Double-target Antisense U1snRNAs Correct Mis-splicing Due to c.639+861C>T and c.639+919G>A GLA Deep Intronic Mutations.

Fabry disease is a rare X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (α-Gal A) enzyme, which is encoded by the GLA gene. GLA transcription in humans produces a major mRNA encoding α-Gal A and a minor mRNA of unknown function, which retains a 57-nucleotide-long cryptic exon between exons 4 and 5, bearing a premature termination codon. NM_000169.2:c.639+861C>T and NM_000169.2:c.639+919G>A GLA deep intronic mutations have been described to cause Fabry disease by inducing overexpression of the alternatively spliced mRNA, along with a dramatic decrease in the major one. Here, we built a wild-type GLA minigene and two minigenes that carry mutations c.639+861C>T and c.639+919G>A. Once transfected into cells, the minigenes recapitulate the molecular patterns observed in patients, at the mRNA, protein, and enzymatic level. We constructed a set of specific double-target U1asRNAs to correct c.639+861C>T and c.639+919G>A GLA mutations. Efficacy of U1asRNAs in inducing the skipping of the cryptic exon was evaluated upon their transient co-transfection with the minigenes in COS-1 cells, by real-time polymerase chain reaction (PCR), western blot analysis, and α-Gal A enzyme assay. We identified a set of U1asRNAs that efficiently restored α-Gal A enzyme activity and the correct splicing pathways in reporter minigenes. We also identified a unique U1asRNA correcting both mutations as efficently as the mutation-specific U1asRNAs. Our study proves that an exon skipping-based approach recovering α-Gal A activity in the c.639+861C>T and c.639+919G>A GLA mutations is active.