Journal
CYTOTHERAPY
Volume 24, Issue 11, Pages 1087-1094Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.jcyt.2022.07.008
Keywords
CAR-NK cells; CRISPR/Cas9; DNA transposon; Genome editing
Categories
Funding
- Irish Clinical Academic Training (ICAT) Programme fellowship
- Wellcome Trust [203930/B/16/Z]
- Health Research Board [203930/B/16/Z]
- Health Service Executive National Doctors Training and Planning
- Health and Social Care, Research and Development Division, Northern Ireland
- Science Foundation Ireland
- Irish Cancer Society (Blood Cancer Network Ireland) [14/ICS/B3042]
- Wellcome Trust [203930/B/16/Z] Funding Source: Wellcome Trust
- Science Foundation Ireland (SFI) [14/ICS/B3042] Funding Source: Science Foundation Ireland (SFI)
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This study reports a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder-mediated NK cell activation and expansion in the current protocol. The approach enabled clinically relevant expansion of NK cells and expression of chimeric antigen receptor (CAR), and the CAR-NK cells effectively targeted AML cells expressing CLL-1. Additionally, the concurrent delivery of CRISPR/Cas9 cargo enhanced the cytotoxicity and altered the phenotype of NK cells by knockout of the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS) gene.
Background: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive antitumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. Aims and Methods: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. Results: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. Conclusions: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols. (c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
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