TY  - JOUR
A1  - Majumder, Snigdha
A1  - Jugovic, Isabelle
A1  - Saul, Domenica
A1  - Bell, Luisa
A1  - Hundhausen, Nadine
A1  - Seal, Rishav
A1  - Beilhack, Andreas
A1  - Rosenwald, Andreas
A1  - Mougiakakos, Dimitrios
A1  - Berberich-Siebelt, Friederike
T1  - Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9\(^+\) T Cells
JF  - Frontiers in Immunology
N2  - 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.
KW  - CRISPR/Cas9
KW  - gRNA-only
KW  - GvHD
KW  - metabolism
KW  - NFAT
KW  - naive T-cell gene editing
KW  - T-cell transfer
KW  - IRF4
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242896
SN  - 1664-3224
VL  - 12
ER  -