Application of CGT-ODN in immune regulation

Abstract

The present invention discloses the application of CGT-ODN in immunoregulation, discloses a CGT-ODN nucleotide sequence, and discloses a vector and a pharmaceutical composition comprising the aforementioned CGT-ODN nucleotide sequence. The present invention also provides the application of the aforementioned GT-ODN nucleotide sequence, vector, and pharmaceutical composition in immunoregulation. The present invention also provides the application of the aforementioned GT-ODN nucleotide sequence and vector in the preparation of a pharmaceutical composition for treating or preventing infectious diseases, allergic diseases, immunodeficiency diseases, and tumors. The present invention also provides the application of the aforementioned GT-ODN nucleotide sequence and vector in the preparation of vaccines, antibody production, or regulating the degree of tRNA degradation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of CGT-ODN in immune regulation. Background Art

[0002] Nucleic acids are the major conserved pathogen determinants detected by pattern recognition receptors (PRRs) to initiate innate immune responses. Extracellular bacterial single-stranded DNA (ssDNA) containing CpG motifs and their synthetic mimics, CpG oligonucleotides (ODNs), can be internalized by immune cells and recognized by Tolllike receptor 9 (TLR9) in endosomes. CpG ODNs have shown great potential to combat cancer, infection, and allergic diseases.

[0003] The first class of PRRs to be identified were Toll-like receptors (TLRs), which are membrane proteins that recognize extracellular or endosomal pathogen ligands. However, some pathogens can evade detection by TLRs and enter the host cytoplasm or nucleus. Therefore, most TLRs have complementary intracellular PRRs that sense similar ligands, such as TLR3 and RIG-I / MDA5 for RNA, and TLR4 and caspases for lipopolysaccharide (LPS). Toll-like receptor 9 (TLR9) is a specific receptor for CpG motifs in ssDNA and is internalized into endosomes, but it is “blind” to cytosolic or nucleic acid ssDNA and is mainly expressed in professional immune cells, suggesting the existence of a widely expressed intracellular ssDNA PRR. However, to date, no such TLR9 complementary receptor that specifically senses intracellular ssDNA has been clearly identified.

[0004] Decades before TLR9 was cloned, bacterial ssDNA was identified as a key active component of Bacillus carinii (BCG) immunotherapy, a strategy closely related to Coley’s toxins. The subsequent discovery that unmethylated CpG motifs are essential for the immunostimulatory activity of ssDNA and synthetic oligonucleotides (ODNs) internalized by certain immune cells has led to various clinical trials of CpG-ODN-based vaccine adjuvants, antitumor therapies, and treatments for allergic and infectious diseases. Notably, the delivery systems used in these applications, as well as antisense oligonucleotide (ASO) therapy, ssDNA donor-based gene editing, and endocytosis can also lead to the accumulation of ssDNA in the cytoplasm and nucleus. However, it is still unclear whether and how intracellular ssDNA stimulates innate immune responses. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides a CGT-ODN nucleotide sequence, and the CGT-ODN nucleotide sequence is as follows:

[0007] 5'-(X) n CGT / A(X) m (G) a (X) b (G) c (X) d -3',

[0008] Wherein, X is any nucleotide base, n represents any integer value ≥ 0, m represents any integer value ≥ 0, a represents any integer value ≥ 0, b represents any integer value ≥ 0, c represents any integer value ≥ 0, d represents any integer value ≥ 0, C represents a number selected from cytosine and 5-methylcytosine, G represents a number selected from guanine, T represents thymine, A represents adenine, and at least one of a and c is an integer ≥ 1;

[0009] Furthermore, at least one of a and c is an integer ≥ 2;

[0010] Further, the flanking sequence of the CGT-ODN nucleotide sequence is rich in consecutive G nucleotides;

[0011] Furthermore, the CGT-ODN nucleotide sequence has a phosphodiester linkage.

[0012] In some embodiments, the CGT-ODN nucleotide sequence does not have thiophosphate (PS) and 2'-O-methyl backbone, which will destroy the immunostimulatory activity of ODN. In some embodiments, the CGT-ODN nucleotide sequence can be a methylated sequence. In some embodiments, the CGT-ODN nucleotide sequence can be a palindromic sequence.

[0013] In some embodiments, the CGT-ODN nucleotide sequence requires at least one CGT / A motif, and the flanking sequence, i.e., the downstream sequence, contains at least two consecutive G nucleotide sequences. The consecutive G nucleotide sequences may be arranged directly after the CGT / A motif, or may be arranged downstream of the CGT / A motif with other sequences interleaved therebetween.

