Systematic optimization of siRNA productive uptake into resting and activated T cells ex vivo.

RNA-based medicines are ideally suited for precise modulation of T cell phenotypes in anti-cancer immunity, in autoimmune diseases and for ex vivo modulation of T-cell-based therapies. Therefore, understanding productive siRNA uptake to T cells is of particular importance. Most studies used unmodified siRNAs or commercially available siRNAs with undisclosed chemical modification patterns to show functionality in T cells. Despite being an active field of research, robust siRNA delivery to T cells still represents a formidable challenge. Therefore, a systematic approach is needed to further optimize and understand productive siRNA uptake pathways to T cells. Here, we compared conjugate-mediated and nanoparticle-mediated delivery of siRNAs to T cells in the context of fully chemically modified RNA constructs. We showed that lipid-conjugate-mediated delivery outperforms lipid-nanoparticle-mediated and extracellular-vesicle-mediated delivery in activated T cells ex vivo. Yet, ex vivo manipulation of T cells without the need of activation is of great therapeutic interest for CAR-T, engineered TCR-T and allogeneic donor lymphocyte applications. We are first to report productive siRNA uptake into resting T cells using lipid-conjugate-mediated delivery. Interestingly, we observed strong dependence of silencing activity on lipid-conjugate-identity in resting T cells but not in activated T cells. This phenomenon is consistent with our early uptake kinetics data. Lipid-conjugates also enabled delivery of siRNA to all mononuclear immune cell types, including both lymphoid and myeloid lineages. These findings are expected to be broadly applicable for ex vivo modulation of immune cell therapies.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Reka A Haraszti reports financial support was provided by German Cancer Aid. Reka A Haraszti reports financial support was provided by Faculty of Medicine, University of Tuebingen. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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