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Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins
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Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins
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Journal Article
Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins
2020
Overview
Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.
Publisher
Frontiers Media SA,Frontiers Media S.A
Subject
/ Antibody-Dependent Cell Cytotoxicity
/ Antigens, Differentiation, T-Lymphocyte - genetics
/ Antigens, Differentiation, T-Lymphocyte - metabolism
/ Cancer
/ Cas9
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Associated Protein 9 - metabolism
/ Genomes
/ GPI-Linked Proteins - genetics
/ GPI-Linked Proteins - metabolism
/ Humans
/ Killer Cells, Natural - immunology
/ Killer Cells, Natural - metabolism
/ Lymphoma
/ mRNA
/ NK-92
/ Proteins
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ RNA, Guide, CRISPR-Cas Systems - metabolism
/ RNP
