Periodic Reporting for period 1 - SNAP-Vax (Engineering a Sugar-targeted Nucleic Acid delivery Polymer to understand and enhance vaccination by self-amplifying RNA)
Okres sprawozdawczy: 2022-04-01 do 2024-03-31
Existing lipid nanoparticle RNA vaccine formulations (LNPs) require high doses and are limited by high production costs, poor storage stability, and significant side effects. Imperial College London nanomaterial engineers (Stevens) and infectious disease RNA vaccinologists (Shattock) invented a polymeric self-amplifying RNA vaccine platform that overcomes these limitations, but these formulations are ~50-fold less potent than state-of-the-art LNPs. We hypothesized engineering a polymeric additive for these formulations would enable us to target saRNA to key cells responsible for generating adaptive immune responses and thereby enhance protective immunity. Thus, we sought to develop more potent polymeric saRNA vaccines and to understand how saRNA targeting impacts vaccine immunogenicity.
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.
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.
This work represents a significant advance in structure-function analysis of ternary complex nanoparticles for RNA delivery and demonstrates their potential as next-generation saRNA vaccine formulations. The discovery of an ideal balance in cloaking polymer properties was enabled by our specialization in high throughput RAFT polymerization, expertise in unique nanoparticle characterization techniques, and access to translationally relevant in vitro, ex vivo, and in vivo physiological systems. The driving concepts behind this fellowship have led to the training of 2 MEng and 5 PhD students, 5 conference presentations, 4 first-author manuscripts in preparation for peer review, and 4 grant applications for follow-on funding. By benchmarking our polymeric TCN formulations against state-of-the-art LNPs at each step, we have identified how biodistribution-altering chemistry can open avenues towards clinical translation. For example, polymeric formulations exceed LNP transfection following intramuscular but not intradermal delivery and result in a milder inflammatory response with fewer side effects. Given their vaccination efficacy, their significantly lower production cost, and their compatibility with lyophilization, we have formed partnerships for clinical translation of pABOL-based formulations in the coming years.