Article

Review Article

Ann Lab Med 2025; 45(2): 146-159

Published online January 8, 2025 https://doi.org/10.3343/alm.2024.0380

Copyright © Korean Society for Laboratory Medicine.

Advancing Natural Killer Cell Therapy: Genetic Engineering Strategies for Enhanced Cancer Immunotherapy

Joo Dong Park , M.S.1*, Ha Eun Shin , Ph.D.2*, Yeon Su An , B.S.1, Hye Jung Jang , M.S.1, Juwon Park , Ph.D.2, Se-Na Kim , Ph.D.3,4, Chun Gwon Park , Ph.D.5,6,7, and Wooram Park, Ph.D.1,7,8

1Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea; 2Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School Medicine, University of Hawai‘i at Manoa, Honolulu, USA; 3Department of Industrial Cosmetic Science, Chungbuk National University, Cheongju, Korea; 4Research and Development Center, MediArk Inc., Cheongju, Korea; 5Department of Biomedical Engineering, Institute for Cross-disciplinary Studies, Sungkyunkwan University, Suwon, Korea; 6Department of Intelligent Precision Healthcare Convergence, Institute for Cross-disciplinary Studies, Sungkyunkwan University, Suwon, Korea; 7Korea Institute of Science and Technology, Seoul, Korea; 8Department of MetaBioHealth, Institute for Cross-disciplinary Studies, Sungkyunkwan University, Suwon, Korea

Correspondence to: Wooram Park, Ph.D.
Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, 2066 Seobu-ro, Suwon 16419, Korea
E-mail: parkwr@skku.edu

* These authors contributed equally to this study as co-first authors.

Received: July 19, 2024; Revised: October 6, 2024; Accepted: December 27, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Natural killer (NK) cells are pivotal innate immune system components that exhibit spontaneous cytolytic activity against abnormal cells, such as infected and tumor cells. NK cells have shown significant promise in adoptive cell therapy because of their favorable safety profiles and minimal toxicity in clinical settings. Despite their advantages, the therapeutic application of unmodified NK cells faces challenges, including limited in vivo persistence, particularly in the immunosuppressive tumor microenvironment. Recent advances in genetic engineering have enhanced the therapeutic potential of NK cells by addressing these limitations and improving their therapeutic efficacy. In this review, we have described various methodologies for the genetic modification of NK cells, including viral vectors, electroporation, and nanoparticle-based approaches. The ongoing research on nanomaterialbased approaches highlights their potential to overcome current limitations in NK cell therapy, paving the way for advanced cancer therapy and improved clinical outcomes. In this review, we also emphasize the potential of engineered NK cells in cancer immunotherapy and other clinical applications, highlighting the expanding scope of NK cell-based treatments and the critical role of innovative genetic engineering techniques.

Keywords: Cell therapy, Genetic engineering, Immunotherapy, Nanoparticle delivery, Natural killer cells

NK cells and their role in immunity

NK cells are cytotoxic lymphocytes of the innate immune system that naturally target and eliminate abnormal cells, such as virus-infected and tumor cells [1]. Clinical studies have shown that NK cells have numerous advantages for adoptive cell therapy, including favorable safety profiles, high therapeutic efficacy, and minimal toxicity. NK cell activity is regulated by an extensive network of integrated signals from activating and inhibitory receptors present on the cell membrane. Abnormal cells are generally lysed by NK cells via the engagement of activating ligands. The major activating receptors in NK cells include natural cytotoxicity receptors and NKG2D, whereas major inhibitory receptors include the KIR/CD158 family and CD94/NKG2A [2-4]. In addition to their cytolytic functions, NK cells play a crucial role in immunomodulation by secreting cytokines and chemokines, such as IFN-γ and MIP-1α [5, 6]. NK cell-mediated target cell elimination also involves a cytokine pathway via the secretion of various cytokines, including TNF-α, which plays a vital role in orchestrating target cell death [1].

Historical and current significance of NK cells in immunotherapy

In 2017, the U.S. Food and Drug Administration approved two chimeric antigen receptor-T (CAR-T) therapies, Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel), for ALL and lymphoma, respectively, marking a significant milestone in adoptive immune cell therapy using genetically engineered effector cells [79]. The Rezvani group studied anti-CD19 CAR-NK cells, which showed potent antitumor activity in patients without exerting significant toxicity [10, 11]. This pivotal finding significantly heightened the interest in the development of NK cell-based cancer immunotherapies. NK cell-based immunotherapy has the benefit of having minimal side effects, such as cytokine release syndrome and central nervous system edema, which can occur with traditional CAR-T cell therapy [12, 13].

Producing CAR-T cells is costly and time-consuming because of the need to isolate CD8+ T cells from the patient. NK cells are being explored as an alternative because they can detect and kill cancer cells without relying on specific antigens or major histocompatibility complex compatibility, which reduces the risk of graft-versus-host disease and associated complications [1, 14]. Consequently, NK cells are a promising off-the-shelf option for allogeneic cell therapies, potentially simplifying production and availability for patients.

