Efficient and transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine--propylenimine) (L-PEI--PPI) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the gold standard carrier. This is especially true for our best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI) demonstrating a higher sustained expression for at least 35 days after injection.