Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia

bioRxiv [Preprint]. 2024 Dec 20:2024.11.27.625322. doi: 10.1101/2024.11.27.625322.

Abstract

Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 (ELP1) gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the ELP1 transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs in vivo. Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain ELP1 splicing defects in a humanized FD mouse model carrying the ELP1 T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.

Publication types

  • Preprint