NK cells in modern therapeutics

With research progress in the field of immunotherapy, NK cells have garnered increasing interest among researchers and medical professionals as potential alternatives to CD8+ T cells in anticancer treatments [15], as indicated by the significant increase in published studies on NK cells (Fig. 1A). A literature review revealed that 44.9% of all NK cell studies are cancer-related, followed by 16.6% being related to viral infections, 5.3% to autoimmune diseases, and 33.2% to “others” (Fig. 1B). These trends highlight the diverse applications of NK cell therapies and their evolving roles in multiple therapeutic domains beyond cancer.

Figure 1. Overview of NK cell research. (A) Yearly numbers of NK cell-related publications from the mid-1970s up to 2024. (B) Proportion distribution of diseases focused on in NK cell studies in 2024. (C) Statistics of clinical trials of NK cell-based therapies (445 trials in total). The image was created with BioRender.com.
Abbreviations: NK, natural killer; CAR, chimeric antigen receptor.

Clinical trials have put forward NK cells as a pivotal player in cell therapy, particularly in cancer treatment [16]. Currently, 79% of NK cell-related clinical trials are focused on oncology, highlighting the potential usability of these immune cells to combat various forms of cancer. The remaining 21% of trials explore the application of NK cells across various other conditions, demonstrating their versatility in therapeutic areas beyond oncology (Fig. 1C). Beyond their use as standalone cellular therapies, NK cells are increasingly being evaluated in combination with other treatment modalities, such as immune checkpoint inhibitors, chemotherapy, and small molecules. This trend reflects a strategic shift in clinical research toward enhancing immunotherapy efficacy via multifaceted approaches.

Therapeutic challenges with non-modified NK cells

Despite the advantages of NK cell-mediated cancer immunotherapy, its application faces several challenges. A key obstacle is the limited durability of infused NK cells without cytokine support, which can compromise in vivo treatment effectiveness [17]. However, adding exogenous cytokines may enhance NK cell proliferation and longevity but may inadvertently activate non-targeted immune subsets such as regulatory T cells [18, 19]. The tumor microenvironment (TME) is characterized by immunosuppressive factors and conditions that significantly hinder NK cell activity, including inhibitory cytokine secretion, various suppressive immune cells, and unfavorable metabolic conditions [20, 21]. This TME condition suppresses NK cell infiltration and impairs their function within tumors.

Benefits of genetically engineered versus native NK cells

To overcome the aforementioned challenges, several strategies aimed at boosting effector responses, improving targeting using CARs, extending in vivo functional persistence via cytokine support, and mitigating suppression in the TME have been investigated [22]. Notably, genetic modification, including integrating CARs, overexpressing immune-activating genes, and using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to knock out immunologically dysfunctional genes, have emerged as promising approaches [2325]. These genetic engineering techniques offer potential solutions to enhance NK cell efficacy and persistence in the TME. However, compared with other immune cells, NK cells are difficult to modify because of the low gene delivery efficiency, which is primarily attributed to the presence of pattern recognition receptors, which impede NK cell entry of foreign genetic material [26, 27]. In this review, we describe various methods for the genetic engineering of NK cells, including electroporation, virus-based transduction, and the use of various nanomaterials. These techniques represent the current state-of-the-art approaches to overcome the inherent resistance of NK cells to genetic modification, allowing the development of more effective NK cell-based immunotherapies.

Global overview of genetic engineering in NK cells

With advancements in genetic modification technologies, various approaches have been employed to engineer NK cells. The two main methods are transfection, which allows for the transient expression of introduced genes, including electroporation and nanomaterial-based gene delivery, and viral transduction using lenti- or retroviruses or transposons, which enables stable gene expression.

In recent years, the engineering of NK cells to express various CARs targeting hematologic and solid tumors has been actively studied. Fig. 2 summarizes 27 CAR-NK cell line studies and 34 primary CAR-NK preclinical studies published in PubMed between 2021 and July 2024. The number of preclinical studies on CAR-NK cells increases annually as efforts to genetically engineer NK cells continue to advance. CD19 is an antigen present on the surface of B cells and is primarily overexpressed in hematologic malignancies such as B-cell lymphomas and leukemias. Most preclinical studies on CAR-NK cells use anti-CD19 CARs to target these malignancies, and most preclinical studies involving engineered NK cell lines and primary NK cells use lentiviral or retroviral vectors, with transduction efficiencies ranging from 13% to >92% [28]. In contrast, non-viral gene delivery methods, such as electroporation used to introduce mRNA encoding CAR to express CAR molecules, have a transfection efficiency of 30%–70% in NK cell lines and of 50%–58% in peripheral blood or umbilical cord blood NK cells [28, 29]. Therefore, current studies are focusing on enhancing stable gene delivery efficiency.