[0014] In some embodiments, the CGT-ODN nucleotide sequence does not limit the length of the nucleotide sequence. Further, the length of the nucleotide sequence includes but is not limited to 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55nt, 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77 nt, 78 nt, 79 nt, 80 nt, 81nt, 82 nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 1000 nt, 10000 nt.

[0015] In the present invention, the term "ODN" refers to an oligodeoxynucleotide capable of activating an immune response, and "ODN" does not specifically limit the nucleotide length in the present invention. In particular, the term "CGT-ODN" refers to an oligodeoxynucleotide having a CGT / A motif and a downstream continuous G nucleotide sequence, which exhibits an immune activation function. The term "continuous G nucleotide sequence" refers to a form in which at least two guanine deoxyribonucleic acids are continuously coupled to CGT-ODN through a phosphodiester bond, and the cytosine deoxyribonucleic acid in the "CGT" may be methylated or non-methylated, and the CGT-ODN nucleotide sequence may have a palindromic sequence or not.

[0016] Furthermore, the CGT-ODN nucleotide sequence has at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 1-15.

[0017] The term "identity" as used in the present invention refers to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences) in a comparison window, wherein for optimal alignment of the two sequences, the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions). The percentage is calculated by determining the number of positions at which the same nucleotide or amino acid residue exists in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce a percentage of sequence identity. A sequence that is identical at each position compared to a reference sequence is said to be 100% identical to the reference sequence, and vice versa.

[0018] Furthermore, the CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-15.

[0019] Furthermore, the CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-9.

[0020] Furthermore, the CGT-ODN nucleotide sequence is shown in any one of SEQ ID NO: 1, 3, 4, 6, and 8.

[0021] Furthermore, the CGT-ODN nucleotide sequence is shown in SEQ ID NO:4.

[0022] Furthermore, the CGT-ODN nucleotide sequence may be of natural origin or artificially synthesized.

[0023] Furthermore, the CGT-ODN nucleotide sequence has a CGT motif, a CGA motif, and a CGT+CGA motif.

[0024] In some embodiments, the CGT-ODN nucleotide sequence is not limited to a specific biological source, and the biological source may include any organism that may have the general structure of the CGT-ODN nucleotide sequence, including but not limited to bacteria, viruses, plants, animals, etc.

[0025] The term "nucleotide" or "nucleic acid" used in the present invention refers to a polydeoxynucleotide (DNA or its analog) composed of bases connected by a backbone. In DNA, the common bases are adenine (A), guanine (G), thymine (T) and cytosine (C), although nucleic acids can contain base analogs (such as inosine) and abasic positions (for example, a phosphodiester backbone lacking nucleotides at one or more positions, U.S. Patent No. 5585481).

[0026] The present invention provides a vector, which comprises the CGT-ODN nucleotide sequence described above.

[0027] Furthermore, the vector includes a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a piggyBac vector or a Sleeping Beauty transposable vector.

[0028] The term "vector" or "expression vector" as used in the present invention includes any nucleic acid capable of directing the expression of a nucleic acid. The term "vector" includes viral vectors, plasmid vectors, and the like. Expression vectors can be delivered to cells using two basic delivery schemes: viral-based delivery systems using viral vectors and non-viral-based delivery systems using, for example, plasmid vectors.

[0029] The present invention provides a pharmaceutical composition, which comprises the aforementioned CGT-ODN nucleotide sequence, the aforementioned carrier, and a pharmaceutically acceptable adjuvant.

[0030] Furthermore, the pharmaceutically acceptable adjuvants include adjuvants required for intravenous drip, subcutaneous injection, intravenous push injection, intravitreal injection, and intramuscular injection.

[0031] In some embodiments, the pharmaceutically acceptable adjuvant includes a diluent, a carrier, an excipient, and a stabilizer, and the pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dosage and concentration used. In some embodiments, the pharmaceutically acceptable adjuvant also includes a buffer, an antioxidant, a preservative, a protein, a hydrophilic polymer, an amino acid, a monosaccharide, a disaccharide, and other carbohydrates, a chelating agent, a sugar, a salt-forming counterion, a metal complex, and a nonionic surfactant.

