Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins

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.

CAR-NK cells: A promising cellular immunotherapy for cancer

“… [72] In NK cells, CRISPR/Cas9 system could be used to augment NK cell anti-tumour functions by targeted CAR insertion and stably knock-outing or integrating genes of interest involved in NK cell exhaustion, activation, tolerance, or memory. [73] …”

Arrested development: suppression of NK cell function in the tumor microenvironment

“…This resulted in superior CAR T cells with uniform CAR expression, reduced tonic signalling, reduced T‐cell exhaustion and improved anti‐tumor efficacy in a humanised murine tumor model 119 . Although high efficiency CRISPR‐HDR has remained difficult in primary NK cells, 120 a recent study has demonstrated a number of optimised genetic engineering approaches in NK‐92 cells 121 . NK‐92 cells were edited to have multiple gene knockouts of inhibitory receptors, knock‐in of a fluorescent protein, and insertion of constitutive promoters to reactivate endogenous CD16 and DNAM‐1 expression.…”

“…NK‐92 cells were edited to have multiple gene knockouts of inhibitory receptors, knock‐in of a fluorescent protein, and insertion of constitutive promoters to reactivate endogenous CD16 and DNAM‐1 expression. CRISPR‐engineered NK‐92 cells demonstrated strikingly enhanced cytotoxicity and could even mediate ADCC against previously hard to kill cancer cell lines 121 . Employing these methods in primary human NK cells could significantly improve adoptive NK cell therapy (Figure 5).…”

Genetic Engineering of Natural Killer Cells for Enhanced Antitumor Function

“…Although CRISPR/Cas9 can be delivered by both viral and non-viral systems, non-viral delivery of a ribonuclear protein (RNP) complex made up by the Cas9 nuclease and the single guide RNA (sgRNA) is preferred, since it limits off-target effects due to viral DNA integration. However, ex vivo non-viral delivery requires optimization, as efficiency is often very limited and viability a concern ( 61 ). Of note, CRISPR/Cas9 can be used to efficiently screen effective synthetic constructs electroporated into T cells ( 66 ), significantly speeding up the discovery of constructs for reprogramming adoptive NK cell functionality and specificity.…”