Engineered Immunoagonist Non‐Coding RNA (incRNA) Activates Dual TLR Pathways for Cancer Immunotherapy

Conventional mRNA therapeutics have focused on optimizing translation and minimizing immunogenicity for vaccine and protein replacement applications. However, immunogenicity, often considered a challenge, can also be harnessed for therapeutic advantage. This work challenges the necessity of extensive mRNA modification as a universal strategy by introducing 'immunoagonist non-coding RNA (incRNA)', a new class of RNA therapeutic that exploits innate immune activation rather than evading it. incRNA is produced through in vitro transcription using a non-coding plasmid DNA template without modified nucleotides or rigorous purification, leveraging the immunotherapeutic potential of single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) dual pattern recognition. Encapsulation and delivery of incRNA in lipid nanoparticles (LNPs) in vitro resulted in robust immunogenic reprogramming, including macrophage repolarization and dendritic cell maturation. These phenotypes are driven by potent dual recognition, exhibited by >20-fold induction of TLR3/interferon (dsRNA) and sixfold upregulation of TLR7/TNF-α (ssRNA) signaling. In vivo, incRNA delivery elicited innate and adaptive immune cell recruitment and significant tumor regression in a melanoma mouse model, observing further heightened immune recruitment in combination with anti-PD-1 checkpoint inhibition. Overall, incRNA demonstrates that the therapeutic efficacy of RNA therapeutics is context-dependent and can be amplified by leveraging, rather than minimizing, RNA's intrinsic immunogenicity.