Harnessing BET-Bromodomain Assisted Nuclear Import for Targeted Subcellular Localization and Enhanced Efficacy of Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics designed to modulate gene expression. Both key mechanisms of action for ASOs operate in the nucleus: splice-switching ASOs modify pre-mRNA, processed in the nucleus, and mRNA-degrading ASOs require RNase H, an enzyme predominantly active in the nucleus. Therefore, to achieve maximal therapeutic efficacy, ASOs require efficient nuclear delivery. In this work, we have synthesized ASO conjugates for active nuclear import, by covalent conjugation with a potent and proven small-molecule nuclear importer, (+)-JQ1. (+)-JQ1 is a well-characterized high-affinity binder for members of the BET bromodomain family of proteins and was recently shown to transport cytoplasmic proteins into the nucleus. Our (+)-JQ1-ASO conjugates outperformed their unmodified counterparts for both splice-switching and mRNA knockdown in the nucleus, across different molecular targets, backbone chemistries, and cell lines. In addition, we show that the improvement in on-target efficacy correlates with increased nuclear localization of the (+)-JQ1-modified ASOs by subcellular fractionation and immunocytochemistry. Notably, we improved the performance of Oblimersen, a BCL-2 ASO drug that failed in phase-III clinical trials. (+)-JQ1-Oblimersen showed increased effectiveness in an acute myeloid leukemia cell model, showing that this therapeutic may merit re-evaluation. This work demonstrates that the covalent modification of ASOs with a small-molecule nuclear importer can significantly improve target engagement and pave the way for more effective therapeutics.