Natural killer (NK) cells engineered with chimeric antigen receptors (CARs) represent a promising alternative to CAR-T cell therapy, offering the potential for off-the-shelf cancer immunotherapy without the risk of graft-versus-host disease. However, despite encouraging preclinical results, CAR-NK cells face significant clinical challenges that have prevented regulatory approval. Advanced gene editing technologies, particularly CRISPR-Cas9, are now being deployed to overcome these fundamental limitations and unlock the therapeutic potential of CAR-NK cell therapy.
Clinical Promise Meets Technical Challenges
More than 60 CAR-NK clinical trials have been registered on ClinicalTrials.gov, representing substantial investment in this therapeutic approach. Yet all registered trials remain in initial phases, with only limited published results available. The primary obstacles include short in vivo lifespan, reduced proliferation capacity, poor trafficking to tumor sites, dependency on exogenous cytokines, and suppression by immunosuppressive tumor microenvironments.
CAR-NK cells offer several theoretical advantages over CAR-T cells. They can be sourced from various allogeneic sources including peripheral blood, umbilical cord blood, NK cell lines, and pluripotent stem cells, enabling industrial-scale expansion. Unlike T cells, NK cells do not require MHC recognition for activation and pose minimal risk of graft-versus-host disease when used in third-party settings. Additionally, CAR-NK cells employ both CAR-dependent and CAR-independent mechanisms for tumor recognition, including antibody-dependent cellular cytotoxicity (ADCC) and natural cytotoxicity receptors.
CRISPR-Enhanced Cytotoxicity and Function
Gene editing is revolutionizing CAR-NK cell engineering by targeting specific inhibitory pathways. CRISPR-Cas9 disruption of the KLRC1 gene, which encodes the inhibitory receptor NKG2A, enhances NK cell cytotoxicity against tumors expressing high levels of HLA-E. Similarly, deletion of TGFBR2 makes CAR-NK cells resistant to TGF-β-mediated immunosuppression without compromising anti-tumor functions.
The cytokine-inducible Src homology 2-containing protein (CIS) represents another critical target. CISH gene disruption enhances proliferation, survival, and metabolic fitness of CAR-NK cells by preventing inhibition of the JAK-STAT signaling pathway. Studies demonstrate that CISH-disrupted CAR-NK cells show superior persistence and tumor control compared to unmodified cells, with effects being particularly pronounced in IL-15-secreting CAR-NK cells.
CD38 disruption addresses both functional enhancement and manufacturing challenges. Beyond serving as an immunometabolic checkpoint that regulates intracellular NAD+ levels, CD38 deletion prevents fratricide during ex vivo expansion when targeting CD38-positive malignancies like multiple myeloma. CRISPR-mediated CD38 disruption reduces fratricide rates from 19% to less than 0.8% while potentially improving oxidative phosphorylation and ATP synthesis.
Overcoming Immunosuppressive Barriers
The tumor microenvironment presents formidable challenges through immunosuppressive metabolites and checkpoint molecules. Gene editing strategies are addressing these barriers through multiple approaches. Disruption of TGF-β signaling can be achieved by deleting TGFBR2 or silencing downstream mediator SMAD3, both of which enhance NK cell cytotoxicity and IFN-γ secretion.
Checkpoint inhibition through genetic modification offers advantages over systemic antibody administration by avoiding immune-related adverse events. CRISPR disruption of PDCD1 (encoding PD-1) leads to better persistence, higher cytokine secretion, and enhanced cytotoxicity. Similarly, targeting TRIM29, which suppresses NK cell IFN-γ secretion, represents an emerging strategy for maintaining proinflammatory states.
The adenosine pathway, which suppresses NK function through CD73-mediated AMP conversion and A2A receptor binding, can be disrupted through genetic ablation of A2AR2. This approach increases CAR-NK persistence within tumor microenvironments where ATP derivatives accumulate due to high rates of apoptosis and hypoxia.
Enhanced Persistence Through Cytokine Engineering
Addressing the short lifespan of CAR-NK cells requires sophisticated cytokine engineering approaches. IL-15 armoring through genetic modification has shown promise in both preclinical models and clinical trials. IL-15-secreting CAR-NK cells demonstrate increased proliferation, persistence, and tumor control, with one clinical trial (NCT03056339) safely utilizing this approach.
