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 out 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 ASOs 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, 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-dependent endocytosis. Mechanistic investigations revealed that B3GALT2 overexpression leads to a significant increase in ASO uptake. These findings highlight B3GALT2 as a novel modulator of ASO cellular entry and establish a new avenue for potentially improving ASO-based therapeutic strategies. Overall design: Comparison of transcriptomic profiles of HEK293 cells under three conditions: wild-type, cells with doxycycline-inducible B3GALT overexpression, and control cells carrying an empty vector. Induction with doxycycline (0 ng/mL) was performed for 72 hours, and RNA was extracted for sequencing. The key variable under investigation is the effect of B3GALT2 overexpression on gene expression.