Pratik Gurnani

Following the successes of the messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines against COVID-19, mRNA-LNPs are being explored for varied multidose applications including cancer immunotherapies and protein replacement therapies. LNPs rely on poly(ethylene glycol)-lipid (PEG-lipid) coatings for stability, but increasing anti-PEG antibody prevalence threatens repeat-dose efficacy and increases pseudoallergic risk, creating an urgent need for alternatives. Here, we introduce poly(acrylamido) lipids (PAM-lipids), a family of polymer lipids that effectively replace PEG-lipids in mRNA-LNP formulations. Systematic variation of monomer chemistry, molar mass, and end group reveals critical structure–activity relationships determining LNP size, polydispersity, and transfection efficiency. Varying PAM monomer chemistry allows minimization of antipolymer antibody cross-reactivity, with multiple PAM-lipids demonstrating no anti-PEG antibody binding. Several PAM-LNPs exhibit superior mRNA transfection efficacy to PEGylated clinical benchmarks in vitro and in vivo across multiple cell types, organs, routes of administration, and mRNA cargos. In particular, lead PAM-LNPs enhance mRNA delivery to dendritic cells (up to 120-fold in vitro and 3-fold in vivo) and macrophages (4-fold in vivo), with 2.5-fold superior lymph node expression, driven in part by increased cellular association and lymphoid targeting. Crucially, PAM-LNPs evade circulating anti-PEG antibodies to rescue lost mRNA efficacy after repeated dosing, resulting in 300% mRNA expression relative to that of PEG-LNPs. These findings demonstrate PAM-lipids as a chemically varied, immunologically compatible family of PEG alternatives for mRNA-LNP therapeutics.

Adjuvanted mRNA polyplex enhances their immunostimulatory effect.

Following the successes of the messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines during the COVID-19 pandemic, mRNA-LNPs are being explored for many critical disease indications including infectious disease vaccination, cancer immunotherapies, and protein replacement therapies. LNPs require a polymer coating to provide stability in storage, and to minimise clearance from the body by reducing protein adsorption after injection. Poly(ethylene glycol)-lipids (PEG-lipids) have fulfilled this role to date, however increasing prevalence of antibodies against PEG in the general population jeopardises the efficacy of future PEGylated LNP doses and increases the likelihood of adverse pseudo-allergic responses. There is, therefore, an urgent unmet need to develop LNPs with new surfaces of PEG-alternative polymers which can evade anti-PEG antibodies, particularly where repeat dosing is required. Here, we present a family of polymer lipids, poly(acrylamido) (PAM) lipids, which effectively replace conventional PEG-lipids in mRNA-LNP formulations. We identify key design parameters to show that PAM-lipid monomer chemistry, molar mass and end-group all have critical effects on LNP size, polydispersity and in vitro transfection efficiencies, while having little impact on LNP morphology or internal structure. We determine that side-group (monomer) chemistry is a key mediator in alleviating anti-polymer antibody cross-reactivity. Compared to clinical benchmark PEGylated LNPs, several PAM-LNPs displayed improved transfection efficacy across multiple mRNA cargos in diverse cell types, organs, and routes of administration, both in vitro and in vivo . In particular, mRNA transfection improved in immune cells both in vitro (up-to 120-fold), and in vivo (up-to 5-fold), including superior mRNA expression in lymph nodes (2.5-fold). In part, this is likely because PAMs increase LNP uptake/association with primary immune cells (BMDCs), and increase biodistribution to the lymphoid tissues (LNs, spleen). Crucially, PAM-LNPs avoid circulating anti-PEG antibodies to recover lost mRNA efficacy after repeated dosing in vivo , 300% higher than PEG-LNPs. Overall, our findings establish the PAM-lipid family as a versatile platform of chemically varied PEG-alternatives, towards the next generation of therapeutic mRNA-LNP technologies. Graphical

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