Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration

Manuel J Carrasco,Ousamah Soliman,Thomas E Cleveland,Mikell Paige,Drew Weissman,Lacey Wright,Alexander Grishaev,Suman Alishetty,Mohamad-Gabriel Alameh,Michael D Buschmann,Hooda Said

doi:10.1038/s42003-021-02441-2

Abstract

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

Highlights

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 Messenger RNA (mRNA) vaccines
Messenger RNA vaccines are accepted modalities following the Emergency Use Approval of two highly efficacious vaccines that are based on lipid nanoparticle (LNP) delivery of nucleoside-modified mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein1,2. mRNA LNPs bear a structural resemblance to viral systems and circulating endogenous lipid-containing chylomicrons in terms of size and lipid envelope[3]
Given the lack of studies that address LNP design principles for IM administration that could lead to a vaccine-optimized mRNALNP, we developed several new theoretical and experimental methods that deepen our understanding of the ionization of the ionizable lipid and of the LNP

Summary

Introduction

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. MRNA LNPs bear a structural resemblance to viral systems and circulating endogenous lipid-containing chylomicrons in terms of size and lipid envelope[3] These features contribute to the application of mRNA-LNPs as delivery vehicles for vaccines and other therapeutics including transcripts encoding antibodies for endogenous translation[4] or enzymes for in vivo gene editing[5]. Once inside the cell in endosomes the pH declines to near 4.5 prior to lysosomal fusion thereby protonating the LNP, which facilitates ion pair formation of the cationic ionizable lipid with anionic endogenous endosomal phospholipids[3] At this point, the second feature that is known to control potency comes into play, namely the molecular shape of the ionizable lipid[11,12]. LNP electric charge is known to control in vivo distribution and expression of mRNA-LNPs for intravascular (IV) administration

Methods

Results

Conclusion