Abstract
Familial dysautonomia (FD) is a fatal inherited congenital neuropathy characterized by both progressive neurological symptoms and systemic abnormalities, and patients have a reduced life expectancy. FD is caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 ( ELP1 ) gene, which affects the ELP1 splicing process by causing tissue-specific skipping of exon 20. Here, we developed a CRISPR base editor (BE) approach capable of precisely correct this mutation. Using Cas9 variants and screening multiple gRNAs, we selected an optimized BE 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 rescue more than 50% of ELP1 transcripts with precise exon 20 inclusion. Moreover, an engineered dual intein-split system was optimized to deliver these constructs in vivo . We packaged these novel constructs in adeno-associated virus (AAV) vectors for further testing in iPSC-derived sympathetic neurons and a mouse model harboring the human ELP1 T6C gene. Our strategy could effectively correct the ELP1 splicing defects in vivo and culminated in phenotypic recovery in human neurons. Minimal off-target editing was observed demonstrating high levels of specificity with these optimized base editors. Therefore, we engineered and validated novel base editor approaches to correct the ELP1 TC6 mutation and ELP1 splicing defect and provided essential proof of concept data towards a permanent treatment for FD.
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