Abstract
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.
Highlights
Natural killer (NK) cells are potent innate effectors capable of targeting and killing virally infected and malignant cells [1]
To enable robust gene knockout (KO) and knock-in (KI) in NK-92, we identified a combination of nucleofection buffer and pulse code for optimal co-delivery of Cas9 RNP and DNA repair template
The genotype and phenotype of genome edited NK-92 were assessed by Sanger sequencing and Inference of clustered regularly interspaced short palindromic repeats (CRISPR) Edit (ICE) tool, generation sequencing (NGS), flow cytometry and cytotoxicity assay (Figure 1A)
Summary
Natural killer (NK) cells are potent innate effectors capable of targeting and killing virally infected and malignant cells [1]. NK cells rely on an array of germline-encoded activating and inhibitory receptors that engage their cognate ligands on the target cells and initiate cytotoxicity [2]. NK cells secrete granzyme B and perforin that lyse the target cells, and cytokines and chemokines to orchestrate the subsequent immune responses [2]. These unique attributes make NK cells an attractive cell type for adoptive immunotherapy. Evidence from clinical studies demonstrates that NK cell immunotherapy is effective and safe [3,4,5,6,7,8], but there are still challenges, in the manufacture of therapeutic NK cells. Ex vivo expansion is necessary to generate clinically relevant levels of primary NK cells for infusion; this process is complicated by telomere shortening and reduced cytotoxicity
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