[0032] In some specific embodiments, the buffer includes phosphate, citrate and other organic acids. In some specific embodiments, the antioxidant includes ascorbic acid and methionine. In some specific embodiments, the preservative includes octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol and meta-cresol. In some specific embodiments, the protein includes serum albumin, gelatin or immunoglobulin. In some specific embodiments, the hydrophilic polymer includes polyvinyl pyrrolidone. In some specific embodiments, the amino acid includes glycine, glutamine, asparagine, histidine, arginine or lysine. In some specific embodiments, the monosaccharide, disaccharide and other carbohydrates include glucose, mannose or dextrin. In some specific embodiments, the chelating agent includes EDTA. In some specific embodiments, the sugar includes sucrose, mannitol, trehalose or sorbitol. In some specific embodiments, the salt-forming counterion includes sodium. In some specific embodiments, the metal complex includes a Zn-protein complex. In some specific embodiments, the nonionic surfactant includes TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some embodiments, the active pharmaceutical ingredient can also be encapsulated in the prepared microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, by coacervation technology or by interfacial polymerization; encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or a coarse emulsion.

[0033] The present invention provides the use of the aforementioned CGT-ODN nucleotide sequence, the aforementioned vector, and the aforementioned pharmaceutical composition in immunomodulation.

[0034] Furthermore, the immune regulation includes activation of immune response, immunostimulatory activity, and regulation of cytokine expression.

[0035] Furthermore, the cytokines include cytokines of JNK, p38, and NFκB signaling pathways.

[0036] Furthermore, the cytokines include TNF, IFNB1, CXCL8, IL-11, IL-12A, IL-32, CCL2, CCL4, ANKRD1, TNFAIP3, JUN, FOS, FOSL1, FOSL2, NFKB1, NFKB2, CASP3, and MAP3K14.

[0037] Furthermore, the cytokines include TNF, IFNB1, and CXCL8.

[0038] The present invention provides the use of the aforementioned CGT-ODN nucleotide sequence and the aforementioned vector in the preparation of a pharmaceutical composition for treating or preventing infectious diseases, allergic diseases, immunodeficiency diseases, and tumors.

[0039] In some embodiments, the immunodeficiency disease types include, but are not limited to, inflammation, antiphospholipid syndrome, systemic lupus erythematosus, rheumatoid arthritis, autoimmune vasculitis, celiac disease, autoimmune thyroiditis, post-transfusion immunity, maternal-fetal incompatibility, transfusion reaction, immunodeficiency such as IgA deficiency, common variable immunodeficiency, drug-induced lupus, diabetes, type I diabetes, type II diabetes, juvenile diabetes, juvenile rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, immunodeficiency, allergy, asthma, psoriasis, atopic dermatitis, allergic contact dermatitis, chronic skin diseases, amyotrophic lateral sclerosis, chemotherapy-induced injury, graft-vs-host diseasediseases), bone marrow transplant rejection, ankylosing spondylitis, atopic eczema, pemphigus, Behcet's disease, chronic fatigue syndrome, fibromyalgia, chemotherapy-induced damage, myasthenia gravis, glomerulonephritis, allergic retinitis, systemic sclerosis, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus including chilblain lupus erythematosus, Sjögren's syndrome, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated blood diseases, lc-SSc (limited cutaneous scleroderma), dc-SSc (diffuse cutaneous scleroderma), autoimmune thyroiditis (AT), Graves' disease (GD), myasthenia gravis, multiple sclerosis (MS), ankylosing spondylitis, transplant rejection, immunosenescence, rheumatic / autoimmune diseases, mixed connective tissue disease, spondyloarthropathies, psoriasis, psoriatic arthritis, myositis, scleroderma, dermatomyositis, autoimmune vasculitis, mixed connective tissue disease, idiopathic thrombocytopenic purpura, Crohn's disease, human adjuvant disease, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, idiopathic inflammatory myopathy, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, immune-mediated renal disease, demyelinating disease of the central or peripheral nervous system, idiopathic demyelinating multiple Neuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, Whipple's disease, autoimmune or immune-mediated skin disease, bullous skin disease, erythema multiforme, allergic rhinitis, atopic dermatitis, food allergy, urticaria, pulmonary immune disease, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, transplant-related disease, graft rejection or graft-versus-host disease, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic sclerosis Skin diseases and sclerosis, responses related to inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult-onset respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, allergic encephalomyelitis, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatous diseases including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, DiamondBlackfan anemia, immune hemolytic anemias including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocytopenia, inflammatory disorders of the central nervous system (CNS), multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid, pemphigus, autoimmune polyendocrine disease, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura ITP, thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune diseases of the testicles and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyendocrine syndrome (or polyglandular endocrine disease) syndrome), Sheehan's syndrome, autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant) to NSIP, Guillain-Barré syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, Berger disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia, and amyotrophic lateral sclerosis (ALS).

