Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery

Abstract

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure–function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure–activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure–activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.

Recent Advances in the Lipid Nanoparticle-Mediated Delivery of mRNA Vaccines

“…The four lipid components listed below (for more information, see Figure 1 and Table 1 ) are the most common building blocks for LNPs, including (i) ionizable lipids, which are primarily required for mRNA complexing; (ii) helper lipids, which enhance the properties of LNPs (stability and delivery efficiency); (iii) cholesterol, which provides structural stability to the LNPs (vehicle); and (iv) surface PEGylation with poly(ethylene glycol) (PEG) or PEG-derivatives, which reduces host-immune recognition and improves systemic circulation [ 9 , 17 , 18 ]. Among all these lipids, ionizable lipids are the essential components of most LNPs, acting as a primary driver to deliver mRNA through high mRNA encapsulation, enhancing stability, and pH-sensitive in vivo delivery [ 18 ]. Each component of LNPs and its function in mRNA delivery will be described in detail.…”

Recent Advances in the Lipid Nanoparticle-Mediated Delivery of mRNA Vaccines

“…The following four lipid components (please see Figure 1 and Table 1 for more details) are majorly used in the synthesis of LNPs -i) ionizable lipids mainly needed to complex with mRNA; (ii) helper lipid to improve the properties of LNPs (stability) and delivery efficiency; (iii) cholesterol to impart structural stability of the vehicle; and (iv) surface PEGylation with poly(ethylene glycol) (PEG) or PEG-derivatives to minimize the hostimmune recognition and enhance the systemic circulation. [17][18][19] Among all these lipids, ionizable lipids are the most essential components of the majority of LNPs acting as a main driver to deliver mRNA through high mRNAs encapsulation, enhance stability and pHsensitive in vivo delivery. 19 The role of each component of LNPs in mRNA delivery is discussed below in detail.…”

“…[17][18][19] Among all these lipids, ionizable lipids are the most essential components of the majority of LNPs acting as a main driver to deliver mRNA through high mRNAs encapsulation, enhance stability and pHsensitive in vivo delivery. 19 The role of each component of LNPs in mRNA delivery is discussed below in detail.…”

“…Ionizable cationic lipids: Ionizable lipids for example OF-Deg-Lin and FTT5 (Table 1) are protonated at low pH which makes them positively charged inside the cell and uncharged/neutral at physiological pH in the bloodstream. 19,20 These positively charged lipids stabilize the nucleic acid and prevent degradation by nucleases. 6 In addition, ionizable lipids facilitate in the cellular uptake of the LNPs and aid in endosomal escape by acquiring the positive charge in the endosomal pH (discussed in detail in next sections).…”

“…6 In addition, ionizable lipids facilitate in the cellular uptake of the LNPs and aid in endosomal escape by acquiring the positive charge in the endosomal pH (discussed in detail in next sections). 19,21 ii). Neutral/helper phospholipids: In addition to cationic ionizable lipids, LNPs also comprise helper lipids, such as phospholipids such as phosphatidylcholine (e.g.…”