Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9\(^+\) T Cells
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- 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 andGene 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.…
Autor(en): | Snigdha Majumder, Isabelle Jugovic, Domenica Saul, Luisa Bell, Nadine Hundhausen, Rishav Seal, Andreas Beilhack, Andreas Rosenwald, Dimitrios Mougiakakos, Friederike Berberich-Siebelt |
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URN: | urn:nbn:de:bvb:20-opus-242896 |
Dokumentart: | Artikel / Aufsatz in einer Zeitschrift |
Institute der Universität: | Medizinische Fakultät / Pathologisches Institut |
Medizinische Fakultät / Medizinische Klinik und Poliklinik II | |
Sprache der Veröffentlichung: | Englisch |
Titel des übergeordneten Werkes / der Zeitschrift (Englisch): | Frontiers in Immunology |
ISSN: | 1664-3224 |
Erscheinungsjahr: | 2021 |
Band / Jahrgang: | 12 |
Aufsatznummer: | 683631 |
Originalveröffentlichung / Quelle: | Frontiers in Immunology (2021) 12:683631. doi: 10.3389/fimmu.2021.683631 |
DOI: | https://doi.org/10.3389/fimmu.2021.683631 |
Allgemeine fachliche Zuordnung (DDC-Klassifikation): | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Freie Schlagwort(e): | CRISPR/Cas9; GvHD; IRF4; NFAT; T-cell transfer; gRNA-only; metabolism; naive T-cell gene editing |
Datum der Freischaltung: | 09.02.2022 |
Datum der Erstveröffentlichung: | 21.07.2021 |
Open-Access-Publikationsfonds / Förderzeitraum 2021 | |
Lizenz (Deutsch): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |