Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations inNEFL

Abstract

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplo-sufficient. We previously edited a dominantNEFLmissense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of differentNEFLmissense mutations cause disease. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis withNEFLdisease mutations for gene excision. We validated this haplotype editing approach for two different missense mutations and demonstrated its therapeutic potential in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent byproduct of excision editing, failed to reliably disrupt mutant allele expression. We deployed alternative strategies and novel molecular assays to increase therapeutic editing outcomes while maintaining specificity for the mutant allele. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.