Engineered stem cells and uses thereof

A method of genetically engineering stem cells to express a synthetic cytokine receptor, comprising:

• contacting a population of stem cells with (i) a guide RNA (gRNA) targeting a target site in an endogenous gene, (ii) an RNA-guided endonuclease, and (iii) a recombinant vector comprising a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand,
• thereby inserting the nucleotide sequence into the endogenous gene;
• wherein the cytokine receptor comprises:
• a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and
• a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and/or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.

A population of induced cytotoxic innate lymphoid (iCIL) cells, wherein the iCILs are mature iCILs expressing CD56 and LFA1, and wherein:

• at least 25% of the iCILs express a cytotoxicity receptor;
• no more than 75% of the iCILs express a dysfunction receptor;
• and/or
• at least 25% of the iCILs are proliferative.