Search for a command to run...
Background CRISPR genome engineering can enhance the antitumor activity of CAR NK cells by increasing their cytotoxicity, overcoming suppression within the tumor microenvironment, or promoting their persistence and homing to tumor sites.Mediator of RNA polymerase II transcription subunit 12 (MED12) is the part of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes.Deletion of the MED12 in human primary CAR T cells has been shown to augment their antitumor potency, cytokine secretion and proliferation.Here we show the potential application of MED12 gene knockout (MED12 KO) in CAR NK cells targeting CD70 or CD19 and expressing a membrane-bound form of IL-15.Methods NK cells were isolated from the peripheral blood of healthy human donors and expanded using IL-2 and stimulation with NKSTIM SM , a modified K562 stimulatory cell line expressing a membrane-bound form of IL-15 (mbIL-15) and 4-1BBL.IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation.Using CRISPR-Cas9 ribonucleoprotein (RNP) systems, we disrupted the MED12 gene in expanded NK cells.FACS and TIDE analysis confirmed successful disruption of the gene.Furthermore, we transduced MED12 KO NK cells to express a CD70-or CD19-targeting CAR and mbIL-15.CAR expression was assessed by flow cytometry.In vitro cytotoxicity was measured using the IncuCyte S3 live cell analysis system.Results MED12 KO CAR NK cells could be produced efficiently and exhibited features of adaptive NK cells.Consequently, CD70 CAR NK cells with MED12 KO exhibited the greatest cytotoxicity after tumor rechallenge with 786-O cells, in comparison with CDK8, CCNC, ID3 and SOX4 KO CAR NK cells.CD19 CAR NK cells with MED12 KO alone, or in combination with CBLB, CISH, or CISH and CBLB KOs demonstrated the highest antitumor activity both in vitro and in vivo in a Nalm-6 xenograft mouse model.Additionally, MED12 KO CAR NK cells displayed improved metabolic fitness characterized by increased basal glycolysis and glycolytic capacity.Furthermore, in the presence of exogenous TGF-b, MED12 KO CAR NK cells showed greater resistance to TGFb inhibition of cytotoxicity.Conclusions In summary, we show MED12 KO CAR NK cells demonstrated increased levels of activation markers indicative of adaptive NK cell features, enhanced antitumor activity against relevant tumor models, and improved resistance to tumor microenvironment inhibition.These data support the further exploration of MED12 KO CAR NK cells for clinical application.