uORF-targeting steric block antisense oligonucleotides do not reproducibly increase RNASEH1 expression

Upstream open reading frames (uORFs) are <i>cis</i>-regulatory motifs that are predicted to occur in the 5' UTRs of the majority of human protein-coding transcripts and are typically associated with translational repression of the downstream primary open reading frame (pORF). Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was previously reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons to inhibit translation initiation, and thereby disrupt uORF-mediated gene regulation. Given the relative maturity of the oligonucleotide field, such a uORF blocking mechanism might have widespread therapeutic utility. Here, we re-synthesized three of the most potent ASOs targeting the <i>RNASEH1</i> uORF described in a study by Liang et al. and investigated their potential for RNASEH1 protein upregulation, with care taken to replicate the conditions of the original study. No upregulation (of endogenous or reporter protein expression) was observed with any of the oligonucleotides tested at doses ranging from 25 to 300 nM. Conversely, we observed downregulation of expression in some instances. We conclude that previously described <i>RNASEH1</i> uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.