V34 - A Universal Polymer-Based Non-Viral Gene and Drug Delivery System with Enhanced Stability

Introduction: : In the last few years, and especially during the COVID-19 pandemic, gene therapy has gained tremendous momentum due to the rapid emergence and large-scale commercial production of non viral mRNA vaccines. The Pfizer–BioNTech and Moderna lipid nanoparticle (LNP) mRNA vaccines exhibited remarkable efficacy against the SARS-CoV-2 virus and demonstrated exceptional safety in clinical trials and throughout their emergency use during the pandemic period. Despite the great achievements of the COVID 19 LNP mRNA vaccines, serious challenges remain regarding their stability and the costs associated with the storage and distribution of these vaccines. While polymeric NPs have been proposed as an alternative, lack of efficacy and concerns about toxicity have largely limited their development. This research aims to develop a novel polymer-lipid hybrid NP platform that employs surface loading of mRNA and can be lyophilized into a shelf stable powder to increase the shelf life of vaccines that utilize these NPs as nucleic acid delivery devices. Furthermore, these NPs can be used to encapsulate therapeutic drugs thus enabling the development of dual therapy approaches in which genetic material and drugs can be co-delivered simultaneously using a single NP delivery system. This dual delivery approach can overcome some of the efficacy issues experienced when designing vaccines to treat certain diseases, such as cancer, by provoking synergistic immune responses. By improving both vaccine efficacy and stability, we believe this new class of gene delivery device represents a significant addition to the ongoing development of the next generation of vaccine technology.

Materials and

Methods: : These lipo-polymeric nanoparticles (NPs) consist of a poly(lactic-co-glycolic acid) (PLGA) core, a polyethyleneimine (PEI) cationic polymer surface coating, and ionizable lipids. For the development of these NPs as an antitumor therapy, we encapsulated trametinib (a chemotherapy to treat melanoma) inside the PLGA core of the NPs. NPs were synthesized via nanoprecipitation and could be mixed with sucrose and a cryoprotectant and lyophilized into shelf stable powders for longer term storage than LNPs. More than 60 different NP formulations and synthesis methods were evaluated. The PEI surface coating gives these NPs a strongly positive surface charge upon reconstitution in water, allowing these NPs to instantaneously bind with mRNA and other inherently negatively charged nucleic acids through simple hand mixing. Utilizing both GFP mRNA and DNA, transfection of cells was easily tracked through fluorescent microscopy and flow cytometry. These NPs could also transfect B16F10 murine melanoma cells to produce large amounts of inflammatory GM-CSF protein. Combined with the encapsulated trametinib, we designed a dual delivery approach to melanoma tumor treatment in which both trametinib and GM-CSF mRNA can be delivered simultaneously on one NP to tumor cells. To evaluate performance in vivo, B16F10 tumors were injected subcutaneously into the flanks of mice and treated with intratumoral injections of NPs with and without mRNA and with or without encapsulated drug. Tumor clearance and mouse survival rates were tracked over time, and key organs were harvested for immunoprofiling and immunophenotyping to elucidate the mechanisms of tumor clearance.

Results, Conclusions, and Discussions:: After more than 60 different NP formulations were created and tested, we were able to formulate NPs with high transfection efficiencies that were able to be reproducible synthesized via nanoprecipitation. These NPs possessed an average size of 150 nm and an average zeta potential of +53 mV as measured through DLS. mRNA and DNA could be instantly complexed to the NPs at room temperature through simple hand shaking and trametinib could be encapsulated in the NPs at an encapsulation efficiency of about 40%. These NPs were able to transfect HEK293T cells with GFP mRNA at the same level as traditional LNP transfection agents, at a transfection efficiency of greater than 95%, while retaining favorable cell viabilities. These NPs could also protect mRNA from degradation from nucleases due to the strong binding activity of the mRNA to the cationic polymer coating on the surface of the NPs. These NPs were also used to transfect B16F10 cells with GM-CSF mRNA to release therapeutically relevant amounts of GM-CSF as measured via ELISA. When applied as an intratumoral therapy in vivo to treat B16F10 melanoma, the NPs possessing the combination of GM-CSF mRNA and encapsulated trametinib were the only treatment that significantly reduced tumor volume and improved overall survival, with an increase of 32% of median survival. Immunoprofiling of the spleen and lymph nodes of the treated mice revealed that the combination of GM-CSF mRNA and trametinib resulted in the greatest increase in the generation of mature immune cells possessing pro-inflammatory cell markers. Overall, these data indicate that the ability for these NPs to simultaneously deliver surface-adsorbed genetic material and encapsulated small molecule drugs can provide synergistic therapeutic benefits in clinically relevant diseases and may possess the potential to overcome the limited efficacy experienced when applying any one of these therapies in isolation. This highly versatile NP platform can be modified to specifically target and treat certain diseases with heightened efficacy, and future research will build upon this foundational work to expand the applicability of this system to a greater array of therapeutic implementations.

Acknowledgements and/or References (Optional):: This work was co-authored alongside Dr. Kareem Ebeid, a postdoctoral scholar in the Desai Lab at Brown University.