Abstract 6694: Multiplex RNAi delivers on-versus-off tumor precision in T cells

Abstract Selective inhibition of graft-versus-host disease (GvHD) while preserving graft-versus-leukemia (GvL) remains a critical challenge in allogeneic stem cell transplantation. More broadly, engineering on-versus-off tumor selectivity represents a central precision medicine problem in T cell-mediated immunotherapies. Evidence suggests that T cell phenotype distributions, defined by transcriptomes and transcriptional regulation, are key determinants of this selectivity. RNA interference (RNAi) therapies, including small interfering RNAs (siRNAs) and microRNA (miRNA) mimics, represent a novel class of precision medicine tools capable of engineering cellular transcriptomes. We identified an siRNA cocktail targeting RAN, WAPAL, and AURKA that achieved GvHD inhibition comparable to approved therapies (35-95% vs. 55-91% reduction in T cell proliferation) while sparing GvL effects (no increase in leukemia proliferation, compared to a 7-20-fold increase with approved drugs). Notably, substituting any siRNA in the cocktail drastically impaired GvHD inhibition (up to a 10-fold reduction), and individual siRNAs against WAPAL and AURKA showed no significant GvHD inhibitory effects. While RAN siRNA alone was highly effective against GvHD in vitro (>90% inhibition of T cell proliferation), it compromised bead-activated T cells. Thus, the RNAi-based transcriptome engineering strategy demonstrated that targeting RAN, WAPAL, and AURKA synergistically inhibits GvHD, while also conferring selectivity to this innovative therapeutic approach. The effect of chemical modifications on miRNA function remains poorly understood. In our study, we screened diverse chemical modification patterns to develop miRNA replacement therapies (miR-146a, miR-181a, miR-374b) within the GvHD/GvL context. Silencing performance of miRNA mimics—whether on fully matched reporter targets or natural targets with mismatches—failed to reliably predict miRNA efficacy in inhibiting GvHD in vitro. Notably, certain miRNA mimics achieved GvHD inhibition comparable to approved therapies (60-95% vs. 55-91% reduction in T cell proliferation) while either sparing or even enhancing GvL effects (up to an 80% decrease vs. up to a 22-fold increase in leukemia proliferation). Generally, miRNA mimics with higher phosphorothioate content demonstrated stronger GvHD inhibition, whereas no such pattern was observed for GvL outcomes. RNA-Seq analysis revealed that the same marker genes could predict responses to miR-374b mimics in both GvHD and GvL assays, highlighting shared molecular pathways influencing these outcomes. Such analyses may serve as target discovery tool to uncover pathways potentially differentiating between GvHD and GvL. Multiplex RNAi therapies, such as siRNA cocktails or miRNA mimics have the potential to rewrite the T cell transcriptional program and enhance on-versus-off-tumor precision of T cell mediated cancer therapies. Citation Format: Reka A. Haraszti, Xavier Segarra-Visent, Anastasia Kremer, Tatyana Ryaykenen. Multiplex RNAi delivers on-versus-off tumor precision in T cells [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 6694.