Engineering immunostimulatory nanocarriers: TLR7- agonist conjugated poly(beta-amino ester) mRNA delivery systems

Messenger RNA (mRNA) technology serves as a powerful foundation for novel vaccines and treatments. Ensuring effective delivery of RNA medicines requires vectors due to the poor cellular association of naked mRNA and susceptibility to degradation by endogenous nucleases. Recently, nanoparticle-based carries have attracted significant attention, particularly poly(beta-amino esters) (PBAEs) which have showed promise as mRNA delivery systems. The first generation of nanoparticle-encapsulated mRNA vaccines have been highly successful in protecting against severe COVID-19 disease, but the durability of immune response remains short and hence there is a need for delivery systems which enhance the robustness of immune response. In this study, we report the development of a TLR7-adjuvanting PBAE through direct conjugation of Loxoribine, a potent TLR7 agonist. We observed that the Loxoribine conjugated PBAE was able to condense self-amplifying RNA (saRNA) efficiently into small (100-200 nm) polyelectrolyte nanoparticles with complete incorporation of the nucleic acid. Formulations specifically at w/w ratio 128 showed high transfection in HEK293T and DC2.4 cell lines with good cytotoxicity profiles. In addition, TLR activation assays using TLR7 reporter HEK-Blue cells showed that Loxoribine conjugated formulations can efficiently agonise TLR7 with some synergy observed between Loxoribine and the delivered saRNA. Although potent activation was observed in model cell lines, evaluation in primary bone marrow derived dendritic cells showed minimal upregulation of CD86 an established activation marker and less than 2% transfection efficiency. Consequently, these findings suggest the potential of adjuvanted PBAE nanoparticles for enhanced RNA vaccines however further development is needed to improve activity in primary immune cells.