Faculty, Staff and Student Publications
Publication Date
12-9-2022
Journal
Nucleic Acids Research
Abstract
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.
Keywords
Humans, Friedreich Ataxia, Iron-Binding Proteins, Oligonucleotides, Antisense, RNA, Messenger, Cells, Cultured, DNA-Binding Proteins, Gene Expression Regulation, Frataxin
DOI
10.1093/nar/gkac1108
PMID
36511872
PMCID
PMC9825156
PubMedCentral® Posted Date
December 2022
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Included in
Bioinformatics Commons, Biomedical Informatics Commons, Medical Sciences Commons, Oncology Commons
Comments
Supplementary Material
PMID: 36511872