Figure 2. Recent research on CAR-NK cells. (A) Overview of published studies on CAR-NK cells during 2021 – 2024. (B) Proportion of studies utilizing primary NK cells versus NK cell lines. The image was created with BioRender.com.
Abbreviations: NK, natural killer; CAR, chimeric antigen receptor.

Methods for stable gene expression in NK cells

Transduction: virus-based NK cell engineering

Viral vector-mediated genetic engineering is commonly employed to introduce DNA into the cellular genome, which facilitates sustained transgene expression. Lenti- and retroviruses have been extensively used in gene therapy for many years because of their ability to efficiently transduce both proliferating and non-proliferating cells. They have proven effective in the treatment of diseases such as human immunodeficiency virus (HIV) infection and cancer in clinical trials [8, 30]. Retroviruses are also widely used as gene delivery vectors in gene therapies [31]. Unlike lentiviruses, retroviruses actively integrate their genome into the host genome in dividing cells [32].

Lentiviral or retroviral vectors are typically primary tools for modifying NK cells, with transduction efficiencies of 10%–60% in cell lines but notably reduced efficiencies in primary NK cells. Lentiviral vectors, which bind to the sodium-dependent neutral amino acid transporter, achieve transduction efficiencies of 38.3%–58.4% post-sorting [33]. The use of lentiviral transduction enhancers such as protamine sulfate, retronectin, BX795, or polybrene can improve NK cell transduction rates from 40% to 100% after two rounds. Adeno-associated viruses, particularly AAV6, have demonstrated 76% efficiency in CAR expression in primary NK cells [34]. Retroviral particles, specifically those pseudotyped with envelope glycoproteins, can achieve up to 65% CAR expression [24]. Other studies involving lenti- or retrovirus-based CAR-NK cells are summarized in Table 1. Despite ongoing research to improve viral gene transduction, significant drawbacks, including high costs, lengthy production processes, safety concerns, and polymorphism risks, persist, which has prompted a shift toward non-viral genetic modification methods for NK cells [35].

Comprehensive overview of viral-based genetic engineering of CAR-NK cells
Molecular targetMalignancyIntracellular signal domainSourceMethodReference
CD70, CD19B-cell lymphoma4-1BB, CD3ζ IL-15CB-NKLentiviral[89]
CD33Acute myeloid leukemia4-1BB/CD137, CD3ζPB-NKLentiviral[63]
CD19B-cell lymphoma4-1BB-CD3ζNK-92Lentiviral[90]
GPC3Hepatocellular carcinomaCD28, 4-1BB, CD3ζ/2B4, DAP10, CD3ζNK-92Lentiviral[91]
cMETCholangiocarcinomaCD28-41BB-CD3ζNK-92Lentiviral[94]
CD276Esophageal squamous cell carcinoma4-1BB-CD3ζiPSC-NKLentiviral[64]
ErbB2 (HER2)Metastatic rhabdomyosarcomaCD28-CD3ζNK-92Lentiviral[95]
GD2Diffuse intrinsic pontine gliomas4-1BB-CD3ζNK-92Lentiviral[69]
HER2, sPD-1Breast cancerCD28-CD3ζNK-92Lentiviral[98]
MesothelinTriple-negative breast cancer4-1BB-CD3ζiPSC-NKLentiviral[100]
CD19ALL, Burkitt’s lymphomaCD28, 4-1BB-CD3ζiPSC-NKLentiviral[89]
CD22Esophageal squamous cell carcinoma4-1BB-CD3ζiPSC-NKLentiviral[63]
DLL3Small cell lung cancer2B4-CD3ζNK-92Lentiviral[91]
NKG2DMultiple myelomaNKG2D-4-1BB-CD3z-CARPB-NKLentiviral[92]
CD70Lymphoma, multiple myeloma, ovarian cancerCD28-CD3ζCB-NKRetroviral[93]
CD19Lymphoblastoid tumorCD28PB-NKRetroviral[94]
CD123Acute myeloid leukemia4-1BB-CD3ζPB-NKRetroviral[96]
EGFRGlioblastomaCD28-CD3ζPB-NKRetroviral[11]

Abbreviations: NK, natural killer; CAR, chimeric antigen receptor; CB, cord blood; PB, peripheral blood; iPSC, induced-pluripotent stem cells; cMET, mesenchymal-epithelial transition factor; EGFR, Epidermal Growth Factor Receptor.



