Non-viral CISH locus-specific integrated IL-15-armored CAR-NK cells achieve potent anti-tumor efficacy via adaptive NK cell differentiation

Chimeric antigen receptor (CAR)-NK therapy has emerged as a highly promising alternative to CAR-T cell therapy, due to its remarkable efficacy and safety in hematological tumors. However, the efficacy of CAR-NK cells currently remains limited in solid tumors. Here, we successfully developed CISH locus-specific integrated CAR-NK cells using a non-viral mini-circular single-stranded DNA (mcssDNA)-based CRISPR/Cas9 targeted genome editing (mcssDNA/CRISPR/Cas9) technology via a single-step electroporation procedure. The developed CISH-knockout (CISH-KO) CAR-NK cells exhibited the enhanced proliferation and cytotoxicity against hepatocellular carcinoma (HCC) compared with conventional lentivirus-transduced CAR-NK (LV-CAR-NK) cells. Single-cell RNA sequencing analysis revealed an important subset of adaptive NK cells that were highly enriched in CISH-KO CAR-NK cells compared to LV-CAR-NK cells and showed the up-regulated JAK/STAT, energy metabolism and activating receptor signaling. Furthermore, non-viral CISH locus-specific integrated IL-15-armored CAR-NK cells were generated and achieved persistent tumor regression and a significant survival advantage in a lung metastatic mouse model of HCC. Collectively, our results demonstrate that non-viral CISH locus-specific integrated CAR-NK cells can be generated by a simplified, single-step manufacturing procedure and achieve potent efficacy against HCC, thus providing an innovative technology for CAR-NK cell therapy against solid tumors. Overall design: The cells are stained with 0.4% trypan blue to assess cell viability under microscopic observation. Cells with greater than 80% viability are qualified for library construction process. Library construction is performed by using DNBelab C Series High-throughput Single-cell RNA Library Preparation Set V3.0 (MGI,China). The single-cell suspension, oil, beads are added into C4 scRNA slide and instrument in order. Following this, the droplets are broken by vacuum pump, releasing the mRNA-bead complexes. The cDNA synthesis is conducted at the suitable temperature. Then the cDNA and Oligo products are amplified and purified. The products are subjected to concentration and size distribution test to make sure in a good range. The Oligo library is constructed and then subjected to amplification, index ligation and purification for circularization. The cDNA is fragmentated, end repair and A tailing to form cDNA products. Then the cDNA is ligated to adaptor at the suitable temperature for a fixed period of time and then purified. The cDNA with adaptor is amplified via PCR and then purified. The cDNA and Oligo products are respectively denatured into single stranded. The reaction system and program for circularization are respectively configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS). The cDNA library was sequenced by PE 47+100 and Oligo library using PE 32+42.