Expression of chimeric antigen receptors (CAR) constitutes a promising approach to enhance therapeutic efficacy of NK cells. Here, we demonstrate that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency.

Primary NK cells were genetically engineered to express a CD19-CAR using lentiviral (LV) or non-viral vectors. For non-viral gene delivery, SB transposons vectorized as minicircles (MC), encoding a CD19-CAR were transfected together with the hyperactive SB100X transposase.  SB-modified NK cells displayed long-lasting CD19-CAR expression with similar viability as non-treated (NT) NK cells during ex-vivo expansion, without a significant change in NK cell phenotype or loss of intrinsic natural cytotoxicity. Assessment of vector integration revealed a significantly higher frequency of insertion into genomic safe harbors (GSHs) for SB-CAR-NK cells compared to LV-transduced CAR-NK cells. SB-CD19-CAR NK cells demonstrated significantly higher cytotoxicity compared to NT-NK cells against CD19-expressing targets. Markedly enhanced antitumor potential was confirmed in a systemic CD19-expressing leukemia xenograft model (NSG-NALM-6/Luc) in vivo. SB-CD19-CAR NK cells resulted in rapid lymphoma eradication in all treated animals, whereas mice receiving NT-NK cells showed progressive lymphoma growth, comparable to untreated mice. In addition, enhanced SB-CD19-CAR NK cell-mediated killing capacity was also observed against primary patient-derived B-ALL blasts. 

The Sleeping Beauty transposon system represents an innovative, safe and relatively cost-effective gene therapeutic approach for non-viral engineering of highly functional CAR-NK cells and can be suitable for a broad range of other cancer immunotherapies.