Nonviral genome engineering of natural killer cells

Abstract

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

Current Perspectives on “Off-The-Shelf” Allogeneic NK and CAR-NK Cell Therapies

“…Another strategy that has been less frequently used for stable non-viral gene delivery is employing DNA transposons to transduce NK cells which is cost effective, has large cargo (ex: CAR in combination with activating receptors or cytokines) deliver capacity with stable integration. Their disadvantages include potential insertional mutagenesis and the transposon must be delivered as DNA ( 130 , 131 ). Despite the limitations described above, the most successful non-viral gene delivery for primary NK cells is still rapid transient expression by electroporation.…”

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development

“…Although viral vector-mediated gene delivery is currently the dominant strategy for most CAR NK cell engineering systems, this approach still deserves much attention, particularly regarding safety issues. Therefore, non-viral systems have received attention for a number of reasons including safety, outstanding design flexibility, and simplicity of large-scale production of therapeutic cells at a reasonable pace and low cost [ 182 ]. In the following, we review the most popular methods applied for CAR cell engineering which are frequently based on membrane permeabilization and carrier-based gene transfer.…”

Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing

“…Non-viral gene editing approaches are increasingly being applied to NK cells successfully and this field has been recently reviewed (8). Electroporation remains the most widely investigated nonviral approach to deliver diverse cargo including mRNA, DNA and protein to NK cells.…”

“…The therapeutic potential of unmodified NK ACT has been most apparent to date in the setting of hematological malignancy (1,5,6). The traditional paradigm that NK cells are more challenging to genetically engineer relative to T-cells is evolving (7,8). Clinical scale gene editing of NK cells is established, most prominently through the addition of a chimeric antigen receptor (CAR) (3).…”