To minimize systemic toxicities associated with secreted cytokines, researchers have developed membrane-bound IL-15 (mbIL-15) that provides autocrine signaling without systemic exposure. The most advanced approach involves IL-15/IL-15 receptor α fusion proteins that provide continuous activation signals. Fate Therapeutics has incorporated this technology into multiple iPSC-derived CAR-NK products including FT522, FT576, and FT596, which are currently in clinical evaluation.
IL-21 engineering represents another promising avenue, with recent studies indicating that IL-21-secreting CD19 CAR-NK cells demonstrate superior persistence, proliferation, and cytotoxicity compared to IL-15-secreting variants. For NK-92 cells, which are highly dependent on IL-2, novel membrane-bound IL-2/IL-2Rα fusion proteins enable survival without systemic toxicities or bystander cell stimulation.
Improving Tumor Trafficking and Homing
Limited migration to tumor sites represents a major bottleneck, particularly for solid tumors. Gene editing is being used to modify chemokine receptor expression patterns to enhance tumor infiltration. CXCR4 overexpression increases CAR-NK cell migration to bone marrow niches, with studies showing up to two-fold improvement in tumor site trafficking for CD19 CAR-NK cells.
Conversely, CRISPR-mediated disruption of CCR5 prevents retention of adoptively transferred NK cells in the liver, increasing their circulation and trafficking to non-hepatic tumor sites. For lymph node targeting, CCR7 overexpression enhances migration in response to CCL19 and CCL21, with studies reporting up to six-fold increases in migration and five-fold improvements in killing efficiency.
Multiple chemokine receptor modifications may be required for optimal solid tumor penetration. Combined CCR7 and CXCR4 modification in NK-92 cells improves migration into colon cancer models, suggesting that multiplexed approaches may yield superior clinical outcomes.
Safety Considerations and Risk Mitigation
Despite their improved safety profile compared to CAR-T cells, CAR-NK therapies require robust safety mechanisms. Inducible caspase9 (iCasp9) safety switches enable selective elimination of CAR-NK cells upon administration of AP1903 dimerizing agent. This approach was successfully incorporated into the first clinical CAR-NK trial, though activation was not required due to absence of adverse events.
Alternative safety strategies include insertion of transgenes encoding CD20 or EGFR epitopes, enabling depletion through rituximab or cetuximab administration. Herpes simplex virus thymidine kinase (HSV-TK) represents another option, converting ganciclovir to a DNA replication inhibitor for selective cell elimination.
Manufacturing and Scalability Solutions
Gene editing is addressing manufacturing challenges that have limited CAR-NK cell production. Transposon-based delivery systems, including Sleeping Beauty and piggyBac, offer cost-effective alternatives to viral vectors with improved safety profiles and larger cargo capacity. While viral vectors remain limited to 8 kb cargo, transposons can deliver up to 100 kb of genetic material.
Site-directed insertion using programmable nucleases and recombinant adeno-associated virus templates enables uniform CAR expression while preventing insertional oncogenesis. Genomic safe harbors including AAVS1, CCR5, and ROSA26 provide ideal integration sites for CAR transgenes.
Future Directions and Clinical Translation
The convergence of advanced gene editing technologies with CAR-NK cell engineering is creating unprecedented opportunities to address longstanding clinical challenges. Multiplexed editing strategies that simultaneously target multiple bottlenecks—such as combining CISH and TGFBR2 disruption with IL-15 armoring—are showing superior efficacy in preclinical models.
However, clinical validation remains limited, with most genetically modified CAR-NK approaches still in preclinical development. The field requires comprehensive clinical studies to evaluate safety profiles and long-term consequences of genetic modifications. Additionally, standardization of manufacturing processes and regulatory frameworks will be essential for successful clinical translation.
The integration of CRISPR gene editing with CAR-NK cell therapy represents a paradigm shift in cancer immunotherapy development. By systematically addressing the fundamental limitations that have prevented clinical success, these approaches are positioning CAR-NK cells as a viable off-the-shelf alternative to personalized CAR-T therapies. Success in ongoing clinical trials could establish gene-edited CAR-NK cells as a cornerstone of next-generation cancer immunotherapy.
