Targeting the 3 splice site by a decoy oligonucleotide attenuates U2AF1 splicing activity and inhibits leukemia
Recurrent mutations in spliceosomal genes are a hallmark of myeloid malignancies, with SF3B1, SRSF2, U2AF1 and ZRSR2 among the most frequently affected. These alterations are typically heterozygous, mutually exclusive missense mutations targeting highly conserved residues, reflecting a selective pressure to maintain a dysregulated yet essential splicing machinery. This constraint suggests that leukemic cells remain dependent on residual splicing activity, exposing a potential therapeutic vulnerability that extends beyond genetically defined subsets. For example, a previously developed therapeutic, Pladienolide B, is a potent cancer cell growth inhibitor targeting the SF3B1 subunit of the spliceosome1,2. Here we present an RNA decoy-based strategy to disrupt 3' splice site recognition by competitively engaging components of the spliceosomal machinery. We engineered a chemically stabilized RNA decoy that mimics the 3 splice site (3'SS decoy), thereby sequestering proteins involved in 3 splice site recognition from endogenous pre-mRNA targets. Although the decoy is expected to engage multiple components of the 3' splice site recognition complex, U2AF1 was used as the primary molecular readout to assess target engagement and downstream effects. To enable intracellular delivery, decoys were encapsulated in lipid nanoparticles (LNPs), facilitating efficient uptake in leukemic systems. We show that LNP-encapsulated decoys are efficiently delivered into leukemic cells, including established cell lines and patient-derived blasts, and directly engage components of the splicing machinery. Decoy treatment induces widespread alterations in RNA splicing programs and impairs leukemic cell fitness in vitro. Importantly, systemic administration of the 3SS decoy significantly reduces leukemia burden in an in vivo xenograft model. Notably, these effects are observed independently of spliceosomal mutational status, supporting a broader dependency of leukemic cells on intact splicing factor function. Together, our findings establish decoy-mediated disruption of splicing factor activity as a mechanistically targeted therapeutic strategy and identify LNPs as an effective platform for the delivery of RNA-based modulators of essential RNA-protein interactions in myeloid malignancies.