Antisense oligodeoxynucleotides (AS-ODNs) are short, synthetic, single-stranded oligodeoxynucleotides that are complementary to specific sequences of target RNA (Gheibi-Hayat and Jamialahmadi 2021). They alter the target RNA and protein expression by interfering with RNA transport, splicing or translation (Kim et al., 2019). Therefore, AS-ODNs have been evaluated as candidate therapeutic agents to silence target genes in viral infections, autoimmune disease, cancer and genetic disorder related diseases due to their high degree of selectivity and low toxicities (Bennett 2019). Moreover, it was also shown to be a powerful tool for gene function analysis in the medical sciences (Dzialo et al., 2017).

DNA aptamers are oligodeoxynucleotide compounds synthesized from single-stranded DNA molecules, usually consisting of 15–100 nucleotides, with the ability to form secondary and tertiary structures that bind specifically to target molecules (Keijzer et al., 2021; Chen et al., 2022). Aptamers are a new class of ligands that can bind to targets in the range of picomoles and are often compared with antibodies (Morita et al., 2018). Both aptamers and antibodies are biomolecules that bind targets with high affinity and can be used to modulate target functions for diagnosis and treatment of diseases. Compared to antibodies, DNA aptamers exhibit significant advantages including small size, mature and rapid screening process, easy synthesis and chemical modification, as well as low immunogenicity (Table 1). These advantages make aptamers as a promising alternative to antibodies (Dehghani et al., 2018; Ahmadyousefi et al., 2019). DNA aptamers can be specifically used to bind with corresponding proteins, peptides, small molecules and other targets, inhibiting their biological functions, affecting their activity, and achieving the purpose of diseases diagnosis and treatment (Chen et al., 2017).

Immunosuppressive oligodeoxynucleotide (iSup ODN) refers to a class of artificially synthesized single-stranded DNA molecules with a length of tens of nucleotide sequence to function the negative regulation of immune response (Yamada et al., 2004). Compared to CpG ODNs derived from exogenous microorganisms, iSup ODN sequences are mostly derived from mammal genomes. For example, the DNA fragments from calf-thymus and human placenta were shown to inhibit bacterial DNA-induced production of interleukin-12 (IL-12) in murine macrophages (Pisetsky 2000). Mammalian telomere derived TTAGGG repeats were found to inhibit CpG ODN induced production of interleukin-6 (IL-6), IL-12, and interferon-α (IFN-α) in mouse spleen cells (Gursel et al., 2003). Similarly, human telomere derived ACCCCTCT repeats were also found to inhibit CpG ODN induced proliferation of human peripheral blood monocytes and type I IFN production (Yang and Yang 2010). These findings suggest that some iSup ODNs may have some self-regulatory mechanism, which can inhibit over-activated immune response in the body by means of protective negative feedback regulation. Thereby, the iSup ODNs have potential applications in the treatment of immune-mediated inflammatory diseases, but many biological functions of iSup ODNs remain to be developed. Further exploration and discussion of iSup ODN can lay a theoretical foundation for the development of a new and effective iSup ODN drug for treating immune-related diseases in the future.

DNA aptamers are developed for cell surface receptors that bind to protein target sites through three-dimensional electrostatic interactions and bag-like structures for targeted delivery. Therefore, cellular internalization of DNA aptamers is an important way to develop targeted drug delivery systems in vivo (Wan et al., 2019). Studies have shown that endocytosis is the main pathway of cell internalization of different aptamers, including phagocytosis, pinocytosis, clathrin-mediated endocytosis (CME) and alveolar protein-mediated endocytosis (Kaksonen and Roux 2018; Wan et al., 2019). The process by which aptamers are taken into cells depends on their targets but is typically clathrin-mediated endocytosis or macropinocytosis. The clathrin-mediated endocytosis depends on actin and dynamin function, whereas macropinocytosis does not (Li et al., 2017; Wan et al., 2022). Depending on the target or cell type, the main internalization mechanism of aptamers is clathrin-dependent (Figure 2). The binding of the aptamer and receptor initiate formation of the clathrin-coated pit, followed by clathrin-coated vesicle budding. Once detached from the membrane, the clathrin coat is disassembled. Clathrin-dependent endocytosis ends in fusion with endosomes and lysosomes. Typical adaptations for demonstrated clathrin-dependent internalization are Burkett lymphoma cell-specific DNA aptamer and anti-protein tyrosine kinase 7 aptamer (Opazo et al., 2015; Wang et al., 2016). So far, there are relatively few studies on the internalization mechanism of aptamers. Therefore, strategies can be adopted to further explore the relevant mechanisms, screen suitable ligands and use them as targeted delivery tools for corresponding cells.

