Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

Abstract

Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin ( FXN ) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.

Elevation of SHANK3 Levels by Antisense Oligonucleotides Directed Against the 3′-UTR of the Human SHANK3 mRNA

“…Li et al . previously reported the use of ASOs to increase and stabilize mRNA levels for the yield of higher protein levels [ 34 ]. We hypothesized that the SHANK3 ASOs also have a stabilizing effect on SHANK3 mRNA levels.…”

“…For transfections, HEK293T were split into 12-well plates in a ratio corresponding to the desired culture time. Transfection times were chosen according to previously published data [ 34 , 41 ].…”

“…Recently, Li et al . could show that targeting the 3′ or the 5′-UTR with ASOs could stabilize Frataxin mRNA and thereby increase Frataxin protein levels [ 34 ]. This finding proves that targeting sequences within the 3′-UTR to impede rapid degradation poses a promising approach toward increasing mRNA stability and by this enhancing mRNA translation.…”

Effects of cocktail combinations of antisense oligonucleotides on potency

“…Although several studies have reported the potency of cocktails composed of non-gapmers or siRNAs [10][11][12], there have been no systematic evaluation of gapmer cocktails. Therefore, we evaluated combination-effect on the potency with 38 gapmer pairs targeting the same pre-mRNA.…”

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects

“…Most of the compounds in clinical development are based on gene silencing or splice switching. Multiple oligonucleotide-based activation mechanisms for gene activation have also been proposed, including promoter-targeted duplex RNAs ( 5–8 ), inhibition of repressive antisense transcripts ( 9 ), reducing noisy splicing ( 10 , 11 ), stabilizing mRNAs ( 12 ), enhancement of translation ( 13–16 ) and reducing nonproductive translation initiation ( 17 ). But to date, gene activation by oligonucleotides has proven to be significantly more challenging than gene silencing.…”