[0040] In some embodiments, the tumor types include but are not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, acute myeloid leukemia in adults, cancer of unknown primary site in adults, malignant mesothelioma in adults, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendix cancer, astrocytoma, childhood cerebellum or cerebral cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, osteosarcoma / malignant fibrous histiocytoma, brain cancer, brain stem glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, cancer of unknown primary site, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood acute myeloid leukemia, childhood primary site Unknown cancer, childhood cancer, childhood cerebral astrocytoma, childhood mesothelioma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, endometrial uterine cancer, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma family of tumors, Ewing's sarcoma in the Ewing's tumor family, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, gallbladder cancer, gastric (stomach) cancer, gastric carcinoid, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, brain stem jelly Gliomas, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, islet cell cancer (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphoblastic leukemia (also known as acute lymphoblastic leukemia), acute myeloid leukemia (also known as acute myeloid leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), leukemia (leukaemia), chronic myeloid leukemia (also known as chronic myeloid leukemia), hairy cell leukemia (leukemia), lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma (AIDS Disease-related), lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, squamous cell carcinoma of the neck with occult metastatic primary lesions, oral cancer, multiple endocrine neoplasms syndrome, childhood multiple myeloma (bone marrow cancer), multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative diseases, chronic myeloid leukemia, myxoma, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumor,Ovarian low malignant potential tumors, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, skin cancer (melanoma) , skin cancer (non-melanoma), Merkel cell skin cancer, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck with occult metastatic primary, gastric cancer, supratentorial primitive neuroectodermal tumor, cutaneous T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, optic pathway and hypothalamic glioma in children, vulvar cancer, macroglobulinemia, and Wilms tumor (kidney cancer). ,

[0041] The present invention provides the use of the aforementioned CGT-ODN nucleotide sequence and the aforementioned vector in the preparation of vaccines.

[0042] Furthermore, the vaccine includes subunit vaccine and viral vector vaccine.

[0043] The term "subunit vaccine" in the present invention refers to a vaccine produced by cloning the CGT-ODN nucleotide sequence into a prokaryotic or eukaryotic expression system using genetic engineering methods to enable its efficient expression.

[0044] The viral vector described in the term "viral vector vaccine" has the ability to replicate and can serve as a carrier of foreign genes while maintaining its own infectivity.

[0045] Furthermore, the vectors in the viral vector vaccine self-assemble into virus-like particles.

[0046] Furthermore, the vector includes a self-inactivating vector.

[0047] Furthermore, the virus is selected from lentivirus, influenza virus, hepatitis virus, alphavirus, filovirus, adenovirus, adeno-associated virus and / or flavivirus.

[0048] In some embodiments, the vaccine is a preparation including a biomolecule, which is, for example, a protein or a nucleic acid molecule encoding a protein, a carbohydrate, a lipid, or a combination of the above. In some embodiments, there are preventive and therapeutic vaccines. The term vaccine generally refers to a product administered to a subject, i.e., comprising an adjuvant (if present), a carrier (if present), an active ingredient (the present invention refers to a CGT-ODN nucleotide sequence), a stabilizer or other excipients. The term vaccine as used herein does not imply that the preparation is effective in preventing or curing a disease.

[0049] The present invention provides the use of the aforementioned CGT-ODN nucleotide sequence and the aforementioned vector in antibody production.

[0050] The term "antibody" as used in the present invention includes heterotetrameric glycoproteins of about 150,000 daltons with identical structural features, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH). This is followed by multiple constant regions. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0051] The present invention provides the use of the aforementioned CGT-ODN nucleotide sequence and the aforementioned vector in regulating the degree of tRNA degradation.

[0052] Furthermore, the tRNA comprises type II tRNA.

[0053] The present invention provides a method for preparing CGT-ODN related antibodies, the method comprising using the aforementioned CGT-ODN nucleotide sequence and the aforementioned vector to immunize non-human animals to obtain antibodies.

[0054] Furthermore, the non-human animal is a mammal.

[0055] Furthermore, the mammals include mice, rats, guinea pigs, rabbits, sheep, cows, horses, and non-human primates.

