An unbiased whole-genome open reading frame overexpression screen identifies B3GALT2, a novel inducer of cellular ASO activity

Antisense oligonucleotides (ASOs) are a promising class of therapeutics made of chemically modified synthetic single-stranded nucleic acid molecules, yet their clinical translation is often hindered by challenges in cellular uptake and delivery. Identifying cellular factors that modulate ASO activity is crucial for overcoming these limitations. Utilizing a whole-genome open reading frame (ORF) overexpression screen with a genetic splice reporter system to identify genes that can increase or decrease ASO activity, we identified B3GALT2 as the most significant hit for increasing ASO activity. Subsequent validation in HEK293 and U2OS cell lines demonstrated that B3GALT2 overexpression consistently and significantly enhances the activity of both splice-switching and mRNA-degrading ASOs across different targets (CD81, MALAT1, and CERS2). Transcriptomic analysis of B3GALT2-overexpressing cells showed upregulation of endocytic scavenger receptors CUBN and SCARA5, and gene set enrichment analysis indicated an enrichment of proteins involved in clathrin-mediated endocytosis. Mechanistic investigations revealed that B3GALT2 overexpression leads to a significant increase in ASO uptake. These findings highlight B3GALT2 as a modulator of ASO cellular entry and offer an avenue for potentially improving ASO-based therapeutic strategies.