NFATC1 isoform editing via RNP-mediated CRISPR/Cas9 in human primary T cells and its \(in\) \(vitro\) effects

Allo-HCT is a valuable and well-established treatment option for patients with hematologic malignancies, however occurring GvHD limits its prospects of success, resembling a severe side effect and significantly increasing morbidity and mortality. While there are different therapy options, they still lack in efficiency, are likely to cause (severe) side-effects themselves, and curtail the desired GvL effect. Hence, better treatment options, especially for steroid-refractory GvHD are required to improve allo-HCT. Selective inhibition of NFAT transcription factors has shown to support Treg expansion and weaken TEFF responses, therefore ameliorating GvHD towards lasting tolerance in a mouse model. Indeed, instead of ablating NFATc1 expression, hindering merely its SUMOylation enabled generation of more tolerogenic T cells causing less GvHD. This motivated us to transfer this concept to human T cells, by generating INDEL mutations in the long isoform specific DNA parts, harboring the SUMOylation sites. In this study, we successfully developed a protocol for isoform-specific gene editing via RNP-mediated CRISPR/Cas9 in unstimulated human T cells, specifically creating NFATC1ΔBC T cells. We were able to achieve a high knockout efficiency, while maintaining good cell viability and proliferation capacities. After the RNP-nucleofection, we cultivated the cells in vitro for three more days. Then, we examined effects regarding T cell subpopulations, surface marker expression, cytokine expression and presence of certain transcription factors. We found that NFATC1deltaBC T cells provoked an enhanced Treg frequency, in line with an enhanced IL-10 secretion. Additionally, the memory T cell population shifted from TEM to more TCM, as well as towards more TSCM at the expense of naïve T cells. Although they still expressed pro-inflammatory cytokines to a similar degree and expressed more CD40L on their surface, overall we see striking parallels to the behavior of murine Nfatc1deltaSUMO and Nfatc1deltaBC T cells. Hereby, we deduct that NFATC1ΔBC expressing human T cells appear to behave more tolerogenic, which should be tested in a more complex in vitro study by co-culturing them with 3D-organoids in order to understand the interaction with the extracellular environment and tendency to infiltrate tissue and cause damage.