[0056] The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1is the result graph of ethidium bromide (EB) staining and SYBR melting curve of ssDNA;

[0058] Figure 2 This is a graph showing the results of TLR9-independent immunostimulatory activity of ssDNA;

[0059] Figure 3 This is the result graph of cell viability and cytokine expression of bacterial ssDNA and human ssDNA;

[0060] Figure 4 This is a flowchart of human whole genome ssDNA screening;

[0061] Figure 5 This is the gel test result of the optimized method for ODN transfection into HEK293 and 293A cells;

[0062] Figure 6 This is the absorbance detection result after ODN transfection of HEK293 and 293A cells;

[0063] Figure 7 It is the crystal violet staining result and cell morphology diagram for detecting the dose-effect relationship of ODN-HEK293;

[0064] Figure 8 This is the result of time course analysis of cell morphology changes after ODN60 transfection in 293A cells;

[0065] Fig. 9 This is a graph showing the results of the immunostimulatory effects of ODN on different cell types;

[0066] Fig.10 The figure shows the morphological changes of HEK293 cells after transfection with different forms of ODN or ORN and the results of cell viability based on ATP; ODNrc is the reverse complementary ODN; ORN is the oligoribonucleotide with the same sequence as ODN; dsODN is the double-stranded ODN obtained by annealing ODN and ODNrc;

[0067] Fig.11 This is the result graph showing the effect of the length of the ODN sequence on the viability of HEK293 cells;

[0068] Fig.12 The figure shows the morphological results of 293A cells electroporated with ODN60 and ODN60rc for 12 h, and the cell viability analysis results of HEK293 cells transfected with different ODNs or directly incubated with ODN outside cells for 48 h;

[0069] Fig.13 It is the Venn diagram and KEGG Orthology database analysis diagram after transfection of ODN60 or ODN60rc in 293A cells;

[0070] Fig.14 It is a heat map of cytokine expression profile after 293A cell treatment and the qPCR validation results of upregulated cytokines;

[0071] Fig.15 This is the qPCR test result of DU145 cells 24 h after ODNs transfection;

[0072] Fig.16 This is a graph of the analysis results of the IPA database and the DEGs analysis results related to the TNFR pathway;

[0073] Fig.17 This is a graph showing the time and dose effects of pathway activation in immunoblotting analysis after HEK293 transfection with ODN60 and ODN60rc;

[0074] Fig.18 Figure 2 is the result of immunoblotting analysis of DU145 cells transfected with the indicated top and bottom ODNs 24 h after antibody infection; UT stands for untreated cells;

[0075] Fig.19 is the result of antibody immunoblotting analysis of the nuclear part of 293A cells transfected with ODNs;

[0076] Fig. 20 This is the expression result of IFNB1 and CXCL8 detected by qPCR after HEK293 cells transfected with different siRNAs were stimulated with ODN60 for 12 h;

[0077] Fig.21 It is the result graph of transfection efficiency test of different ODNs;

[0078] Fig. 22 is a graph showing the effect of the exchange of the indicated adjacent nucleotides in ODN60 on the stimulatory activity;

[0079] Fig.23 This is the result diagram showing the effect of 3' end truncation of ODN60 on the stimulatory activity;

[0080] Fig.24 This is a graph showing the results of immunostimulatory activity testing after different mutations in the CGT motif of ODN60 were transfected into HEK293 cells;

[0081] Fig.25 Yes Figure 6 The percentage of CGT / A containing ODN in the sorting interval shown is divided into results;

[0082] Fig.26The results of ATP cell viability assay, cell morphology and cytokine activation of top ODNs carrying CGT / A motif mutations in HEK293 cells transfected with the wild-type sequence; AGT, CGT→AGT mutation; CCG, CGT→CCG mutation; CGG, CGT (CGA for ODN86) mutated to CGG mutation;

[0083] Fig. 27 is the statistical map of the position of CGT / A motif in the highest and lowest ODNs;

[0084] Fig.28 It is a graph showing the characteristic analysis results of the CGT motif flanking sequences of the top and middle ODNs and the statistical results of the percentage of different bases;

[0085] Fig.29 The specific sequences of ODN-H and ODN-L synthesized according to the characteristics of ODN sequences and the cytokine activation results after transfection of HEK293 cells;

[0086] Fig.30 This is a result diagram showing the effect of the distribution of continuous G or T bases in the flanking sequence of the CGT / A motif in the ODN sequence on the cytokine activation efficacy in cells;

