Allele-specific inactivation for dominant negative NEFL Mutations

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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.