# Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL

## TL;DR

Researchers developed a gene-editing strategy to treat a genetic disorder by targeting common DNA variants linked to disease-causing mutations in motor neurons.

## Contribution

The study introduces haplotype editing as a mutation-agnostic approach to inactivate dominant NEFL mutations in CMT2E.

## Key findings

- Haplotype editing rescued disease phenotypes in iPSC-derived motor neurons with different NEFL missense mutations.
- Gene inversion, a common editing by-product, failed to reliably disrupt mutant allele expression.
- Population genetics analysis showed haplotype editing can benefit the largest number of patients with CMT2E.

## Abstract

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. 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.

Judge and colleagues demonstrated mutation-agnostic therapeutic gene editing using induced pluripotent stem cell (iPSC) models of Charcot-Marie-Tooth type 2E. They targeted common variants to inactivate dominant NEFL missense mutations and rescue phenotypes in iPSC motor neurons. This haplotype editing approach could streamline therapeutic development for CMT2E and other genetic diseases.