[0087] Fig.31 It is a statistical result graph of the percentage of CGT / A and CGT / A flanking sequences containing more than 3 G in ODN sequences;

[0088] Fig.32 It is a statistical result diagram of the more stimulating ODN of the viral genome;

[0089] Fig.33 The figure is the effect of 2'-O methylation modification, RNA base substitution, phosphorothioate (PS) bond and cytidine methylation on stimulatory activity in CGT / A ODN and the comparison result between CGT / A ODN and classic CpG ODN, where capital letters, phosphodiester linkage; lowercase, phosphorothioate linkage; underline, palindromic sequence;

[0090] Fig.34 This is a typical result of qPCR detection of IFNB1 expression level after CpG-ODN and ODN-H were transfected into lung fibroblasts of WT and TLR9 knockout mice respectively;

[0091] Fig.35 This is a schematic diagram of the genome-wide CRISPR screening of essential genes for ODN60 response;

[0092] Fig.36It is the gene abundance ranking of the average hits of multiple gRNAs, the abundance fold change between ODN60 and ODN60rc treated cells, and the gRNA enrichment ranking result graph of ODN60 treated cells;

[0093] Fig.37 This is a statistical result graph of cytokines and cell viability triggered by ODN60 and other top-ranked ODNs after the loss of SLFN11;

[0094] Fig.38 This is the result of immunoblotting to detect the effect of complementary expression of SLFN11 on the activation of p38 and JNK induced by ODN60;

[0095] Fig.39 Different ODNs were transfected into WT and SLFN11 - / - HEK293 cells post-qPCR detection of TNF and CXCL8 expression results;

[0096] Fig.40 is the result of immunoblotting analysis of the expression level of SLFN11 in cells;

[0097] Fig.41 This is a statistical graph showing the expression of cytokines induced by different types of nucleotides in SLFN11 knockout DU145 cells;

[0098] Fig.42 is a schematic diagram of luminescent ODN immunoprecipitation (ODIP) and biotin-ODN pull-down assay;

[0099] Fig.43 This is a graph showing the results of ODIP assay for the binding activity of endogenous and re-expressed SLFN11 to ODN60 and ODN60rc;

[0100] Fig.44 It is a graph showing the results of ODIP and biotin-ODN pull-down experiments to detect the binding of SLFN11 and ODN in cells;

[0101] Fig.45 This is a result diagram of the effect of the "CGT" motif obtained in ODN60rc on binding to SLFN11 in biotin pull-down assay in HEK293 cells;

[0102] Fig.46 It is a schematic diagram of the predicted structures of the three subgroups of the SLFN family;

[0103] Fig.47 Schematic diagram of using full-length and truncated SLFN11 and SLFN11 expressing full-length and truncated SLFN11 - / -ODIP analysis results of full-length and truncated SLFN11 and ODN60 after HEK293 cells were transfected with ODN60;

[0104] Fig.48 This is the result of the in vitro experiment of ODN binding activity of SLFN11, which includes Coomassie blue staining, electrophoretic mobility shift assay, fluorescent ODN GST pull-down assay, and BLI assay.

[0105] Fig.49 is the result of immunoblot analysis of MAPK pathway activation induced by truncated SLFN11 and ODN;

[0106] Fig.50 This is the distribution map of SLFN subgroups among taxa in the NCBI database;

[0107] Fig.51 This is a comparison diagram of the gene trees of the third subgroup SLFN, AIM2 and cGAS in the TreeFam 9 database;

[0108] Fig.52 This is the expression level of SLFN family proteins in the NCBI human and mouse redundant protein sequence database and the GTEx database, as well as the SLFN11 homologous protein search results;

[0109] Fig.53 This is a graph showing the results of an experiment with biotin-ODN60, a member of SLFN subgroup III;

[0110] Fig.54 This is the staining result of SLFN11 and ODN60 co-aggregating in the cell nucleus;

[0111] Fig.55 This is a picture of the immunoblotting detection result of SLFN11 distribution in HEK293 cytoplasm and nucleus;

[0112] Fig.56 This is the result of immunoblotting detection of the distribution of SLFN11 in the cytoplasm, perinuclear area, and nucleus of HEK293 cells before and after stimulation with ODN60;

[0113] Fig.57 It is a diagram showing the subcellular localization of ODN-bound SLFN11 and the ESMA assay results of the cytoplasmic-perinuclear-nuclear components of ODN-bound SLFN11;

