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

• Published in: Nucleic acids research 2022-12, Vol.50 (22), p.12657-12673
• Main Authors: Wang, Feng, Calvo-Roitberg, Ezequiel, Rembetsy-Brown, Julia M, Fang, Minggang, Sousa, Jacquelyn, Kartje, Zachary J, Krishnamurthy, Pranathi Meda, Lee, Jonathan, Green, Michael R, Pai, Athma A, Watts, Jonathan K
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Chemical Biology and Nucleic Acid Chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Frataxin; Friedreich Ataxia - genetics; Gene Expression Regulation; Humans; Iron-Binding Proteins - genetics; Iron-Binding Proteins - metabolism; Oligonucleotides, Antisense - genetics; Oligonucleotides, Antisense - metabolism; Oligonucleotides, Antisense - pharmacology; RNA, Messenger - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkac1108

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.