UW-Madison researchers have developed a non-viral method of developing CAR NK cells. The inventors tested many parameters along established processes for genetically modifying immune cells without virus. They found that using feeder cells to support the growth and proliferation of the modified NK cells was important. They also learned that the timing and pulse used for electroporation of the cells affected gene editing efficiency. Finally, using two different small molecules during and after the transfection step positively impacted gene editing efficiency. Two previously reported methods of non-viral gene knock-in methods for modifying NK cells resulted in 7.5% gene editing efficiency and 16% gene editing efficiency in a modified method. The UW researchers report gene editing efficiency >20% using their method.
The inventors knocked out the NKG2A on the NK cells and added a CAR that recognizes GD2 receptor on tumor cells (the target of CAR T cells). NKG2A inhibits the activity of NK cells when the tumor produces an HLA-E ligand that binds that NK cell receptor. The removal of this receptor and addition of the CAR happens through a single CRISPR/Cas event. Other CAR NK cells have been manipulated like this but have used two genetic events which is time consuming and weakens the cells.