Antisense oligonucleotide rescue of CGG expansion-dependent <i>FMR1</i> mis-splicing in fragile X syndrome restores FMRP.

Abstract

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the <i>FMR1 (Fragile X Messenger Ribonucleoprotein 1)</i> gene is transcribed in >70% of the FXS tissues. In all <i>FMR1</i>-expressing FXS tissues, <i>FMR1</i> RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known <i>FMR1</i>-217 RNA isoform, which is comprised of <i>FMR1</i> exon 1 and a pseudo-exon in intron 1. <i>FMR1</i>-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced <i>FMR1</i>, antisense oligonucleotide (ASO) treatment reduces <i>FMR1</i>-217, rescues full-length <i>FMR1</i> RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, <i>FMR1</i> gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases <i>FMR1</i>-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length <i>FMR1</i> expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing <i>FMR1-217</i>, ASO treatment may offer a therapeutic approach to mitigate the disorder.