Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells

Abstract

Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3+ T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9+CD3+ T cells, CD4+ and CD8+ conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9+CD3+ T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+T cells

“…Using selective NFAT inhibitors (60) instead of broadspectrum immune suppression might therefore improve virus control while ensuring survival of transplanted organs. For the setting of allo-HSCT, we found that CRISPR/Cas9-mediated single NFAT member ablation in BM-co-transplanted T cells is sufficient to ameliorate GvHD (61). When we now extrapolate our new findings we predict that single NFAT ablation will not only preserve the anti-tumor effect (Vaeth, 2015), but also CMV reactivation.…”

Challenges of CRISPR-Based Gene Editing in Primary T Cells

“…While human T cells can survive this period without any cytokines added to the medium, mouse T cells require IL-7, which upregulates the expression of the anti-apoptotic protein Bcl-2 and maintains their viability [81]. During this time, unstimulated T cells can be targeted with CRISPR/Cas to some extent to induce gene knockouts [82][83][84].…”

Challenges of CRISPR-based Gene Editing in Primary T Cells

“…While human T cells can survive this period without any cytokines added to the medium, mouse T cells require IL-7, which upregulates the expression of the anti-apoptotic protein Bcl-2 and maintains their viability [81]. During this time unstimulated T cells can be targeted with CRISPR/Cas to some extend to induce gene-knockouts [82][83][84].…”