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Review
. 2024 Sep 10;22(1):552.
doi: 10.1186/s12951-024-02746-4.

Non-viral approaches in CAR-NK cell engineering: connecting natural killer cell biology and gene delivery

Emma M McErlean  1 , Helen O McCarthy  2   3   4
Affiliations
Review

Non-viral approaches in CAR-NK cell engineering: connecting natural killer cell biology and gene delivery

Emma M McErlean et al. J Nanobiotechnology. .

Abstract

Natural Killer (NK) cells are exciting candidates for cancer immunotherapy with potent innate cytotoxicity and distinct advantages over T cells for Chimeric Antigen Receptor (CAR) therapy. Concerns regarding the safety, cost, and scalability of viral vectors has ignited research into non-viral alternatives for gene delivery. This review comprehensively analyses recent advancements and challenges with non-viral genetic modification of NK cells for allogeneic CAR-NK therapies. Non-viral alternatives including electroporation and multifunctional nanoparticles are interrogated with respect to CAR expression and translational responses. Crucially, the link between NK cell biology and design of drug delivery technologies are made, which is essential for development of future non-viral approaches. This review provides valuable insights into the current state of non-viral CAR-NK cell engineering, aimed at realising the full potential of NK cell-based immunotherapies.

Keywords: CAR-NK engineering; Cancer immunotherapy; Cell therapies; Drug delivery technologies; NK-specific CAR design; Nanoparticles; Natural killer cells; Non-viral gene delivery.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic detailing the progression of CAR-T construct design elements. First generation CARs which consisted of a single-chain variable fragment (scFv) antigen recognition domain, transmembrane domain, and an intracellular T-cell activation domain derived from CD3 zeta chain (CD3ζ) progressed to second generation CAR designs, which have an additional costimulatory domain (CD28 or 4-1BB) that enhances T cell proliferation and cytotoxicity, utilised by commercial products. Further third generation CAR constructs incorporate two distinct costimulatory domains (e.g., CD28 and 4-1BB). “Armoured” fourth generation CARs (also referred to as T-cells redirected for universal cytokine-mediated killing (TRUCKs)) incorporate inducible expression components such as cytokines (e.g., IL-12) which can increase activation of CAR-T cells and tumour killing and fifth generation or “next generation” CARs contain a truncated interleukin-2 (IL-2) receptor β chain (IL-2Rβ) with a binding site for the transcription factor STAT3 to fully exploit CAR activation signals via JAK-STAT3/5 signalling; enhancing T cell activation, proliferation and persistence. Created with BioRender.com
Fig. 2
Fig. 2
Simplified schematic of commonly expressed NK cell A activating and B inhibitory receptors and corresponding ligands. NK receptors act in synergy to provide a mechanism of self-regulation to prevent damage to healthy cells and may identify abnormal cells through downregulation of human leukocyte antigens (HLA) synonymous with major histocompatibility complex (MHC), or upregulation of stress-induced ligands, such as major histocompatibility complex class 1 chain-related protein A and B (MICA, MICB). Upon recognition of a target cell, NK cells are activated through a complex signalling pathway involving a variety of receptors, co-receptors and intracellular proteins including natural cytotoxicity receptors (NCRs), Killer Ig-like receptors (KIRs), NKG2D and DNAM-1. Created with BioRender.com
Fig. 3
Fig. 3
NK cell maturation and interaction with other immune cells. A Changing receptor status according to maturation state of NK cells from immature CD56bright/CD16− cells to mature CD56dim/CD16+ and terminally differentiated NK cells which also may be described as “memory-like” NK cells. B NK cell signalling and interaction with myeloid cells and target cells following activation. NK cells mediate cytotoxicity against target cells via direct killing (activating receptors including NKG2D, NKp30, NKp44, NKp46, KIRs), apoptosis (TRAIL and FasL death receptors) and/or ADCC (CD16) and produce inflammatory cytokines and chemokines which stimulate and recruit other immune cells. Created with BioRender.com
Fig. 4
Fig. 4
Schematic comparing CAR construct design elements for CAR-T and CAR-NK therapy. Initial studies investigating CAR-NK cells used the same constructs used for third generation CAR-T therapies, which are designed to mimic the T cell receptor. NK specific CAR constructs have since been developed to incorporate NK transmembrane signalling domains along with co-stimulatory receptors (e.g., DAP10, 2B4, CD28 or 4-1BB), to enhance NK cell effector functions. The activating receptor NKG2D has also been incorporated into NK-specific CAR constructs; although not a classical “CAR” as NKG2D is not an scFv, but they are referred to as CARs in the literature. Created with BioRender.com
Fig. 5
Fig. 5
Non-viral genetic engineering of “off-the-shelf” CAR-NK cells requires efficient transfection and stable expression of the CAR transgene. 1. Introduction of CAR constructs encoded in DNA and/or mRNA or genetic editing using CRISPR Cas9 or DNA transposon technologies such as PiggyBac and Sleeping Beauty to give stable gene expression. 2. Nucleic acids encoding the CAR construct may be delivered to NK cells via several non-viral delivery strategies including electroporation, lipid nanoparticles, cell penetrating peptides or multifunctional nanoparticles. 3. Following transfection, NK cells expressing the CAR construct are expanded in vitro to produce an “off-the-shelf” CAR-NK product which may be used to treat multiple patients
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