During the 36-month implementation period, CANCERNA undertook a wide range of interlinked research and development activities. High-throughput sequencing of tumour samples from AML, MDS, and uveal melanoma patients led to the identification of hundreds of aberrant splicing events, many of which were linked to known splicing factor mutations such as SF3B1, SRSF2, and U2AF1. These events were mined using novel computational tools and machine learning classifiers to identify peptide sequences likely to bind to MHC class I molecules. A subset of these peptides was synthesised and validated for immunogenicity in vitro, demonstrating T-cell activation and tumour-specific cytotoxicity. These results formed the basis for a set of dendritic cell-based and mRNA-formulated vaccines targeting splice-derived neo-antigens.
The project also developed several therapeutic platforms. These included antisense oligonucleotides (ASOs) and CRISPR-Cas9 guides designed to modulate splicing in T cells and tumour cells, targeting FAS, CXADR, LRRN1, and LRRN3. T-cell receptors (TCRs) recognising specific splice variant-derived peptides were cloned and tested for function, with preliminary evidence of tumour cell killing. In parallel, the consortium optimised lipid nanoparticle (LNP) formulations to deliver a broad range of RNA payloads, including siRNAs, mRNAs encoding tumour antigens, and nanobody constructs. These LNPs were shown to achieve highly efficient transfection of dendritic cells and macrophages, offering a scalable and reproducible platform for clinical translation.
Importantly, the project demonstrated proof-of-concept for mRNA-based checkpoint inhibition. Nanobodies targeting PD-L1 were encoded in mRNA and delivered into suppressive immune cells, with functional evidence of restored immune activation. These results offer a new route to intracellular immunomodulation. The project also actively engaged with regulatory stakeholders and began drafting ATMP-aligned documentation to support the future clinical development of these modalities.
