Targeting a Pathogenic Variant Creating an Upstream AUG in the ENG 5' Untranslated Region with Antisense...

Pathogenic variants creating upstream open reading frames (uORFs) in the 5' untranslated region (5'UTR) of the ENG gene can disrupt translation from the main ORF and contribute to hereditary hemorrhagic telangiectasia (HHT). This is the case of the ENG c.-79C>T that introduces a uAUG shown to decrease endoglin expression and associates with HHT. Here, we investigated whether 2'-O-methyl (2'OMe) antisense oligonucleotides (ASOs) could restore protein levels by masking this aberrant uAUG or by targeting predicted secondary structures within the ENG 5'UTR. Several ASOs of varying lengths and backbone chemistries (full phosphodiester or full phosphorothioate) were designed to target the mutant region. Their effects were evaluated in HeLa cells transfected and in HUVECs transduced with wild-type or mutant ENG constructs. Transfection efficiency was verified by MALAT1 knockdown via qPCR, and endoglin protein levels were assessed by Western blot. Despite efficient ASO delivery and optimized experimental conditions, no reproducible increase in endoglin expression was observed upon ASO treatment. These findings highlight the limitations of steric-blocking ASOs targeting 5'UTR variants and underscore the need for deeper mechanistic understanding of uORF-mediated translational regulation.