Novel Split-GFP Based Sensors for in vivo RNA Delivery (SPLIDS)

Major advancements in RNA therapeutics, such as genome editing and mRNA vaccines, are paving the way for personalized medicine approaches. However, persistent challenges including off-target effects, toxicity, and difficulties bypassing biological barriers highlight the urgent need for improved screening and delivery efficiency strategies. This doctoral work presents the development of SPLIDS (SPLit GFP-based Delivery Reporter System), a novel platform for monitoring and quantifying vehicle delivery efficiency in targeted cells, tissues, and organs. The system uses a β barrel protein split into GFP1-10 β strands and a β strand 11 that only complement to emit fluorescence when they are in a proximity. The versatility of the three spectral variants of the protein and the tunability of the signal intensity by increasing the tandem repeats of β strands 11 make it an ideal tool for screening delivery events for mRNA, peptide, plasmid both in vitro and in vivo. After generation of stable monoclonal HeLa cell lines expressing various levels of GFP1-10 and challenging them with different repeats of plasmid β strand 11 or as a synthetic peptide, the sensitivity of our system was successfully tested for screening potential on and off signal mutant s11 peptides. We then transitioned to an in vivo model, by generating a GFP 1-10 transgenic Danio rerio line. Simultaneous transfections of organelle targeted FP1-10 of different spectral variation (nucleus targeted CFP1-10, plasma membrane targeted YFP1-10, cytosolic untargeted GFP1-10) revealed organelle specific complementation without spectral interference. A future perceptive includes the generation of a triple transgenic Danio rerio line to further expand the capabilities of RNA drug delivery screening. In parallel with my PhD project, pioneering SPLICS (SPLit GFP-based Contact Sites) reporters were developed to monitor nucleus-mitochondria SPLICSNU-MT and lysosome plasma membrane SPLICSLYSO-PM communication. To sum up, pioneering SPLIDS and SPLICS reporters may serve as valuable tools for in vitro and in vivo high-throughput screening, optimizing drug delivery and investigating inter-organelle communication, unlocking novel therapeutic avenues.