Abstract 483: RNA-missplicing dependent nucleic acid therapeutics for SF3B1-mutant cancers

Cancer therapies face the challenge of specificity-limiting toxicity to normal cells and maximizing it in malignant ones. The last two decades have seen significant advancements in therapeutics that increase specificity; however, these remain elusive for the many cancerous aberrations that affect nonenzymatic, intracellular proteins. One such protein, SF3B1, mediates branch point selection in pre-mRNA splicing as part of the U2snRNP complex. Its mutation in various cancers-from hematologic malignancies to pancreatic carcinomas-creates conserved missplicing events, including alternative 3’ splice acceptors, exon exclusion/inclusion, and intron retention. Here, we sought to create nucleic acid therapeutics that leverage missplicing to express targetable or intrinsically toxic cargo in SF3B1-mutant cancers. We previously reported a highly specific missplicing event caused by mutant SF3B1: the inclusion of a cryptic exon in the UBA1 gene. Using a fragment containing this exon, we engineered a plasmid to switch between dsRed with canonical splicing and eGFP with cryptic splicing. We also created a splicing dependent iCaspase9 plasmid that induced mutant-specific cell death with transient transfection. However, when using more clinically relevant delivery with viral vectors, we discovered that expression was too low to produce a significant effect. We therefore engineered more efficient and potent plasmids that could induce toxicity even with low expression. We first tested a variety of constitutive promoters driving expression of our fluorescent switch. Across separate transient transfections in triplicate, flow cytometry consistently showed that the promoter from human elongation factor 1 alpha (EF1a) resulted in the highest ratio of cryptic to canonical fluorescence. In isogenic acute myeloid leukemia (K562) and uveal melanoma (Mel202) models, we further optimized plasmids with our synthetic UBA1 gene fragment and CD19 or diphtheria toxin A subunit (DTA) cargo. For CD19, we created a fusion of the CD44 signal peptide and CD19 extracellular domain that demonstrated high specificity for SF3B1-mutant clones through anti-CD19 staining and flow cytometry. For DTA, our plasmid with an attenuated version of the toxin induced statistically significant, highly mutant-specific cell death, both through transient transfection and delivery with adeno-associated viral (AAV) vectors. Using the cryptic exon inclusion event from UBA1, we have selectively killed SF3B1-mutant cells in 2 distinct isogenic cell models in vitro. Next, we hope to further optimize plasmid delivery and splicing specificity, especially in vivo. While recent literature has shown the efficacy of a different cryptic intron inclusion event in inducing SF3B1 mutant-specific death, our approach may result in greater splicing specificity. Combined, our results underscore the promise of mutant-SF3B1-targeting nucleic acid therapies. Citation Format: Benjamin G. Dubner, Jian Xiang Peng, Rebecca Foerstch, In-Young Choi, Panagiotis Tsakiroglou, Ilias Sinanidis, Fatemeh Rajaii, Ben Park, Jonathan Ling, William Dalton. RNA-missplicing dependent nucleic acid therapeutics for SF3B1-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 483.