Activating frataxin expression by repeat-targeted nucleic acids

Abstract

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

“…However, some of these compounds demonstrate selectivity by increasing FXN expression in certain cell types without affecting FXN transcription in other lineages [15]. Similarly, in our study, compounds capable of reactivating FXN expression during somatic cell reprogramming of FRDA fibroblasts did not have an effect on FXN expression in differentiated FRDA fibroblasts.…”

“…Although from a therapeutic perspective, any increase in the FXN level is beneficial to the patients. Recent work on GAA-specific oligonucleotides reported a higher, three-to four-fold increase in FXN mRNA and protein levels [15]. These results indicate that sequence-specific secondary structures (R-loops formed at the GAA repeats) are the primary instigators of transcriptional silencing, and targeting the initial steps not only corrects pathological chromatin changes in FRDA cells but also improves efficacy [15].…”

“…Recent work on GAA-specific oligonucleotides reported a higher, three-to four-fold increase in FXN mRNA and protein levels [15]. These results indicate that sequence-specific secondary structures (R-loops formed at the GAA repeats) are the primary instigators of transcriptional silencing, and targeting the initial steps not only corrects pathological chromatin changes in FRDA cells but also improves efficacy [15]. Perhaps combining both approaches, targeting the genomic structure and enhancing epigenetic remodeling of the FXN locus, would result in a synergistic effect and increase the level of FXN reactivation, as well as prolong the therapeutic window between administration of the drugs, thus reducing potential side effects.…”

“…Chromatin changes, resembling the epigenetic silencing landscape at repetitive elements, are a hallmark of the molecular pathogenesis of FRDA [7][8][9][10][11]. Current models postulate that expanded GAAs block initiation and progression of transcription by formation of noncanonical DNA or DNA-RNA hybrid structures, leading consequently to the recruitment of silencing machineries and establishing heterochromatin-like landscape at the FXN locus [12][13][14][15].…”

Non-coding RNAs as drug targets

“…Our laboratory has targeted steric block locked nucleic acid (LNA) ASOs and duplex RNAs to the expanded GAA repeat in FA patient-derived cells (Figure 2b) 78 . These compounds reduce R-loop formation and increase FXN mRNA and protein levels to those found in wild-type cells.…”