Identification of c.146G > A mutation in a Fabry patient and its correction by customized Cas9 base editors in vitro

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to reduced alpha-galactosidase (alpha-Gal A) activity. Current treatments, like enzyme replacement, have limitations affecting efficacy and patient outcomes. CRISPR/Cas9 genome editing tools may offer the potential to develop therapeutic strategy via correcting GLA mutations. In this study, we diagnosed a female FD patient with a missense mutation in exon 1 of the GLA gene (c.146G > A, p.R49H). Bioinformatic predictions and biochemical analyses in GLA-knockout cells revealed that this mutation significantly reduced alpha-Gal A stability and activity, confirming its pathogenicity. To correct this, we used adenine base editing. The mutation, along with a nearby bystander A, was efficiently edited by the traditional N-terminal adenine base editor. To avoid unwanted bystander editing, we developed a series of domain-inlaid base editors with the aim of narrowing editing window. The most effective variant, with deaminase inserted between the 947th and 948th residues of the RUVC3 domain, was further optimized by modifying linker rigidity. These adjustments shifted the editing window, eliminating bystander editing. Our findings clarify the pathogenic nature of a novel GLA mutation and demonstrate the potential of a customized base editor for therapeutic application in FD.