Reprogramming TFs PU.1 IRF8, and BATF3, together with a GFP reporter, were cloned as monocistronic and polycistronic cassettes into established in vitro transcription (IVT) templates for the production of linear, circular, and self-replicating RNAs. In vitro analyses across human dermal fibroblasts, as well as mouse and human cancer cell lines, demonstrated that all three tested RNA modalities enabled transgene expression with distinct expression levels and kinetics, consistent with their structural properties. GFP expression from linear mRNA and circular RNA (circRNA) peaked at day 2, with linear mRNA inducing strong but transient expression, whereas circRNA provided more sustained expression over time. Self-replicating RNA (srRNA) enabled durable expression but was associated with increased cellular toxicity.
All RNA platforms successfully induced cDC1 reprogramming, as illustrated by the upregulation of antigen-presentation markers HLA-ABC/MHC-I and CD40 across fibroblasts and cancer cell lines. Notably, circRNA induced enhanced reprogramming efficiencies, whereas srRNA promoted lower efficiency but more durable reprogramming.
In primary patient-derived tumor samples, particularly head and neck carcinomas, we observed the induction of antigen-presenting phenotypes and functional T cell activation, with circRNA-LNPs showing the most pronounced effects, as measured by IFN-γ production and CD69 upregulation following co-culture of T cells with reprogrammed cells.
Intratumoral injection of GFP-encoding RNAs formulated in LNPs into established MC38 colon carcinoma tumors enabled transgene expression within solid tumors in vivo, with circRNA exhibiting the most persistent expression compared to mRNA and srRNA. Furthermore, in situ reprogramming in xenograft models resulted in the upregulation of antigen-presentation and co-stimulatory markers in tumor cells, supporting the feasibility of RNA-mediated induction of tumor immunogenicity in vivo. Importantly, circRNA-reprogrammed tumors exhibited significantly delayed tumor growth and achieved complete tumor remission in a subset of animals, outperforming both mRNA and srRNA.
Collectively, these findings demonstrates that RNA-mediated cDC1 reprogramming induces anti-tumor immunity in vivo, paving the way for the development of a non-viral, scalable, and potentially safer immunotherapy strategy. Furthermore, we effectively disseminated and communicated the project’s outcomes, resulting in two peer-reviewed publications (with an additional manuscript currently under revision), as well as a published protocol on in vivo cDC1 reprogramming and presentations at international conferences. In parallel, our close collaboration with Asgard Therapeutics and clinical collaborators ensured strong innovation and exploitation outputs. This led to the submission of one provisional patent application, a comprehensive competitive landscape analysis, and a targeted search for potential partners to support future development. These efforts also enabled the establishment of an initial Target Product Profile for RNA-mediated in vivo reprogramming.