Transposons: a non-viral strategy for stable ectopic expression in NK cells

To bypass the limitations of viral-based genetic engineering, transposon systems have emerged in the genetic engineering of NK cells (Table 2). Transposons are naturally occurring repetitive DNA sequences that can move within the genome. Representative transposon systems include piggyBac (PB), TcBuster (TcB), and Sleeping Beauty (SB). A recent report described a novel hyperactive mutant TcB (TcB-M) transposase that was engineered using structure-guided and in vitro evolution approaches [36]. The CD19-CAR expression level in human primary NK cells was ~50% with TcB-M, relative to ~25% with TcB [36]. According to Robbins, et al. [37], the SB11 and SB100X systems achieve transfection efficiencies of ~9% and 15.2%, respectively, in human primary NK cells. Recently, Bexte, et al. reported that human primary NK cells achieved ~44% CD19-CAR expression when they were simultaneously transfected with SB100X transposase mRNA [38]. Although transposon systems overcome the limitations of viral-based systems, transposon-mediated genetic engineering of NK cells still faces challenges, including large cargo size, reduced cell viability in the initial days because of electroporation, and a low percentage of cells expressing the ectopic gene when compared with the use of viral systems.

Transposon-based gene delivery efficiency into NK cells
Cell sourceGenetic modification systemGenetic materialTransfection efficiencyReference
Human primary NK cellsTcB transposon systemCD19-CAR DNA~25%[36]
Human primary NK cellsTcB-M transposon systemCD19-CAR DNA~50%[37]
Human primary NK cellsSB11 transposon systemEGFP mRNA~9%[37]
Human primary NK cellsSB100X transposon systemEGFP mRNA~15.2%[37]
Human primary NK cellsSB100X transposon systemCD19-CAR DNA~44%[38]

Abbreviations: NK, natural killer; TcB; TcBuster; TcB-M, mutant TcB; CAR, chimeric antigen receptor.



CRISPR/Cas9 system: precise gene editing

Recent advances in gene editing techniques, particularly the CRISPR/Cas9 system, have paved the way to precise NK cell modification. Gene editing offers a broad approach to enhancing NK cell antitumor efficacy by enabling the knockout (KO) of disruptive genes or knockin (KI) of CAR, activating cytokines, or chemokines. Gong, et al. [39] recently reported that a therapeutic approach based on KO of the inhibitory receptor NKG2A in human primary NK cells improved the antitumor response compared with that observed with therapy using NKG2A monoclonal antibodies. Because electroporation was used to introduce the ribonucleoprotein (RNP) system into NK cells, 13.8% of cells were NKG2A before sorting. Gurney, et al. [40] reported that a CLL-1 CAR and CISH-targeting CRISPR/Cas9 system were co-delivered into ~50% of human primary NK cells [40]. Another study revealed that NK cells with KO of CALHM2, a calcium homeostasis modulator, showed enhanced cytotoxicity and tumor infiltration in mouse and human primary NK cells [41]. The KO efficiency in human primary NK cells, NK92 cells, and mouse primary NK cells was ~40%, ~60%, and ~70%, respectively. However, CRISPR/Cas9 introduction into NK cells relies on electroporation, which continues to pose significant challenges in terms of reduced cell viability and the necessity for sorting cells that have successfully incorporated the genetic material. Table 3 summarizes the above applications.

Transfection efficiency of CRISPR/Cas9 system in NK cells
Cell sourceGenetic modification systemGenetic materialTransfection efficiencyReference
Human primary NK cellsCRISPR/Cas9 (electroporation)RNP for NKG2A KO13.8%[39]
Human primary NK cellsCRISPR/Cas9 (electroporation)RNP for CALHM2 KO~40%[41]
NK92CRISPR/Cas9 (electroporation)RNP for CALHM2 KO~60%[41]
Mouse primary NK cellsCRISPR/Cas9 (electroporation)RNP for CALHM2 KO~70%[41]
Human primary NK cellsCRISPR/Cas9 and TcB transposon system (electroporation)RNP for CISH KO~50%[40]

Abbreviations: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9); NK, natural killer; RNP, ribonucleoprotein; KO, knockout; TcB; TcBuster.



Methods for transient gene expression in NK cells

Transient transfection is an effective technique for the targeted introduction of nucleic acids into cells, facilitating the delivery of genome editing tools for gene KO or the introduction of exogenous transgenes. A significant concern associated with viral NK cell modification is the potential for insertional mutagenesis because of the high viral titers required for efficient transduction. Non-viral transfection methods mitigate this risk, rendering them more advantageous from a safety standpoint, particularly in the context of innovative immunotherapy development. However, compared with viral-based approaches, non-viral methods typically result in rapid but transient transgene expression. The most prevalent gene transfer techniques using transfection are lipofection and electroporation.

Electroporation: overview and applications

Electroporation, a widely used non-viral transfection strategy, induces the formation of temporary pores on the recipient cell surfaces to facilitate gene entry. When performed in the presence of activating cytokines, such as IL-2 or IL-15, NK cell electroporation can achieve efficiencies up to 90% [4244]. However, transfection efficiency varies by cell type, and transfected cell viability depends on the cargo and sometimes necessitates the presence of cytokines. In NK cells, transfection efficiencies are often diminished because of reduced cell viability, which negatively impacts the ability of the cells to uptake genes and survive the electroporation process. Despite these challenges, electroporation remains a valuable technique for NK cell genetic modification, particularly when viral methods are impractical or undesirable. However, this technique requires a substantial quantity of genetic material, a specialized electroporator, and appropriate buffer solutions to achieve effective transfection.

