Abstract
mRNA therapeutics are promising platforms for protein replacement therapies and gene editing technologies. When delivered via non-viral gene delivery systems, such as lipid nanoparticles (LNPs), mRNA therapeutics are easy to produce and show low toxicity and immunogenicity. However, LNPs show limited delivery efficiency and tissue specificity in certain applications. To overcome this, we designed RGD peptide (Arg-Gly-Asp) based ionizable lipids, which can be formulated into LNPs for integrin binding on cells and targeted mRNA delivery. RGD-LNPs were formulated using microfluidic devices and screened in vitro for size, mRNA encapsulation efficiency, transfection efficiency, and cell viability. A lead candidate, 1A RGD-based hybrid LNP, showed effective mRNA encapsulation and transfection, and was selected for further testing, including the co-delivery of Cas9 mRNA and sgRNA for gene editing applications. In vitro, 1A RGD-based hybrid LNP outperformed a non-targeted control LNP and showed GFP knockout efficiencies up to 90%. Further, the improved cellular uptake was reversed in the presence of soluble RGD, supporting the hypothesis that this improved uptake is RGD-dependent. In vivo, 1A RGD-based hybrid LNPs showed comparable mRNA delivery to the liver and spleen, when compared to a non-targeted control, and had increased expression in the whole body. Overall, this RGD-based hybrid LNP system is a promising platform for targeted mRNA delivery, which may allow for mRNA-based protein replacement and gene editing in a more efficient and specific manner with reduced off-target effects.