iSup ODNs can be divided into two categories based on sequence and functional characteristics (Figure 3) (Fehér 2019). The first type is the guanine (G)-rich ODNs. Usually, G-rich iSup ODN can inhibit the activation of TLR9, especially inhibit the immune responses induced by CpG ODNs, such as B cell proliferation, the production of IL-6, IL-12, IFN-γ, and IFN-α (Stunz et al., 2002; Barrat et al., 2005). Moreover, the increasing of poly-G enhances its immunosuppressive activity (Stunz et al., 2002). As a typical inhibitory ODN with poly G, A151 with tandem repeat sequence “TTAGGG” derived from the telomeres of mammalian chromosomes was well investigated (Khan et al., 2022). It has been reported that A151-pretreated dendritic cells and macrophages have reduced their ability to produce type I IFN and tumor necrosis factor α (TNF-α) when activated by cytoplasmic dsDNA. Moreover, A151 blocked IFN-γ, IL-12, IL-6, and other signaling pathways by inhibiting the phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT4, thereby downregulating immune activation (Peter et al., 2008). To clarify the mechanism of iSup ODNs blocking STAT phosphorylation, it was found that A151 was highly specific for intracellular binding to STAT1, STAT3, and STAT4, but did not bind to nuclear factor kappa B (NF-κB) or other molecules in the signaling cascade. Also, A151 inhibited macrophage and dendritic cells from producing mature IL-1β and IL-18 by binding to the AIM2 inflammasome in competition with the irritating ODN (Kaminski et al., 2013). Notably, A151 ODN requires G-tetramer formation to maintain its broad immunosuppressive activity (Klinman et al., 2003).

Some non-G-rich ODN also can inhibit the activation of immune response. For example, SAT05f was designed by our laboratory, which can selectively inhibit the activation of TLR7/9 signaling pathway, subsequently downregulate the production of IFN-α production. Moreover, the inhibition of TLR9 activation is closely related with the blockade of endoplasmic reticulum transmembrane protein Unc93B1 intracellular transport induced by SAT05f (Zhang et al., 2014). MT01, another iSup ODN designed in our lab, can inhibit the proliferation of human peripheral blood mononuclear cells (PBMCs) induced by CpG ODN, and can inhibit the production of type I IFN and B cell activation induced by TLR agonists (Zheng et al., 2020). Similarly, it was showed that MT01 activated Runx2 phosphorylation through MAPKs signaling pathway, delayed the aging rate of dental pulp stem cells (DPSCs), and inhibited the development of pulpitis (Shen et al., 2012). However, the mechanism of iSup ODN is not yet fully understood and needs to be further explored and clarified by researchers.

AS-ODN is of great significance for the treatment of inflammatory diseases, and its mediated intervention has become an important therapeutic method for targeting gene expression operations (Zamecnik and Stephenson 1978; Keating 2014; Crooke et al., 2017). Some recent studies have displayed LNA modified AS-ODNs have considerable therapeutic effects for spinal cord injury, pulmonary fibrosis (Liu et al., 2010) and osteoarthritis (Nakamura et al., 2019). Some AS-ODNs also have therapeutic effects in the field of inflammatory bowel disease (IBD). As we known, IBD is a chronic, recurrent, inflammatory gastrointestinal disease (Seyedian et al., 2019). ISIS 2302 (Alicaforsen) is an RNase H-dependent 20-base-long antisense thiophosphoric oligonucleotide that inhibits human intercellular adhesion molecule-1 (ICAM-1), and it also was the first AS-ODN to be used for the treatment of IBD (Glover et al., 1997). Moreover, a specific AS-ODN targeting Smad7 has been reported to restore smad2/3 phosphorylation, leading to a reduction in inflammatory cytokines in IBD mucosal cells (Monteleone et al., 2001).

In addition, AS-ODN directly acts on viral genomic RNA or transcripts and can be rationally designed for the treatment of human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and HCV), herpes viruses or any new virus (Panda et al., 2021). Fomivirsen (Vitravene), an anti-cytomegalovirus (CMV) agent, is the first AS-ODN to be approved by the FDA as an antiviral therapy. Fomivirsen is an oligonucleotide complementary to the major immediate early region 2 (IE2) of CMV mRNA. In the treatment of CMV retinitis in immunodeficient HIV patients, Fomivirsen can be used to help patients who are resistant to other treatments for CMV (Stein and Castanotto 2017). Thus, AS-ODN can be used as an antiviral agent against acute and chronic inflammation caused by virus infections.

