A Robust Delivery System for RNA Therapeutics

The field of RNA therapeutics is currently undergoing both transformation and expansion. Specifically, research in lipid nanoparticle (LNP) based RNA therapeutics is gaining significant traction. Other research into mechanisms of gene regulation and manipulation, including siRNA and the CRISPR/Cas9 system have demonstrated the potential of RNA-based disease treatment. This work identifies a delivery system which can regulate expression of green fluorescent protein (GFP) in human embryonic kidney cells (HEK293) stably expressing GFP. Analysis of siRNA-induced gene knockdown demonstrates that the current siRNA-LNP formulation is equally as effective as a commercially available transfection reagent, Lipofectamine RNAiMAX (RNAiMAX), which is designed specifically for plate-based transfection experiments. The siRNA-LNP formulation can reduce GFP expression to 30% with cytotoxicity slightly lower than RNAiMAX. CRISPR formulations also resulted in significant knockdown, though further research is needed to optimize and verify loss of GFP expression. Particle characterization of both siRNA-LNP and CRISPR RNA-LNP formulations reveals that particles of low size (~100 nm) and low polydispersity index (PDI \u3c 0.10) can be achieved, demonstrating their in vivo applicability and potential for further pharmaceutical development. This study also outlines a route for continued formulation development through evaluation of a complex processing stream. An RNase detection assay reveals that RNA-LNPs can be produced using a continuous processing system without risk of degradation