Periodic Reporting for period 1 - DART (Driving tumour antigen presentation by RNA-mediated transdifferentiation)
Periodo di rendicontazione: 2024-08-01 al 2026-01-31
Immunotherapy has revolutionized cancer treatment. However, effectively targeting refractory solid tumors remains a challenge due to poor T-cell priming and activation. This impairment is largely driven by downregulation of antigen presentation pathways and lack of professional antigen-presenting cells in the tumor microenvironment, which contribute to immune evasion. To counteract this, we have established the direct reprogramming of tumor cells into type 1 conventional dendritic cells (cDC1) by overexpression of the transcription factors (TFs) PU.1 IRF8, and BATF3 (PIB), and is currently moving towards clinical application via a viral gene therapy. Despite its promise, this approach has limitations, including inefficient targeting of solid tumors in vivo, and scalability. As such, the DART project arose from the ERC-funded project TrojanDC, aiming to develop in vivo reprogramming of tumor cells into cDC1-like cells using RNA vectors. To achieve this, we first aimed at evaluating the capacity of different RNA modalities encoding PIB to reprogram fibroblasts and cancer cells in vitro. Secondly, we evaluated RNA-mediated reprogramming of cancer cells and assessed anti-tumor immunity in vivo.
Experimental activities with industrial collaborators and clinicians were coupled with exploitation plans, ensuring commercialization through novel intellectual property, broad dissemination, and product development. Ultimately, this project would set the stage for a new, off-the-shelf, safe, and scalable immunotherapy solution that also has the potential to enhance current immunotherapy approaches.
Experimental activities with industrial collaborators and clinicians were coupled with exploitation plans, ensuring commercialization through novel intellectual property, broad dissemination, and product development. Ultimately, this project would set the stage for a new, off-the-shelf, safe, and scalable immunotherapy solution that also has the potential to enhance current immunotherapy approaches.
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.
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.
The DART project pioneered the use of RNA for the direct in vivo reprogramming of tumor cells into cDC1-like cells, addressing key limitations associated with viral gene therapy approaches. DART provided proof-of-concept that different PIB-RNA modalities can induce antigen-presenting phenotypes in fibroblasts and cancer cells, including patient-derived tumor samples. Importantly, this approach restored antigen presentation in the tumor microenvironment, which led to anti-tumor immunity.
Moving forward, we will further validate this approach across additional tumor models and patient-derived samples, while also optimizing systemic RNA delivery strategies. Continued collaboration with Asgard Therapeutics, will support intellectual property protection, regulatory development, and advancement toward clinical translation.
Moving forward, we will further validate this approach across additional tumor models and patient-derived samples, while also optimizing systemic RNA delivery strategies. Continued collaboration with Asgard Therapeutics, will support intellectual property protection, regulatory development, and advancement toward clinical translation.