Allergic asthma is also as typical IMIDs. Adenosine A1 receptor has been proposed as a therapeutic intervention target for asthma (Haskó et al., 2008; Bhalla et al., 2020). EPI-2010 is a 21-mer PS-modified respiratory AS-ODN developed by Epigenesis Pharmaceuticals, which reduces airway inflammation and bronchoconstriction by selectively reducing the expression of adenosine A1 receptors in vivo, and increases the level of surfactant in experimental models of allergic asthma (Ball et al., 2004). As we known, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 play a key role in allergic inflammation. They mediate their effect via receptors with a common beta subunit (betac) that transduces cell signaling. TPI ASM8 is an AS-ODN targeting betac with PS modification. It has been confirmed that TPI ASM8 downregulated the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Moreover, TPI ASM8 has been shown to be safe and well tolerated in human trials (Gauvreau et al., 2011; Imaoka et al., 2011). Although EPI-2010 and TPI ASM8 have not been associated with any serious side effects, these AS-ODN safety studies need to last longer because asthma, COPD, and fibrosis are chronic conditions requiring long-term medication (Liao et al., 2017).

Importantly, the continued enrichment of human transcriptomic and proteomic data could facilitate the identification of promising nucleic acid targets for AS-ODNs. With the validation of new targets, and the optimization of nucleic acid-based drug delivery and modification strategies, AS-ODN has the potential to be a promising therapeutic strategy for the treatment of inflammatory diseases in clinical application.

DNA aptamers can change the distribution of intracellular substances and inhibit biological functions by specifically binding to corresponding proteins, peptides and small molecules, which indicates their potential in the treatment of diseases (Dantsu et al., 2021). DNA aptamers, especially those with targeting inflammatory cytokines, have a therapeutic effect on “hyperactive” immune diseases at the onset of the disease (Zhu and Chen 2018). For example, IL-1α is an essential cytokine that contributes to inflammatory responses, a naphthyl-modified DNA aptamer specifically targeting IL-1α was developed to inhibit the inflammatory signaling pathway (Ren et al., 2017). Similarly, TNF-α is also one of the most important inflammatory cytokines. The developed aptamer targeting TNF-α (Apt-TNF-α) could eliminate acute lipopolysaccharide (LPS)-induced acute lung injury (ALI) and associated acute liver failure (ALF) in mice (Lai W. Y et al., 2019). IL-17A is a pro-inflammatory factor produced by Th 17 cells, which acts as a chemical inducer that recruits immune cells, including monocytes and neutrophils, to inflammatory sites. It is reported that DNA aptamer RA10-6, which binds to IL-17A receptor, can inhibit IL-17A binding and reduce synovial inflammation in mice with osteoarthritis (Chen et al., 2011). Meanwhile, studies have shown that DNA aptamer IL-23 can detect and control brain inflammation (Shahdadi Sardou et al., 2020). Ceria nanoparticle gelatin hydrogel coated with aptamers targeting IL-17 can significantly reduce the level of inflammation in brain tissue by reducing the expression and serum concentration of IL-17, IL-10 and IL-6 (Hekmatimoghaddam et al., 2019).

Various receptors or angiogenesis are also involved in controlling the process of inflammatory diseases. Activation of the TLR4 pathways may cause inflammation, infection, and chronic disease. Thus, the development of TLR4-specific DNA aptamers has the potential for its neutralization as a therapeutic intervention (Shirey et al., 2021). Moreover, the TLR4 aptamers were highly effective in alleviating the brain damage after cerebral hemorrhage (Fernández et al., 2018). Presence of CD4 receptor on target cells is critical for productive HIV infection (Parrish et al., 2012; Zhang et al., 2021). Blocking CD4 using synthetic CD4 aptamer showed comparable cell-binding specificity as standard CD4 antibody, resulting in inhibition of viral entry and subsequent inflammatory response (Zhang et al., 2010; Fellows et al., 2020). Angiogenesis is a widely observed process in the progression of rheumatoid arthritis, IBD and neovascular age-related macular degeneration (AMD) (Aguilar-Cazares et al., 2019; Jeong et al., 2021). The increased expression of vascular endothelial growth factor A (VEGF-A) in the eye choroid promotes the formation of neovascularization by binding to its homologous receptor VEGFR2 (Yang et al., 2016). Pegaptanib is a selective anti-VEGFA aptamer that acts in the extracellular space to inhibit the 165 isoform (VEGF165), was approved by the FDA in 2004 for the treatment of all types of neovascular AMD (Xie et al., 2019). With the development of aptamer screening technology, it is believed that more and more new nucleic acid aptamer drugs will be developed and eventually used to treat diseases.

