Tumor-Targeted CRISPR/Cas9 Nanoparticles Disrupt CD73 to Reverse Adenosine-Mediated Immunosuppression and Enhance Chemotherapy

The efficacy of cancer immunotherapy is often compromised by low tumor immunogenicity and an immunosuppressive tumor microenvironment (TME), a phenomenon partly driven by the reprogramming of adenosine metabolism within tumors.

Recently, researchers published a study in Nature Communications entitled “CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy.”

In this study, the researchers developed biodegradable CRISPR/Cas9 nanoparticles capable of durably and precisely downregulating CD73 expression across multiple cellular populations within the TME, including tumor cells, endothelial cells, cancer-associated fibroblasts, and tumor-infiltrating immune cells. This approach effectively disrupted the adenosine metabolic pathway.

The nanoparticle system also co-delivered the chemotherapeutic agent doxorubicin (DOX) to induce immunogenic cell death. The combination synergistically enhanced tumor immunogenicity and reprogrammed the TME into an immune-activating state. This remodeling of the immune microenvironment suppressed tumor progression and prolonged median survival in female mice bearing preclinical models of melanoma and triple-negative breast cancer (TNBC).

The study presents a precise and durable strategy for regulating tumor immunometabolism, thereby enhancing the synergistic antitumor efficacy of combined chemotherapy and immunotherapy.

Adenosine Metabolism as a Driver of Tumor Immunosuppression

Cancer immunotherapy, which restores the body’s antitumor immune response, is an increasingly important component of clinical cancer treatment. However, tumor cells evolve multiple metabolic reprogramming mechanisms that not only support their proliferation, survival, and physiological functions but also establish an immunosuppressive TME, substantially weakening the effectiveness of immunotherapy. Extracellular adenosine (ADO), which accumulates at high levels within the TME, is a key metabolite responsible for reducing therapeutic efficacy.

Extracellular adenosine is primarily generated through the sequential hydrolysis of extracellular ATP by the cell-surface enzymes CD39 and CD73. Whereas extracellular ATP acts as both a “danger signal” and a “chemotactic signal” that activates immune responses, extracellular adenosine binds to adenosine receptors on immune cells. This interaction suppresses the activity of effector immune cells and promotes the expansion of immunosuppressive populations, including regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), ultimately creating an immunosuppressive TME.

Numerous studies have demonstrated that CD73 is upregulated in a variety of cancers, including melanoma and breast cancer. Elevated CD73 expression is closely associated with tumor progression, distant metastasis, resistance to immunotherapy, and poor prognosis. CD73 is therefore a highly promising therapeutic target. Its inhibition could prevent the transition from an ATP-mediated immune-stimulatory environment to an ADO-mediated immunosuppressive environment, providing an effective means of intervening in tumor progression.

Some preclinical studies have used small-molecule inhibitors to suppress the ectonucleotidase activity of CD73 and reduce the conversion of ATP into ADO. However, these agents often have limited selectivity, may cause adverse effects, and can induce drug resistance following repeated administration. Although monoclonal antibodies can generate favorable antitumor immune responses, their relatively short duration of action and potential off-target effects restrict their broader application.

RNA interference can downregulate the expression of disease-associated genes and has considerable potential in cancer therapy. Nevertheless, low transfection efficiency and transient gene silencing make it difficult to achieve durable therapeutic outcomes. New strategies are therefore urgently needed to inhibit CD73 expression precisely and persistently and to reverse adenosine-mediated immunosuppression.

Combining CRISPR/Cas9 Editing with Chemotherapy

CRISPR-Cas9 technology can permanently disrupt tumor-associated genes and therefore holds substantial promise for cancer treatment. Targeting the metabolic immune checkpoint CD73 with CRISPR-Cas9 could overcome many of the limitations associated with small-molecule inhibitors, antibodies, and small interfering RNA therapies. Permanent disruption of CD73 has the potential to relieve adenosine-mediated immunosuppression and produce a potent antitumor response.

However, the effectiveness of therapies that reprogram adenosine metabolism is strongly influenced by the pre-existing immune landscape. Most tumors are immunologically “cold,” characterized by defective antigen presentation, a lack of endogenous T-cell infiltration, and extensive accumulation of immunosuppressive cells. Such tumors respond poorly to therapies that target adenosine metabolism alone. Combining these strategies with other treatments may help convert “cold” tumors into immunogenic “hot” tumors and improve the overall response to immunotherapy.

Multiple preclinical studies have shown that combining chemotherapy with adenosine-metabolism blockade can produce synergistic effects. Immunogenic cell death induced by chemotherapy complements the inhibition of adenosine-mediated immunosuppressive signaling, resulting in substantially greater suppression of tumor growth. Integrating CRISPR/Cas9-mediated adenosine metabolic reprogramming and a chemotherapeutic agent within a single delivery system may therefore offer a more effective cancer treatment strategy.

Figure 1. Schematic illustration of genome-editing nanoparticles to reprogram ADO-mediated immunosuppressive microenvironment and amplify DOX-induced anti-tumor immunity for cancer immunotherapy. (Sun X, et al., 2026)

To address these challenges and achieve an optimal therapeutic effect, the researchers developed a biodegradable CRISPR/Cas9 nanodelivery system that co-delivered CRISPR/Cas9 ribonucleoprotein complexes and DOX to remodel the TME. The nanoparticles, designated iRGD-DCCD73@CaP, reduced CD73 expression in multiple cell populations within the TME and markedly decreased adenosine accumulation.

Comprehensive Remodeling of the Tumor Immune Microenvironment

In poorly immunogenic mouse models of 4T1 triple-negative breast cancer and B16F10 melanoma, the nanoparticle system combined with DOX comprehensively activated antitumor immunity. The abundance of effector immune cells, including CD8⁺ T cells, natural killer cells, M1-like macrophages, and dendritic cells, increased significantly. At the same time, the proportions of immunosuppressive populations, including MDSCs, Tregs, and M2-like macrophages, decreased.

Through these dual and complementary mechanisms, the nanoparticles converted the TME into an immune-stimulatory environment, significantly inhibited primary tumor growth in both models, and prolonged mouse survival.

The multifunctional iRGD-DCCD73@CaP gene-editing delivery platform offers several advantages, including straightforward preparation, high drug-loading capacity, tumor-microenvironment-responsive cargo release, and effective tumor targeting. It represents a promising therapeutic strategy for reversing adenosine-associated immunosuppression, activating antitumor immunity, and restricting tumor progression.