Nanoparticles: advanced technology for NK cell engineering

Engineered particles made from various materials to encapsulate and transport genetic material into cells can be used for NK cell engineering. Nanoparticle-based gene transfer methods utilize endocytosis to effectively deliver genetic materials, offering safety, biocompatibility, and immunological control comparable to viral-based approaches. Furthermore, nanoparticles can function as immunomodulators, enhancing NK cell activity and providing additional functionality beyond gene delivery. Therefore, nanoparticle-based gene delivery into NK cells is actively being researched worldwide (Table 4). Multifunctional nanoparticles (MF-NPs) coated with a cationic polymer such as polyethyleneimine can transfect plasmids into NK cells with up to 60% efficiency [45]. MF-NPs not only facilitate the genetic manipulation of NK92MI cells but also enable in vivo tracking via non-invasive magnetic resonance and fluorescence optical imaging. Adjei, et al. [46] developed manganese dioxide nanoparticles (MnO2-NPs) that deliver small interfering RNA (siRNA) targeting TGF-β receptor 2 (TGFBR2) into NK92 cells to restore their activity against cancer cells. MnO2-NPs were synthesized by reducing potassium permanganate (KMnO4) with the cationic polyelectrolyte poly(allylamine) hydrochloride and achieved ~90% gene silencing efficiency.

Comprehensive overview of nanoparticle-based gene delivery systems for NK cells
Cell sourceDelivery systemGenetic materialsTransfection efficiencyAdditional functionReference
NK92MIMultifunctional nanoparticlesAnti-EGFR-CAR-encoding plasmid DNA~60%In vivo tracking (non-invasive magnetic resonance and fluorescence optical imaging)[45]
NK92Manganese dioxide nanoparticlesTGFBR2 siRNA~90%-[46]
NK92LipofectamineIL-15-encoding plasmid DNA--[47]
NK92LipofectamineStem cell factor-encoding plasmid DNA--[48]
Human primary NK cellsLipofectamineCy3 dye-labeled pre-miR negative control~30%[49]
Human primaryNK cells from a hepatocellular carcinoma patientHiPerfect transfection reagentIGF siRNA or miR-486-5p~40%[50]
Human primaryNK cellsCAR-T polyplexesEGFP mRNA~30%[53]
NK92YSK12-multifunctional envelope-type nanodeviceGAPDH siRNA~80%-[54]
NK92CL1H6-LNPGAPDH siRNA~60%-[55]
NK92CL1H6-LNPEGFP mRNA and Fluc mRNA~90%-[56]
NK92MI, mouse primary NK cells, human primary NK cellsDOTAP-LNPAnti-GPC3 CAR mRNA~45%NK cell activation via ERK/MAPK signaling and mitochondrial dynamics[57]

Abbreviations: NK, natural killer; IGF, insulin-like growth factor; CART, charge-altering releasable transporter; DOTAP; 1,2-dioleoyl-3-trimethylammonium-propane, siRNA, small interfering RNA.



Lipofection or liposome encapsulation, another well-established nanoparticle-based method, involves the encapsulation of genes within liposomes, which are composed of lipid bilayers and designed to facilitate gene transfer into target cells. Specifically, liposomes containing 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxythyl ammonium bromide/dioleoyl phosphatidyl ethanol-amine as cationic lipids are commonly used [37]. Zhang, et al. [47, 48] used lipofectamine to transfect NK92 cells with the IL-15 or stem cell factor gene. Regis, et al. [49] used lipofectamine to transfect a miR-27a-5p inhibitor into human primary NK cells derived from healthy donors. Another study used HiPerfect transfection reagent, a blend of cationic and neutral lipids, to transfect miR-486-5p or insulin-like growth factor siRNA into primary NK cells isolated from patients with hepatocellular carcinoma [50].

Charge-altering releasable transporters (CARTs) offer a novel approach to lipid-based gene delivery. CARTs are multiblock oligomers consisting of lipid blocks and a charge-altering block [51]. Unlike traditional lipofection methods, which use persistently cationic lipids, CARTs are initially cationic to complex with nucleic acids but biodegrade under physiological conditions, releasing neutral products that facilitate cargo release [51, 52] This unique property may enhance transfection efficiency while reducing cytotoxicity compared with those observed using conventional cationic lipids. Wilk, et al. [53] demonstrated that the approach using CARTs achieved ~30% transfection efficiency in primary NK cells. This approach represents a promising advancement in lipid-based gene delivery, potentially offering improved efficiency and reduced toxicity compared with those of traditional lipofection techniques.