iSup ODN has been used in a bunch of experimental studies on the treatment of IMIDs. In our previous study, an AAAG-rich ODN designed based on the sequence of human microsatellite DNA, named as MS19, could inhibit the production of inflammatory factors in the lungs of mice caused by influenza virus or LPS, and exhibiting its therapeutic role on acute lung injury (ALI) (Gao et al., 2017; Zhang et al., 2022). Interestingly, MS19 significantly reduced the expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines not only via inhibiting the nuclear translocation of interferon regulatory factor 5 (IRF5), but also associated with NF-κB signaling (Gao et al., 2017; Zhang et al., 2022). In addition, MS19 was proved to alleviate the myocarditis induced by coxsackievirus B3 (CVB3) infection in mice (Nie et al., 2019). A close relationship was existed between the virus-induced ALI and the level/activity of IRF7 in local infectious sites. Thus, IRF7-rODN M1 was designed to alleviate influenza virus-induced ALI, and led to decreased mRNA levels of IFN-α, reduced neutrophil infiltration in the lungs and prolonged survival of mice (Yang et al., 2019). Another iSup ODN MT01, based on the sequence of human mitochondrial DNA, could inhibit the proliferation of PBMCs and production of type I IFN induced by influenza virus, CpG DNA and herpes simplex virus (HSV) (Yang et al., 2010). Meanwhile, MT01 could also induce differentiation of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis (Shen et al., 2012). In addition, other iSup ODNs also have been screened to treat inflammatory diseases. ODN SAT05f is an oligonucleotide with CCT repeats derived from human microsatellite. It was suggested that SAT05f can recruit surface TLR9⁺ (sTLR9) neutrophils to play a protective role in the development of systemic inflammatory response syndrome (SIRS) in locally inflammatory areas (Meng et al., 2017). YW002 could alleviate the pathological changes of alcoholic hepatitis by downregulating the inflammatory factors TNF-α, IL-1β, and IL-6 (Wang et al., 2012).

In addition to above inflammatory diseases, autoimmune disease is another type of IMIDs. Systemic lupus erythematosus (SLE) is an autoimmune disease with glomerulonephritis and multifocal terminal organ damage caused by many pathogenic autoantibodies and immune complexes (Mortezagholi et al., 2017). The mammalian telomere-derived A151 can significantly reduce glomerular and tubular injury, basement membrane proliferative changes, monocyte infiltration, IgG deposition and vascular lesions in SLE model mice. Also, A151 can delay the production of proteinuria caused by glomerulonephritis, reduce the grade of proteinuria and improve the survival rate of mice (Dong et al., 2005; Yazar et al., 2020). Moreover, A151 can significantly reduce the levels of two key inflammatory factors, monocyte chemotactic protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1), in the inflammatory process of atherosclerosis (Cheng et al., 2008). Similarly, iSup ODN IRS954 could reduce the level of serum nucleic acid specific autoantibody and the grade of proteinuria, relieve the symptoms of glomerulonephritis and increase the survival rate of SLE mice (Barrat et al., 2007). Besides the inhibition of SIRS, SAT05F can also effectively reduce the deposition of immune complexes and delay the onset of lupus nephritis in mice with chronic graft-versus-host disease (GVHD), and inhibit the activation of TLR7/9 pathway in vitro (Zhang et al., 2014). Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory joint disease. It was found that the PS modified ODN1411 could competitively inhibit the signal transduction of TLR8 by interacting with TLR8, that is, it inhibited the production of inflammatory factors in RA model by inhibiting the phosphorylation of signal molecules and the activation of NF-κB, which provided a possible new therapy for the treatment of RA (Sacre et al., 2016). In general, iSup-ODNs showed a good application prospect in IMIDs, especially the diseases associated with over-activation of immune response induced by TLR signal transduction (Li et al., 2022).