While mRNA therapies represent a rapidly advancing clinical frontier, significant challenges remain in realizing their full potential. A primary hurdle is achieving targeted cell-type specificity, which is particularly critical in the cancer field. This limitation has historically been addressed using antibody-targeted delivery vehicles that recognise upregulated cancer-specific surface markers, or by using the inherent tendency for certain delivery vehicles to be directed to specific organs. In the Fischer lab, we propose a novel intracellular approach: utilising the cell’s own transcriptome as a molecular barcode. My work has focussed on developing an RNA-sensing molecular switch that can generate an orthogonal protein output strictly upon the detection of a specific RNA trigger. The core of this RNA-based switch relies on a group I intron, a small RNA that can catalyse splicing (a ribozyme). Catalysis is conferred by a complex secondary and tertiary structure. As such, when this intron is split into two parts, each part is catalytically inactive due to misfolding. To achieve splicing, the parts must be reunited to fold into the correct conformation. Co-localisation and reunification are driven by guide sequences complementary to an ‘activator RNA’. Upon splicing, the coding sequence of a therapeutic protein resolves into a continuous open reading frame and is translated. My PhD program has been developing this general design and including therapeutically relevant optimisations.
As the basis of the switch, we utilised a group I intron from the Fuligo septica myxomycete (Fse.L1898), providing the first in vivo characterisation of splicing activity outside its native host. This was done in yeast models and, more crucially, in various human cell lines. This ribozyme demonstrated superior activity compared to the model group I intron Tth.L1925 (from Tetrahymena thermophila). Using this Fse.L1898, we created a split-ribozyme system that has been further enhanced by engineering certain sequences within the intron. Furthermore, we engineered an in vivo circularising design centred around an internal ribosome entry site (IRES) capable of rolling circle translation (RCT). This optimisation significantly increased protein output in our experimental systems compared to the linear Fse.L1898-based split system. Finally, to validate the molecular switch’s transcript-dependent activation and therapeutic potential, we are directing this optimised switch to use the EML4-ALK fusion transcript as the activator RNA, being prevalent in many non-small cell lung cancers. Ultimately, this work demonstrates a robust RNA-based tool that can be used for precise, targeted production of a chosen therapeutic protein strictly within designated cells.