cGAS/STING Innate Immune Signaling
cGAS (cyclic GMP-AMP synthase) is a nucleotide transferase consisting of an unstructured, non-conserved N-terminal (130-150 residues) and a C-terminal containing an Mab21 domain and a highly conserved zinc-binding motif. The N-terminal of cGAS has sequence-independent DNA binding activity, allowing it to recognize almost all dsDNA (exogenous DNA) by detecting the phosphoribose backbone structure. The C-terminal catalytic domain contains three DNA binding sites, known as “site A,” “site B,” and “site C.” Site A plays a major role in inducing conformational changes in cGAS during interaction with dsDNA, while sites B and C assist in dsDNA binding and cGAS dimerization. The zinc-binding motif in the C-terminal binds Zn2+ to maintain the higher-order structure and physiological function of cGAS, thereby promoting the dsDNA-cGAS interaction and stabilizing the cGAS-dsDNA complex.
STING (stimulator of interferon genes) is a transmembrane protein consisting of a transmembrane domain (TMD), a cytoplasmic ligand-binding domain (LBD), and a C-terminal tail (CTT). STING exists as a dimer, maintaining its stability through intramolecular and intermolecular interactions between its domains. The C-terminal part of STING, containing LBD and CTT, faces the cytosol, forming an “open” V-shaped binding pocket. The LBD can bind to the two phosphate groups of cGAMP, inducing a conformational change in the C-terminal. Subsequently, the V-shaped dimer adopts a tighter “closed” conformation and forms a “cap” that covers the cGAMP binding site. This conformational change triggers the translocation of STING from the ER-Golgi intermediate compartment (ERGIC) to the Golgi apparatus. Upon reaching the Golgi, STING undergoes palmitoylation at two cysteine residues (Cys88 and Cys91) in its N-terminal, facilitating its interaction and activation with TANK-binding kinase 1 (TBK1).
TBK1 (TANK-binding kinase 1) is a central kinase that directly phosphorylates interferon regulatory factor 3 (IRF3), thereby upregulating IFN-I expression. Eight amino acid residues (369-377) in the CTT of STING serve as the TBK1 binding motif, which is crucial for promoting the interaction between STING and TBK1. The higher-order oligomerization of the STING-TBK1 complex further enhances the cis-autophosphorylation of TBK1 and phosphorylation of nearby STING. The phosphorylated LXIS motif (363–366) in STING forms an IRF3 binding site, recruiting IRF3 and enabling TBK1 to phosphorylate IRF3. After phosphorylation, IRF3 forms a tightly bound dimer structure. The dimerized IRF3 then translocates to the nucleus, where its N-terminal DNA-binding domain attaches to DNA, initiating the transcription of downstream genes and driving IFN-I expression and secretion. Meanwhile, TBK1 activates NF-κB, a transcription factor with various regulatory functions. Once translocated to the nucleus, NF-κB collaborates with IRF3 to stimulate the gene expression of various cytokines, including IFN-I, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α).
cGAS-STING Pathway in Cancer Immunotherapy
Encapsulation of STING Agonists to Activate the STING Pathway
A schematic diagram of in situ gel vaccination for sustained release of cGAMP nanoparticles, doxorubicin (Dox), and immune modulators to generate persistent STING activation and effective anti-cancer immunity. Damage-associated molecular patterns (DAMPs) refer to endogenous molecules released during cell death, also known as endogenous danger signals, which are activated by immune cells from damaged or necrotic tissues.
An in situ gel vaccine based on thermoresponsive silk fibroin (SF) hydrogel encapsulates nanoscale STING agonists (cGAMPnp), immunogenic cell death (ICD) inducers, and immune modulators, providing a local reservoir for controlled, sustained release of immunotherapeutic agents. The in situ gel vaccine effectively triggers STING-driven tumor vascular normalization and activates dendritic cells (DCs) and effector CD8 T cells both within the tumor and in draining lymph nodes and distant metastases. This results in the inhibition of tumor spread and prevention of immune-related tumor recurrence.
Nanoscale STING agonists, cGAMPnps, facilitate the efficient uptake of cGAMP and induce the activation of type I IFN responses in DCs. Hydrogel formulations containing the ICD inducer Dox trigger the release of tumor antigens and enhance and prolong STING activation by cGAMPnps. Additionally, incorporating checkpoint inhibitors (e.g., anti-PD-1 antibodies) or immune modulators (such as OX40L) into the hydrogel reprograms the immunosuppressive tumor microenvironment (TME), thereby improving the efficacy of in situ cancer vaccination. This vaccination approach leads to STING-induced tumor vascular normalization, with significantly increased DC activation and effector CD8+ T cells in both the tumor and associated lymph nodes, indicating improved anti-tumor immunity.
Metal Ion Activation of the STING Pathway
Mn2+ can directly activate cyclic GMP-AMP synthase (cGAS), catalyzing the production of cyclic adenosine monophosphate (cGAMP), which can bind to the STING dimer. Subsequently, TANK-binding kinase 1 (TBK1) is recruited, and interferon regulatory factor 3 (IRF3) is phosphorylated, leading to the secretion of type I interferons (IFN), which enhances innate immunity.
