Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers

Abstract

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Effect of vorinostat on protein expression in vitro and in vivo following mRNA lipoplex administration

“…The thin film was hydrated by adding sterilized water at 60˚C, followed by vortexing and subsequent sonication for 10 min at 42 kHz, 100 W and at room temperature in a bath-type sonicator (Bransonic 2510J-MTH; Branson Ultrasonics Corporation). To prepare the mRNA lipoplexes, the cationic liposome suspension was combined with the mRNA solution at a charge ratio (+:-) of 4:1, as described in a previous study (8), thorough vortexing for 10 sec and subsequently left undisturbed at room temperature for 15 min. Particle size distribution and ζ-potential of the mRNA lipoplexes were assessed using a light-scattering photometer (ELS-Z2; Otsuka Electronics Co., Ltd.), as previously described (15).…”

“…Following incubation at 37˚C for 24 h, EGFP mRNA lipoplexes (0.5 µg mRNA) were diluted in 1 ml of culture medium (EMEM and DMEM for HeLa and HepG2 cells, respectively) supplemented with either 1 or 10 µM vorinostat (final mRNA concentration: 0.5 µg/ml) and subsequently introduced to the cells. At 24 h post-transfection, EGFP expression in the cells was observed through fluorescence microscopy, as previously described (8).…”

“…Therefore, the development of efficient and safe mRNA delivery systems is imperative (5)(6)(7). Previously, we demonstrated that cationic liposomes, comprising N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and poly(ethylene glycol) cholesteryl ether (PEG-Chol), induce high protein expression in the lungs and spleen following intravenous administration of mRNA/cationic liposome complexes (mRNA lipoplexes) (8).…”

Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems

“…The PEI is a cationic polymer that has been on the spotlight for nucleic acid delivery as an alternative to viral vectors. The PEI and PEI-derived polymers (>20 kDa) in a high-molecular-weight form display multivalent binding to nucleic acids that lead to stable nanoparticles that can withstand membrane penetration. , However, they also display significant toxicity on sensitive human cells, although PEI cytotoxicity has been successfully reduced by modifying it with peptides, vitamins, amino acids, etc. − The low-molecular-weight PEI (<2 kDa) is relatively nontoxic due to weaker cell membrane interactions but also unable to act as a carrier. Our studies have shown that lipid modifications on low-molecular-weight PEI can convert the noneffective small-molecular-weight PEI into an effective carrier for a range of nucleic acids due to the increased uptake of resultant nanoparticles, so that it can be safely used to deliver siRNA, pDNA, and mRNA in vitro . ,− However, the in vivo delivery efficiency of the modified PEIs for nucleic acids remained to be explored.…”

“…The PEI and PEI-derived polymers (>20 kDa) in a high-molecularweight form display multivalent binding to nucleic acids that lead to stable nanoparticles that can withstand membrane penetration. 10,11 However, they also display significant toxicity on sensitive human cells, although PEI cytotoxicity has been successfully reduced by modifying it with peptides, vitamins, amino acids, etc. 12−14 The low-molecular-weight PEI (<2 kDa) is relatively nontoxic due to weaker cell membrane interactions but also unable to act as a carrier.…”