Lipid nanoparticles (LNPs), recently highlighted by their use in COVID-19 mRNA vaccines, have emerged as promising mRNA delivery tools. LNPs offer significant advantages, including high efficiency, the ability to preserve genetic material, and biocompatibility. Leveraging these benefits, studies have attempted LNP-based gene delivery to NK cells. For example, a multifunctional envelope-type nanodevice incorporating YSK12-C4 lipid, an ionizable cationic lipid, delivered siRNA into ~80% of NK92 cells [54]. Nakamura, et al. [55] developed an LNP composed of CL1H6 (CL1H6-LNP), a pH-sensitive cationic lipid, to efficiently deliver mRNA and siRNA into NK92 cells. CL1H6-LNP encapsulating siRNA showed an ~60% delivery efficiency in NK92 cells [55]. mRNA-loaded CL1H6-LNP resulted in high mRNA expression levels and a 90% transfection efficiency in NK92 cells [56]. Recently, LNPs have been developed to deliver genes into NK cells while also enhancing the antitumor effects of NK cells via the properties of the LNPs. LNPs functionalized with 1,2-dioleoyl-3-trimethylammonium-propane, a cationic lipid, effectively delivered CAR-encoding mRNA into ~45% of NK cells [57]. This approach not only facilitates gene delivery but also enhances NK cell-mediated cancer-killing via extracellular signal-regulated kinase/mitogen-activated protein kinase pathway modulation and alterations in mitochondrial dynamics [57].

Potential of improving genetic engineering via nanoparticles

Research on nanoparticles facilitating efficient genetic engineering in NK cells is progressively expanding and gaining significance. Studies have indicated that cationic substances, which have a positive charge and serve as efficient nucleic acid condensers, hold significant promise for genetic engineering in NK cells [45, 57]. However, further research is required to fully understand the mechanisms underlying their effectiveness. The physicochemical properties of these cationic materials, such as their size, composition, and surface modifications, should be better characterized, particularly in terms of their impact on endocytosis and cellular uptake. Moreover, future studies should investigate the molecular biology of their effects and mechanisms on NK cells, including examination of their interactions with cellular components and potential impacts on gene expression and cell function.

Although conventional viral-based and electroporation methods are commonly used for gene delivery into NK cells, nanoparticles have unique multifunctional capabilities that set them apart from these traditional methods [58, 59] Nanoparticles can be designed to not only deliver genetic material but also to provide additional functionalities, including cell tracking and controlled release of therapeutic agents. Therefore, nanoparticle-based genetic engineering of NK cells can be further explored with a focus on conferring these functionalities using different approaches.

Potential areas for further investigation include developing nanoparticles that allow for real-time monitoring of the NK cell distribution and persistence in the body, nanoparticles that can deliver genes to enhance NK cell function while simultaneously providing immunomodulatory effects, nanoparticles with surface modifications that can specifically target NK cells or guide them to tumor sites, and nanoparticles that can provide sustained or triggered release of genetic material or supportive factors to maintain NK cell function over time. By leveraging these multifunctional capabilities, nanoparticle-based approaches have the potential to overcome current limitations in NK cell genetic engineering and pave the way to more effective and versatile NK cell-based therapies.

Genetic engineering of NK cells based on functional genetic materials

NK cells primarily kill target cells via three mechanisms [60]. First, they can directly kill target cells by releasing cytoplasmic granules containing perforin and granzymes. Second, they can release cytokines that induce tumor cell apoptosis by interacting with the corresponding receptors on the tumor cell surface. Third, the Fc receptor CD16 binds to the Fc region of the antibody, which can trigger antibody-dependent cell-mediated cytotoxicity to kill target cells. These diverse killing mechanisms are the basis for the antitumor activity of NK cells; hence, genetic modification strategies can be used to improve the efficacy of NK cell-based cancer immunotherapy. With advances in this field, NK cell genetic manipulation has opened up possibilities to study numerous pathways involved in NK cell tumor targeting and the ability to genetically modify NK cells to improve their tumor cytotoxicity. Gene modification strategies can augment in vivo persistence and expansion, tumor tissue migration, and the tumor-targeting capacity of adoptively infused NK cells via autocrine stimulation, cellular homing-related receptors, adhesion molecules, CARs, activating NK cell receptors, or silencing of inhibitory NK cell receptors (Table 5). In what follows, we discuss the enhancement of NK functionality in the function of the gene introduced, i.e., CARs and non-CARs.

