Genetic and epigenetic modification of human primary NK cells for enhanced antitumor activity

Abstract

Cancer immunotherapy using genetically modified immune cells such as those expressing chimeric antigen receptors has shown dramatic outcomes in patients with refractory and relapsed malignancies. Natural killer (NK) cells as a member of the innate immune system, possessing both anticancer (cytotoxic) and proinflammatory (cytokine) responses to cancers and rare off-target toxicities have great potential for a wide range of cancer therapeutic settings. Therefore, improving NK cell antitumor activity through genetic modification is of high interest in the field of cancer immunotherapy. However, gene manipulation in primary NK cells has been challenging because of broad resistance to many genetic modification methods that work well in T cells. Here we review recent successful approaches for genetic and epigenetic modification of NK cells including epigenetic remodeling, transposons, mRNA-mediated gene delivery, lentiviruses, and CRISPR gene targeting.

Keywords: CRISPR; Cancer immunotherapy; Genetic modification; Natural killer cells; Transposons; Viral vectors.

Figure 1.

Methods for genetic modification. a) Transposase-mediated genetic modification. b) Viral transduction. c) DNA-free engineering by electroporation of Cas9/RNP. d) Combination of Cas9/RNP and AAV for targeted gene insertion.

Figure 2.

Generation of CAR-NK cells by electroporation of mRNA.

Figure 3.

Epigenetic modification of NK cells. a) Cytokine expansion utilizing soluble and feeder cell-presented cytokines lead to epigenetic changes in the primary NK cell – such as the depicted activation of JAK and STAT by binding of the common-gamma chain cytokines IL-21 and IL-2 – to drive NK cell expansion as well as functional and phenotypic shifts making them an optimized adoptive immunotherapeutic; b) The addition of TGF beta to feeder cell expansion leads to epigenetic downregulation of the SMAD pathway, imprinting the NK cells to be resistant to further TGF-beta exposure; c) Histone deacetylase inhibitors such as valproic acid can alter chromatin conformation, maintaining accessibility of genes favorable to NK cell activation, like NKG2D; d) Modification of histone methylation as with inhibition of EZH2, can similarly enhance expression of activating receptors such as NKG2D and the high-affinity IL-2 receptor; e) Alteration of histone ubiquitination, such as through gene products of the Mysm1 gene, have been shown to have a critical role in activation of loci crucial for NK cell maturation, and could thus be a target of epigenetic NK cell modification favorable for immunotherapy.