Therapeutic Potential of Rescued FMR1 Mis-Splicing in Fragile X Syndrome - Fragile X Syndrome (FXS) is a neurodevelopmental disorder that is the most common inherited form of
intellectual impairment and most prevalent single gene cause of autism. FXS is caused by an expansion of 200
or more CGG triplets in the 5’ untranslated region (UTR) of the FMR1 gene, which leads to FMR1 methylation
and transcriptional silencing. The loss of the FMR1 gene product FMRP results in excessive protein synthesis
in the brain, which likely contributes to several manifestations of the disorder.
We performed deep sequencing of RNA from white bloods cells (WBCs) of individuals with FXS as well
as age-matched typically developing (TD) individuals. We found that hundreds of RNAs were up or down
regulated in FXS WBCs compared to TD. We also detected hundreds of RNAs that were mis-spliced in FXS
compared to TD. These mis-regulated RNA events were statistically significant and may constitute a robust
biomarker for FXS individuals. To our surprise, we also found that in 50% FXS WBC samples, FMR1 RNA was
synthesized. In these cases, FMR1 RNA was mis-spliced such that an exon was spliced to a “pseudo-exon”
within an FMR1 intron. This mis-spliced RNA is polyadenylated and could encode a small polypeptide whose
function, if any, is unknown. We generated 2‘-O-methoxyethyl (2’-MOE) antisense oligonucleotides (ASOs) that
tiled across the intron, the intron-exon junction, and into the pseudo-exon. When transfected into FXS WBC
lines, we found that two ASOs blocked improper FMR1 splicing, rescued proper FMR1 splicing, and most
importantly, restored FMRP to TD levels. We also detected FMR1 mis-splicing in FXS postmortem brain,
indicating the widespread nature of FMR1 mis-splicing in FXS individuals. These and other data suggest that
ASO correction of FMR1 mis-splicing and restoration of FMRP may provide an innovative therapy to treat FXS.
To assess the therapeutic potential of ASO treatment of FXS, we will investigate the mechanism of CGG-
dependent FMR1 mis-splicing, determine whether additional ASOs with different linkages, lengths, or sequences
more efficiently inhibit FMR1 mis-splicing and restore FMRP, examine whether ASO rescue occurs in iPSC-
derived FXS neurons, and assess ASO toxicity, pharmacokinetics, and brain targeting following
intracerebroventricular (ICV) injection into mice.
