These studies will also provide proof-of-concept for a strategic approach that can be generalized to other dominant neurogenerative diseases, such as the other forms of CMT2, and ALS.