We hypothesized that direct transfection and activation of antigen presenting cells (APCs) would evoke a stronger innate and adaptive immune response than untargeted saRNA delivery. We organized our approach in four work packages (WP), which are illustrated in the attached figure.
Through WP1, I synthesized a layered polymeric saRNA vaccine delivery platform with neutralized charge and mannose targeting. With my MEng and PhD students, we performed a detailed structure-function investigation of a library of novel PEGylated anionic polymers we synthesized to cloak the surface of cationic pABOL-saRNA polyplexes. Using specialized characterization techniques measuring dynamic light, fluorescence, Raman, and neutron scattering, we discovered that variables like hydrophilicity, hydrophobicity, polymer length, and charge density must all be balanced to form stable ternary complex nanoparticles (TCNs) with neutral surface charge. We then chemically engineered stable TCNs for APC targeting by polymerizing our lead cloaking polymer from a mannose-based ligand.
In WP2-3, we critically evaluated whether our engineered TCNs were able to target saRNA to APCs in a series of increasingly complex biological environments. Only our lead cloaking polymer prevented non-specific polyplex uptake in cultured cell lines, confirming that an optimal amount of PEG and hydrophobicity are needed to enable ligand-directed uptake. We demonstrated that mannose on the surface of TCNs increased uptake and transfection in APCs, both in simple 2D cultures and in a microfluidic lymph node co-culture model. Transfection in primary tissue explant cultures revealed that the densely negatively charged extracellular matrix composition of human skin inhibits TCNs but not LNPs. Finally, our ongoing work related to WP3-4 investigates the biodistribution and immunogenicity of intramuscularly injected mannose-cloaked TCNs with and without adjuvants in mice.
