RegulatingPCCAgene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia

Abstract

Pseudoexons are nonfunctional intronic sequences that can be activated by deep intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. ThePCCAc.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder, propionic acidemia, by deficiency of the propionyl-CoA carboxylase enzyme encoded byPCCAandPCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. ThePCCAc.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. ThePCCApseudoexon can be exploited as a gene-regulatory switch, as healthy tissues show relatively high levels of inclusion. By blocking inclusion of the non-activated wild type pseudoexon, we increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels not only in patient fibroblasts harboringPCCAmissense variants, but also those harboringPCCBmissense variants. This could be a potential treatment strategy for propionic acidemia.