An improved approach to generate IL-15+/+/TGFβR2−/− iPSC-derived natural killer cells using TALEN

We present a TALEN-based workflow to generate and maintain dual-edited (IL-15^+/+/TGFβR2^-/-) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFβR2 in immune cells can enhance resistance to the suppressive TGF-β signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFβR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15^+/+/TGFβR2^-/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-β signaling.