Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming.
MicroRNAs (miRNAs) regulate gene expression and generate isoforms (isomiRs) with distinct targeting properties, but their roles in cellular specification and reprogramming remain poorly understood. Here, we investigated miRNAs and isomiRs during direct reprogramming to conventional type 1 dendritic cells (cDC1s), identifying miR-124 and miR-142 as facilitators of the process. The canonical miR-124-3p strand induced a permissive chromatin landscape enriched for cooperative transcription factor motifs, thereby transiently increasing reprogramming efficiency. In contrast, miR-142 enhanced lineage fidelity by repressing fibroblast identity and activating cDC1-specific programs through cooperating isomiRs that drove XCR1 expression and increased type 3 interferon production. In addition, miR-142 mimics enhanced cancer cell reprogramming and anti-tumor immunity in vivo. Finally, we developed RNA-based cDC1 reprogramming using transcription factor mRNAs combined with miRNA mimics. Together, these findings uncover miRNA isoform diversity as a programmable regulatory layer in immune cell specification and establish RNA-driven reprogramming as a non-viral strategy for cancer immunotherapy.