[0114] Fig.58 This is a graph showing the co-localization results between SLFN11 and the top-ranked ODN in the cytoplasm of DU145 cells;

[0115] Fig.59 This is a diagram showing the screening results for SLFN11 homologs in mice;

[0116] Fig.60 is the schematic diagram of the construction of SLFN9-KO mice;

[0117] Fig.61 is the result of qPCR detection of cytokines in WT and SLFN9-KO mice;

[0118] Fig.62 WT, SLFN9 - / - and tr19 - / - The results of qPCR detection of Ifnb1 mRNA expression level and ELISA detection of IFNα secretion in mouse primary cells after transfection of HIV6441 and classical TLR9 CpGODN respectively;

[0119] Fig.63 Figure 1 is a graph showing the immune response of SLFN9 in mice induced by CGT-ODN, where AF, CGT-ODN-induced immune response in newborn (B, C) and adult (DF) mice; schematic diagram of intravenous injection of CpG ODNs encapsulated in LNPs and luciferase mRNA-LNPs in the liver (A); qPCR and immunoblotting were used to detect the activation of cytokines (B) and immune pathways (C), respectively; qPCR and HE staining were used to detect the expression levels of cytokines 6 h after injection (D) and histological changes 3 d after injection (E), respectively, and the survival was monitored for 4 days; H and I, AAV2 single-stranded DNA genome was encapsulated in LNPs and intravenously injected into mice to detect the 4-day survival and the expression of verification factors in the liver; JL, WT or SLFN9- / - B16-F10 cells were inoculated into WT C57BL / 6 mice were subcutaneously injected with HIV6441 encapsulated with transfection reagents and then injected intratumorally. The treatment schedule was shown in J. The average tumor volume of each group (K) and the tumor volume of a single mouse (L) were measured at each time point;

[0120] Fig.64 This is the expression result of SLFN9 and TRL9 in the liver of B16-F10 and WT mice;

[0121] Fig.65 This is a graph showing the changes in cell morphology, ATP-based cell viability, and cytokine expression in SLFN9- / - and Trl9- / - B16-F10 mice;

[0122] Fig.66It is a result diagram of the reduction of tRNA cleavage caused by CGT ODN and pathogenic ssDNA such as bacteria or AAV; wherein, A is PAGE gel electrophoresis staining, B is dose-effect relationship analysis, CD is the effect of SLFN11 knockout on tRNA cleavage caused by CGT-ODN stimulation, EF is the cell survival result, G is the cytokine expression result, H is the effect of ssDNA from other sources on the tRNA degradation of SLFN11, I is the cell survival result in ssDNA from other sources, J is the tRNA degradation of HeLa cells with wild-type SLFN11 when stimulated by CGT ODN, K is the downstream cell survival result when exogenous SLFN11 is expressed in HeLa cells, and L is the inflammatory response result when exogenous SLFN11 is expressed in HeLa cells. DETAILED DESCRIPTION

[0123] Example 1 Intracellular ssDNA reduces cell viability in a sequence-specific manner

[0124] 1. Acquisition of human and bacterial ssDNA and preliminary analysis of ssDNA reducing cell viability

[0125] To obtain ssDNA, human and bacterial genomic DNA was heat denatured and identified using dsDNA-preferred ethidium bromide staining and SYBR green melting curve analysis. Figure 1 To test the TLR9-independent immunostimulatory activity of ssDNA, ssDNA and dsDNA were transfected into TLR9 pathway-deficient HEK293 cells, and the results are shown in Figure 2 As shown, the results showed that bacterial ssDNA reduced cell viability to a greater extent than bacterial dsDNA or human ssDNA. Bacterial ssDNA activated greater expression of TNF and CXCL8 than bacterial dsDNA or human ssDNA. The results are shown Figure 3 As shown, this indicates that intracellular ssDNA can also activate TLR9-independent immune responses in a sequence-dependent manner.

