Chapter 4 - Delivery systems for mRNA

Abstract

Synthetic mRNAs for therapeutic purposes require a suitable delivery system to protect the labile mRNA from premature enzyme degradation, improve its pharmacokinetic profile, facilitate its entry into cells, and promote its subsequent endosomal escape for translation by the cell's ribosomes. This chapter describes the development of delivery systems for IVT mRNA, beginning with liposomes and an overview of liposomal versatility, composition, and methods of synthesis. This is followed by a description of passive and active targeting strategies for nanocarrier delivery systems, including the use of PEGylated stealth carriers, the exploitation of the enhanced permeability and retention (EPR) effect for tumor targeting, as well as the use of specific targeting ligands and stimuli-sensitive systems. The discussion then moves to the development of cationic lipids and lipoplexes for mRNA delivery, followed by the development of “neutral-ionizable” lipids for the encapsulation of antisense oligonucleotides in stabilized antisense-lipid particles. Numerous optimization studies for neutral-ionizable lipids are described, culminating in the development of lipid nanoparticle (LNP) delivery systems for small interfering RNA (siRNA). The discussion then focuses on the molecular shape hypothesis, which provides a theoretical underpinning for lipid interactions, lipid nanoparticle associations and self-assembly, and the lipid membrane destabilization effects that promote endosomal escape. LNP synthesis via the ethanol dilution method using T-chamber and microfluidic mixing is described, with a focus on different microchip designs. This prior foundational knowledge was leveraged for the development of LNP delivery systems for mRNA, and the commerically available mRNA COVID-19 vaccines. The structure and function of typical lipid constituents used in mRNA-LNP formulations are described, as well as mRNA-LNP morphology, which can include spherical nanoparticles, mRNA-LNP constructs with bleb protrusions, and other architectures.