Novel Efficient Lipid-Based Delivery Systems Enable a Delayed Uptake and Sustained Expression of mRNA in Human Cells
Artem G Fedorovskiy1,2, Denis N Antropov3, Anton S Dome3
1Belozersky Institute of Physico-Chemical Biology, Department of Materials Science, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia.
Abstract:
Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.
