Allele-specific inactivation for dominant negative NEFL Mutations

抽象的。 导致脊髓运动神经元变性的显性突变是最常见的致残性疾病之一。 遗传性疾病，没有有效的治疗方法。基因组编辑正迅速走向临床应用， 然而，要将这种潜力转化为显性神经系统疾病的现实，仍然存在许多挑战。特别是， 显性突变的治疗性编辑需要精确到只靶向突变等位基因， 通常与野生型等位基因仅相差一个核苷酸。此外，对于许多基因， 显性突变可导致病理学。为每个人设计和测试治疗试剂 突变是令人生畏的，但靶向疾病突变的顺式杂合变异的共同位点， 克服这一挑战并治疗大部分患者。腓骨肌萎缩症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性疾病，并且已知是由>30引起的 NEFL基因的不同突变，每年都有更多的报道。这项建议的主要目的是 开发并验证显性运动神经元疾病的治疗性基因编辑平台，使用CMT2E作为 一个测试案例NEFL基因中罕见的功能丧失突变以隐性方式遗传， 证明杂合子携带者是健康的，强有力的证据表明，一个单一的功能性拷贝的基因 就足够了这表明靶向灭活疾病等位基因将是一种有效的治疗方法 战略事实上，我们的初步数据表明，特异性靶向严重的CMT2E突变对 在体外预防病理学。我们已经建立了一个基于人诱导多能干细胞的CMT2E模型， 细胞（iPSC），并在从突变iPSC分化的运动神经元中观察到严重的表型。我们人类 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。在第一个目标中， 我们将仔细测试两种不同CMT2E突变的突变特异性编辑，并开发严格的表型 在人iPSC衍生的运动神经元中的治疗效果的测定。在第二个目标中，我们将通过实验 确定控制NEFL表达的重要非编码调控序列，其中大部分 在第三个目标中，我们将系统地筛选常见的杂合子， 可以通过等位基因特异性编辑靶向以切除蛋白质编码或关键调控区的变体， 致病等位基因。这些目标的实现将为临床前开发奠定基础。 CMT2E的基因编辑疗法。这些研究还将为一种战略方法提供概念验证， 可以推广到其他显性神经变性疾病，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.