A screening of typical bioactive metal anions (GeO32-, MoO42-, WO42-, VO3, and VO43-) for necroptosis inducers revealed that vanadate exhibited significant cytotoxicity. By inhibiting ATPase activity, vanadate disrupted intracellular K+ and Ca2+ ion homeostasis, inducing necroptosis. After confirming the bioactivity and necroptosis-inducing efficacy of several metal anions, polyethyleneglycol (PEG)-based bimetallic vanadate-manganese nanoparticles (MnVOx) were synthesized. On the one hand, MnVOx depletes glutathione (GSH) and upregulates reactive oxygen species (ROS). The degradation product, vanadate anion, inhibits Na+/K+ ATPase and Ca2+ ATPase activities. The subsequent K+ efflux and Ca2+ overload lead to inflammasome activation, mitochondrial damage, and endoplasmic reticulum (ER) stress, ultimately resulting in rapid cell death and the production of pro-inflammatory mediators IL-1β and IL-18.
On the other hand, in vitro and in vivo, the released Mn2+ ions activate the STING pathway by promoting DC maturation and downstream IFN-β secretion. This synergistic strategy inhibits tumor growth and reprograms the tumor microenvironment (TME) by recruiting CD8+ T cells and M1 macrophages. Additionally, the increase of regulatory T cells (Tregs) in combination with α-CTLA-4 (similar to immune checkpoint blockade (ICB) therapy, where CTLA-4 is an early immune checkpoint in T cell activation and PD-1 tends to regulate T cell differentiation upon activation) triggered a robust immune response, effectively defeating both primary and distant tumor metastases.
Finally, a MnVOx-lipiodol dispersion (MnVOx-Lip) was developed for interventional arterial embolization (TAE) treatment of liver cancer in rabbits, and it was shown that this MnVOx-Lip formulation provided better therapeutic effects compared to the clinically widely used lipiodol.
cGAS-STING Pathway in Antibacterial and Antiviral Therapy
Encapsulation of STING Agonists to Clear Viruses
A previously developed Salmonella-based RNA nuclease (MazF)-regulated delivery system, known as the Salmonella mRNA interferase regulation vector (SIRV) system, mediates enhanced expression of exogenous antigens in Salmonella cells through differential regulation of mRNA degradation. It also actively creates membrane pores, with or without the ACA base triplet, to release intracellular materials. Using the SIRV system’s Salmonella strains as carriers, a new pure biological nanoparticle vector (PBNV) system was developed. This new system improves cargo-targeting specificity, thereby enhancing the self-assembly of nanoparticles (NPs). Additionally, it simultaneously biosynthesizes STING agonists, specifically targeting their exposure to the host innate immune system.
The platform consists of two modules:
(1) Self-assembled nanoparticle antigen: A plasmid containing an exogenous gene is introduced into a Salmonella vector. Under the control of the SIRV system, the gene is highly expressed and secreted into the periplasm, where it effectively self-assembles into nanoparticles (NPs).
(2) Biosynthesis of the STING agonist cyclic di-adenosine monophosphate (CDA): The gene encoding di-adenosine cyclase (DacA) from Gram-positive bacteria is integrated into the recombinant Salmonella genome, coding for an enzyme that catalyzes the conversion of ATP into the STING agonist CDA, providing adjuvant activity. CDA is released in large quantities from the SIRV vector, activating the host cell’s STING-IFN-β-ISGs pathway, inducing a broad-spectrum antiviral response. The activation of the STING pathway enhances the activation of antigen-presenting cells (APCs) and T cells, thereby boosting the adaptive immune response to viruses.
Sonodynamic Therapy Combined with the STING Pathway
A new iron-based covalent organic framework (COF) nanoadjuvant doped with curcumin and platinum (CFCP) has been developed for the effective treatment of repetitive implant-related infections (IRI), a destructive complication in orthopedic surgeries. Curcumin is a natural sonosensitizer that generates high-energy singlet oxygen (1O2) under ultrasound (US) irradiation. The loaded platinum (Pt) exhibits catalase (CAT) activity, catalyzing the generation of oxygen (O2), which enhances the sonodynamic therapy (SDT) of curcumin. CFCP-mediated SDT disrupts the biofilm structure of IRIs and synergizes with the metabolic interference of iron ions to accelerate bacterial death. Subsequently, immunogenic bacterial-related double-stranded DNA (dsDNA) released from the disrupted bacterial biofilm upregulates the STING pathway, inducing dendritic cell (DC) maturation and activating neutrophils’ innate antibacterial immune response. Moreover, the iron ions increase the expression of CD206 on DCs, further promoting the presentation of bacterial-associated antigens (BAAs). DCs increase the presentation of BAAs to T cells and B cells, thus stimulating adaptive immunity characterized by specific antibody secretion and memory immunity.
The enhanced sonodynamic therapy (SDT) of CFCP, combined with metabolic interference by iron ions, increases the release of bacterial-associated double-stranded DNA (dsDNA). The immunogenic dsDNA promotes DC maturation via the activation of the STING pathway and amplifies the immune stimulation of neutrophils through interferon-β (IFN-β). Meanwhile, enhanced BAA presentation in B cells and T cells induces humoral immunity, leading to long-term resistance to recurrent infections.