Overview of in vitro and in vivo strategies evaluated based on CAR or non-CAR genetic materials for improving NK functionality in cancer immunotherapy
CAR/non-CARGenetic moleculeStrategyNK cell sourceMalignancyGenetic manipulation methodReference
CARαEGFR CARCytotoxicity plus targetingNK92MIBreast cancerMultifunctional nanoparticles[45]
αGPC3 CARCytotoxicity plus targetingNK92MI, mouse primary NK cells, and human primary NK cellsLiver, breast, renal, and colon cancer cells, hepatocellular carcinomaDOTAP-based lipid nanoparticle[57]
αCD19 CARCytotoxicity plus targetingHuman primary NK cellsHuman patients (CD19+ B-cell malignancy)Retrovirus[11]
αCD33 CARCytotoxicity plus targetingHuman primary NK cellsAcute myeloid leukemiaLentivirus[63]
αCD73 CARCytotoxicity plus targeting, infiltration, and cytokine productionHuman primary NK cellsLung adenocarcinomaElectroporation and lentivirus[71]
αGD2 CARCytotoxicity plus targetingNK92Patient-derived diffuse intrinsic pontine gliomas (DIPG) cells, DIPGLentivirus[64]
Soluble PD-1 with αHER2 CARCytotoxicity plus targeting and immune activationNK92Breast cancer cells,
mammary carcinoma
Lentivirus[62]
HER2/synNotch GAL4 αCEA CARCytotoxicity plus targetingNK92Breast adenocarcinoma cell, colorectal carcinoma cell, colorectal adenocarcinoma cell, colorectal adenocarcinomaLentivirus[65]
αEPCAM CAR with IL-15Cytotoxicity plus targeting and proliferationNK92Leukemia cells, triple-negative breast cancer cells, melanoma cellsLentivirus[61]
αNKG2D CARCytotoxicity plus targetingHuman primary NK cellsMultiple myelomaLentivirus[66]
αNKG2D CARCytotoxicity plus targetingHuman primary NK cellsMetastatic colorectal cancerElectroporation[67]
αNKG2D CAR with IL-15Cytotoxicity plus targeting and persistenceHuman primary NK cellsAcute myeloid leukemiapiggyBac system[68]
αDelta-like ligand 3 (αDLL3) CARCytotoxicity plus targeting and cytokine productionNK92Small cell lung cancerLentivirus[69]
αMesothelin/αCD19 CARCytotoxicity plus targeting and cytokine productionNK92Gastric cancerLentivirus[70]
CCR5Chemoattractive capacityHuman primary NK cellsColon cancerLentivirus[77]
CCR7Chemoattractive capacityNK92B-cell lymphomaElectroporation[44]
CXCR1Chemoattractive capacityHuman primary NK cellsPancreatic cancerLentivirus[78]
CXCR2Migration and calcium fluxHuman primary NK cellsRenal cell carcinomaRetrovirus[25]
CXCR2Chemoattractive capacity and cytotoxicityNK92Colon cancerCRISPR-Cas9[74]
TGFBR2Activation and cytotoxicityHuman primary NK cellsNeuroblastomaRetrovirus[75]
TGFBR2 (siRNA)Infiltration and cytotoxicityNK92Lung cancer cellsManganese dioxide nanoparticles (MnO2 NPs)[46]
IGF-1 siRNA or miR-486-5pCytolytic functionHuman primaryNK cellsHepatocellular carcinomaHiPerfect transfection reagent[50]
IL-15 with CD8 α transmembrane domain (mbIL-15)Survival and cytotoxicityHuman primary NK cellIn vitro (leukemia, lymphoma, and solid tumor cells)
In vivo (sarcoma)
Retrovirus[72]
IL-21Autocrine (persistence and antitumor activity)Human primary NK cellsIn vivo (glioblastoma)Retrovirus[73]

Abbreviations: NK, natural killer; IGF, insulin-like growth factor; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; EGFR, epidermal growth factor receptor; NK92, allogeneic NK cell line (IL-2 dependent NK cells); PD-1, programmed cell death protein 1; GAL4, galactose-responsive transcription factor; CEA, carcinoembryonic antigen; NP, nanoparticle



Genetic manipulation based on CARs

The effects of CARs on NK cells differ depending on which CAR structures are expressed in NK cell lines or primary NK cells. As mentioned previously, CARs are engineered receptors that have extracellular targeting specificity combined with an intracellular signaling domain [22]. The CAR-arming of NK cells grants specific antigen recognition in a major histocompatibility-independent manner and delivers an activation signal within NK cells to trigger cytotoxicity and/or cytokine secretion. With CAR development, additional benefits can be incorporated, for example, to increase NK cell proliferation [61] or modulate the TME [15, 62]. Recently, CAR-engineered NK cells targeting various tumor targets, including EGFR [45], GPC3 [57], CD19 [11], CD33 [63], GD2 [64], CEA [65], EPCAM [61], NKG2D [6668], DLL3 [69], and mesothelin [70], have been generated. Unlike CAR-T cells, CAR-NK cells are effective against solid tumors such as liver cancer [57], renal cancer [57], glioblastoma [64], lung cancer [69, 71], and breast cancer [45, 57, 61, 62, 65].

