Functionalized nanowires for miRNA-mediated therapeutic programming of naïve T cells

Cellular programming of naïve T cells, in contrast to activated T cells, can enhance the efficacy of adoptive T cell therapy. The differentiation of naïve cells into memory T cells impairs adoptive immunotherapy. Most current strategies for ex vivo engineering of T cells require pre-activation of T cells, with CD3/CD28 crosslinking antibodies or cytokines, prior to the delivery of genetic material, and therefore T cells lose the naïve state. Here, using a cationic polymer-functionalized nanowire platform, we pre-programmed the fate of primary, naïve CD8+ T cells to achieve the therapeutic response in vivo. We demonstrate the delivery of single versus multiple microRNAs (miRNAs) to primary naïve mouse and human CD8+ T cells without pre-activation, allowing for T cell programming in user-specific ways, as well as delivery of large, whole lentiviral particles with potential for long-term integration. Because CD8 T cell differentiation is a continuous process where fate decisions require the interplay of multiple miRNAs, we demonstrate how a combination of deletion and overexpression of miR-29 and miR-130 impact ex vivo T cell differentiation fate from the naïve state. The programming of CD8+ T cells using nanowire-delivered co-delivery of miRNAs resulted in the modulation of T cell fitness by altering T cell proliferation, phenotypic and transcriptional regulation, and secretion of effector molecules. Importantly, the in vivo adoptive transfer of murine CD8+ T cells programmed through nanowire-mediated dual delivery of miRNAs provided enhanced immune protection against different types of intracellular pathogens (influenza, Listeria monocytogenes). Nanowires open up therapeutic possibilities for adoptive T cell transfers where the amounts of effector differentiation in CD8+ T cells can be fine-tuned to achieve the therapeutic response in vivo. Overall design: Splenic CD8 T cells incubated with functionalised Nanowire dually coated with miR-29 ASO and miR-29 mimic or inert controls for 3 days in vitro