Ionizable Polymeric Micelles with Phenylalanine Moieties Enhance Intracellular Delivery of Self-Replicating RNA for Long-Lasting Protein Expression In Vivo.

mRNA-based therapeutics are revolutionizing the landscape of medical interventions. However, the short half-life of mRNA and transient protein expression often limits its therapeutic potential, demanding high treatment doses or repeated administrations. Self-replicating RNA (RepRNA)-based treatments could offer enhanced protein production and reduce the required dosage. Here, we developed polymeric micelles based on flexible poly(ethylene glycol)-poly(glycerol) (PEG-PG) block copolymers modified with phenylalanine (Phe) moieties via biodegradable ester bonds for the efficient delivery of RepRNA. These polymers successfully encapsulated RepRNA into sub-100 nm micelles assisted by the hydrophobicity of the Phe moieties and their ability to π-π stack with the bases in RepRNA. The micelles made from Phe-modified PEG-PG (PEG-PG(Phe)) effectively maintained the integrity of the loaded RepRNA in RNase-rich serum conditions. Once taken up by cells, the micelles triggered a pH-responsive membrane disruption, promoted by the strong protonation of the amino groups at endosomal pH, thereby delivering the RepRNA to the cytosol. The system induced strong protein expression in vitro and outperformed commercial transfecting reagents in vivo, where it resulted in enhanced and long-lasting protein expression.

Block catiomers with flanking hydrolyzable tyrosinate groups enhance in vivo mRNA delivery via π-π stacking-assisted micellar assembly.

Messenger RNA (mRNA) therapeutics have recently demonstrated high clinical potential with the accelerated approval of SARS-CoV-2 vaccines. To fulfill the promise of unprecedented mRNA-based treatments, the development of safe and efficient carriers is still necessary to achieve effective delivery of mRNA. Herein, we prepared mRNA-loaded nanocarriers for enhanced in vivo delivery using biocompatible block copolymers having functional amino acid moieties for tunable interaction with mRNA. The block copolymers were based on flexible poly(ethylene glycol)-poly(glycerol) (PEG-PG) modified with glycine (Gly), leucine (Leu) or tyrosine (Tyr) via ester bonds to generate block catiomers. Moreover, the amino acids can be gradually detached from the block copolymers after ester bond hydrolyzation, avoiding cytotoxic effects. When mixed with mRNA, the block catiomers formed narrowly distributed polymeric micelles with high stability and enhanced delivery efficiency. Particularly, the micelles based on tyrosine-modified PEG-PG (PEG-PGTyr), which formed a polyion complex (PIC) and π-π stacking with mRNA, displayed excellent stability against polyanions and promoted mRNA integrity in serum. PEG-PGTyr-based micelles also increased the cellular uptake and the endosomal escape, promoting high protein expression both in vitro and in vivo. Furthermore, the PEG-PGTyr-based micelles significantly extended the half-life of the loaded mRNA after intravenous injection. Our results highlight the potential of PEG-PGTyr-based micelles as safe and effective carriers for mRNA, expediting the rational design of polymeric materials for enhanced mRNA delivery.

Polymer-Based mRNA Delivery Strategies for Advanced Therapies.

Messenger RNA (mRNA)-based therapies offer great promise for the treatment of a variety of diseases. In 2020, two FDA approvals of mRNA-based vaccines have elevated mRNA vaccines to global recognition. However, the therapeutic capabilities of mRNA extend far beyond vaccines against infectious diseases. They hold potential for cancer vaccines, protein replacement therapies, gene editing therapies, and immunotherapies. For realizing such advanced therapies, it is crucial to develop effective carrier systems. Recent advances in materials science have led to the development of promising nonviral mRNA delivery systems. In comparison to other carriers like lipid nanoparticles, polymer-based delivery systems often receive less attention, despite their unique ability to carefully tune their chemical features to promote mRNA protection, their favorable pharmacokinetics, and their potential for targeting delivery. In this review, the central features of polymer-based systems for mRNA delivery highlighting the molecular design criteria, stability, and biodistribution are discussed. Finally, the role of targeting ligands for the future of RNA therapies is analyzed.