Genetic modification based on non-CARs

Cytokines and chemokines are key non-CARs used in NK cell genetic engineering to expand NK cell proliferation [72], persistence [73], and tumor cytotoxicity [46, 50, 72, 74, 75]. The major benefit of NK cells is that they can kill a wide range of tumor cells without the requirement for specific antigen recognition. Although adoptively infused NK cells have induced clinical responses in a few patients with solid tumors [76], a potential shortcoming for the accomplishment of adoptive NK cell therapy is their low persistence in the body and inefficient migration into the tumor site, partially explaining the overall poor clinical responses in cancer patients [15]. Cytokines and chemokines control the tumor immune cell recruitment to promote intratumoral NK cell infiltration and tumor regression. Genetic alteration based on non-CARs can enhance NK activation related to infiltration and cytotoxicity based on the basic target-killing function of NK cells. non-CARs targeted in engineered NK cells include CCR5 [77], CCR7 [44], CXCR1 [78], CXCR2 [25], TGFB2 [75], IL-15 [72], and IL-21 [73]. Non-CAR-NK cells have shown efficacy against colon cancer, pancreatic cancer, renal cancer, neuroblastoma, lung cancer, and lymphoma. With various functional enhancements of CARs or non-CARs, functionally boosted NK cells have emerged as a next-generation cell therapy for clinical applications beyond cancer immunotherapy.

NK cells are promising immune effectors for cancer and infectious diseases. Current techniques allow the creation of precisely engineered NK cells and may enable a shift from CAR-T to CAR-NK therapy, enhancing safety and reducing the processing time for allogeneic treatments. Beyond cancer, NK cells are vital in innate immunity against viral infections such as cytomegalovirus, HIV-1, and SARS-CoV-2 infections [79], aiding early inflammatory responses and controlling viral replication [80]. As tissue-resident NK cells are crucial for antiviral defense via cytokine production and targeting infected cells, genetically engineered NK cells may hold promise for enhancing immune defenses against viruses [81, 82]. Engineered NK cell lines have undergone preclinical evaluations, with some progressing to clinical trials, and umbilical cord blood- and stem cell-derived NK cells have shown potential for adoptive transfer therapies. Despite challenges in maintaining viability and functionality post-modification, modern genetic techniques hold promise for expanding NK cell applications in clinical settings.

Two main challenges in NK cell-based cancer immunotherapy are the limited durability of NK cells in vivo in the absence of cytokines and diminished function in vivo. Advancements in genetic engineering, such as CRISPR/Cas9 and nanoparticle delivery systems, may enhance the efficacy of NK cell-based therapies by enabling precise modifications that improve NK cell persistence and resistance to the immunosuppressive TME. A recent study used retroviruses to create cytokine-producing NK cells to enhance their durability [73]. Creating cytokine-producing NK cells via CRISPR/Cas9 delivered via nanoparticles offers a promising, non-viral approach. Secondary genetic modifications using CRISPR/Cas9 can help CAR-NK cells overcome the suppressive TME. Nanoparticles capable of carrying both genes and drugs, such as metformin, can enhance NK cell function and reduce PD-L1/PD-1 expression, potentially improving efficacy in the TME [83, 84]. Combining genetic engineering with immunomodulatory approaches, such as checkpoint inhibitors or cytokine therapies [85, 86], may boost NK cell efficacy. However, nanoparticle-based NK cell gene delivery faces challenges, including the need for efficient endosomal escape after internalization [87, 88]. Enhancing endosomal escape efficiency is crucial for optimizing nanoparticle-based gene delivery systems. Moreover, as this delivery method often results in transient expression, further research into controlled release mechanisms is needed to prolong gene expression. Addressing these challenges is crucial for improving the longevity and efficacy of genetically engineered NK cells in cancer immunotherapy.

As research in this field advances, several critical aspects demand attention to fully realize the potential of genetically engineered NK cells in both cancer immunotherapy and infectious disease treatment. Addressing potential safety concerns remains paramount, necessitating thorough investigation and mitigation strategies. Concurrently, optimizing manufacturing processes is essential to ensure the scalability, consistency, and cost-effectiveness of these cell therapies. Rigorous, well-designed clinical trials will be crucial for validating the efficacy and safety profiles of engineered NK cells in various disease contexts. By focusing on these key areas, the scientific community can pave the way for the widespread clinical application of genetically engineered NK cells, potentially revolutionizing treatment paradigms in oncology and infectious diseases.

Park JD and Shin HE equally contributed to the writing, literature review, and overall conception of this review article. An YS and Jang HJ created and visualized the figures. Park W conducted the final modifications and proofreading of the entire article. Park J, Kim SN, and Park CG critically reviewed the manuscript and provided valuable feedback. All authors have read and agreed to the published version of the manuscript.

This work was supported by Basic Science Research Program grants (RS-2024-00350878, RS-2023-00218648, and RS-2023-00242443) through the National Research Foundation of Korea (NRF) grants, the KIST Institutional Program (2E32351-23-130), and Regional Innovation Strategy (RIS) through the NRF funded by the Ministry of Education (MOE) (2021RIS-001).

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