Overexpression of adapted U1snRNA in patients' cells to correct a 5' splice site mutation in propionic acidemia.

Splicing defects account for 16% of the mutant alleles in the PCCA and PCCB genes, encoding both subunits of the propionyl-CoA carboxylase (PCC) enzyme, defective in propionic acidemia, one of the most frequent organic acidemias causing variable neurological impairment. Most of the splicing mutations identified affect the conserved 3' splice (3' ss) or 5' splice (5' ss) sites, the latter predictably through lowering the strength of base pairing with U1snRNA. Among the 5' ss mutations we have focused on the c.1209+3A>G (IVS13+3A>G) mutation in the PCCA gene, identified in four patients (three homozygous and one heterozygous) of common geographical origin and causing exon 13 skipping. To study the potential of splicing modulation to restore PCC function, we analyzed the effect of transient transfections in patients' cells with modified U1snRNA adapted to compensate the mutant change and other mismatches at different positions of the 5' ss. Using this strategy normal transcript could be efficiently recovered with the concomitant disappearance of the aberrant exon skipping transcript, as observed after standard RT-PCR and sequence analysis or using fluorescent primers and semiquantitative RT-PCR. Different efficiencies with up to 100% exon inclusion were observed depending on the transfection conditions and specifically on the adapted U1snRNA used, confirming previously reported dependencies between nucleotides at the 5' ss for its correct recognition by the spliceosome. The reversal of the splicing defect did not result in a significant increase in enzyme activity, suggesting other factors must be taken into account for the application of overexpression of splice factors such as U1 as therapeutic strategy for splice defects.