T Cell Targeting Biomimetic Polymeric Nanoparticles for mRNA Delivery and Stimulation

Abstract

T cell immunotherapies have demonstrated robust clinical success in treating some cancers but are not without their challenges. Engineering T cells, such as chimeric antigen receptor (CAR) strategies, has been shown to be a powerful approach to direct the adaptive immune response in cancer. However, these approaches are expensive, time consuming, and inefficient given the need for T cell extraction from the patient, ex vivo engineering, then reinfusion. Genetically modifying T cells in situ would significantly simplify the process and reduce costs. Non-viral polymeric nanoparticles offer a potential vehicle for in situ gene engineering for their well-established efficacy and safety in multiple cell types. Here, we developed a modular, targeted polymeric/lipid NP that can deliver mRNA cargo and effectively stimulate T cells in situ. Using synthetic poly(beta amino ester) (PBAE), mRNA nanoparticles were synthesized by self-assembly in buffer. Particles were subsequently conjugated with either anti-CD3 or both anti-CD3 and anti-CD28, a costimulatory signal, to transfect primary murine T cells isolated from C57BL/6. Our results suggest that both in vitro and in vivo, mRNA-NPs with anti-CD3 conjugated to the surface significantly outperformed unconjugated NPs. In vitro, transfection efficacy of NPs + anti-CD3 reached ~17%, a 3-fold increase over the unconjugated particles. Likewise, particles conjugated with both anti-CD3 and anti-CD28 were able to induce ~5-fold T cell proliferation with minimal toxicity. For in vivo studies, transgenic Ai9 mice were injected systemically with mRNA-NPs encoding a Cre mRNA molecule for tdTomato expression analysis. NPs + anti-CD3 achieved significantly higher transfection in the spleen and lymph nodes than unconjugated particles, and preferentially transfected CD4+ T cells. Unconjugated particles had no significant preference for transfection in CD8+ or CD4+ T cells. Studies are currently underway to further investigate antibody conjugation and apply this T lymphocyte targeted gene delivery platform in situ for immunoengineering applications. </p