Development of a novel splitGFP-based sensor for testing RNA delivery and efficiency in vitro and in vivo

RNA-based therapies represent a promising frontier for personalized medicine, yet key challenges such as instability and target accessibility limit their success. While nanoparticle carriers have been developed to overcome these barriers, their optimization is hindered by a lack of quantitative methods to assess functional cytosolic delivery. To address this, we developed SPLIDS (SplitGFP-based RNA Delivery Sensor) by exploiting the splitGFP technology, which relies on the spontaneous complementation of two non-fluorescent fragments, GFP1-10 and GFP11. In this setup, the large GFP1-10 fragment is constitutively expressed in the host model, while the small GFP11 tag is fused to the RNA cargo. Consequently, fluorescence is reconstituted exclusively when the RNA reaches the cytosol in an active form, providing a direct readout of endosomal escape. We established a range of GFP1-10-expressing models, from cell lines and 3D spheroids to hiPSCs and a novel transgenic mouse. Validation using Lipofectamine-mediated delivery of TagRFP-GFP11 mRNA confirmed a robust, dose-dependent fluorescence response in 2D and 3D cultures. Importantly, successful splitGFP reconstitution was also demonstrated in hiPSCs and primary fibroblasts derived from the transgenic model. Furthermore, we enhanced the platform’s versatility by engineering GFP11 variants for signal amplification (tandem repeats) and gene silencing assays (mutant forms). Finally, SPLIDS effectively discriminated the delivery efficiency of lipid nanoparticles (LNPs) across different cell models. These results establish SPLIDS as a robust tool to correlate nanoparticle engineering with functional RNA delivery validation.