[0126] 2. Screening of human and bacterial ssDNA and detailed analysis of ssDNA reducing cell viability

[0127] To further investigate the stimulatory activity of ssDNA on the human genome, a genome-wide 24-nucleotide (nt) ODN library was prepared to screen for stimulatory sequences. Figure 4 As shown. Use Figure 5 The optimized ODN transfection method shown in Figure 2 was used to transfect the library into HEK293 and 293A cells. Figure 6As shown in the results, it was observed that 14 out of 200 ODNs reduced the average cell viability of these cells to less than 40%. The top 11 highly active ODNs were validated in a concentration gradient, and the results are shown in Figure 7 As shown in Figure 2, ODN60 was found to exhibit the highest immunostimulatory efficacy. Therefore, in subsequent studies, ODN60 was used as a representative of ODN. In addition, the immunostimulatory efficacy of ODN60 was as low as 24 nM, while its reverse complementary ODN (ODN60rc) did not reduce cell viability even at 120 nM, as shown in Figure 2. Figure 8 As shown, it is shown that the immunostimulatory activity is determined by the sequence rather than its targeted genomic location. The ODN sequences used in the experiment are shown in Table 1.

[0128] Table 1

[0129]

[0130]

[0131] To explore whether the response to ssDNA is cell type restricted, the top-ranked ODNs were transfected into multiple human cell types, and the results were Fig. 9 As shown in Figure 2, ODN-responsive cell types included DU145, T47D, U251, and A549 cells as well as HUVECs, but not 293T cells. The morphological characteristics exhibited by these responsive cells clearly indicated cell death after stimulation. Fig.10 As shown in the results, only ODN, but not oligonucleotides (ORN) or double-stranded ODN (dsODN), reduced cell viability. This immunoreactivity was not limited to the 24 nt length, as the 96 nt ODN containing the highest sequence also reduced cell viability, as shown in Fig.11 Finally, both electroporation and transfection (but not incubation) rendered the top-ranked ODNs active, as shown in Fig.12 This indicates that the presence of ssDNA in cells is essential for its immunostimulatory activity.

[0132] Example 2 Intracellular ssDNA activates cytokine expression and immune-related signaling pathways

[0133] Since cell viability is not a specific indicator for evaluating immune response, in order to further determine the immunostimulatory activity of ODN, 293A cells were stimulated with ODN60 or ODN60rc for 10 and 24 hours, and then RNAseq was performed on the cells. Fig.13As shown, compared with the expression of immune-related genes in the untreated control group, only ODN60 triggered their expression, while ODN60rc did not. Importantly, ODN60 stimulated the expression of many cytokines and chemokines (hereinafter referred to as "cytokines"), and the specific cytokine results are shown in Fig.14 This further suggests the immunostimulatory activity of ssDNA, as transcriptional activation of cytokines is a major component of the innate immune response induced by pathogen-associated molecular patterns (PAMPs). Similarly, experiments were performed in DU145 cells, and all of the top-ranked ODNs activated the expression of cytokines, as shown in Figure 2. Fig.15 As shown in Figure 2, these activated cytokines also include IFNB1, which cannot be triggered in HEK293 cells. Fig.14 shown.

[0134] Furthermore, in addition to the innate immune response revealed by cytokine activation, most of the apically enriched signaling pathways in ODN60-treated cells were related to immune responses, as shown in the results Fig.16 As shown in Figure 2, the results showed that these genes contribute to the enrichment of TNFR1 and TNFR2 pathways. ODN60 also activated the JNK, p38, and nuclear factor-κB (NFκB) signaling pathways, as shown in Figure 2. Fig.17 Consistently, all the top-ranked ODNs activated the JNK, p38, and NFκB pathways in the DU145 cell line, as shown in Fig.18 Given that the reduction in IκBα levels indicates activation of the NFκB pathway, we further evaluated the nuclear translocation of NFκB and found that the translocation of p65 and NF-κB1 to the nucleus was significantly increased after stimulation with the top-ranked ODNs, as shown in Figure 2. Fig.19 Knockdown of p65 and NF-κB1 partially reduced the activation of IFNB1 and CXCL8 induced by ODN60 in DU145 cells. Fig. 20 In summary, the decrease in cell viability, increase in cytokine expression, and activation of immune pathways revealed the immunostimulatory activity of intracellular ODN.

[0135] Example 3 CGT / A with consecutive Gs is the core motif for intracellular ODN immunostimulatory activity

[0136] Before investigating the immunostimulatory sequence patterns in the top-ranked ODNs, we excluded the possibility that the differences in immunoreactivity between the top- and bottom-ranked ODNs were caused by different transfection efficiencies, as shown in the results Fig.21 Next, we designed a series of ODN60 mutations and truncation constructs. ODN60 was inactivated using a position switch of three pairs of adjacent bases from the 5′ terminal region instead of the 3′ terminal region. The specific schematic diagram is shown in Fig. 22Consistently, 3′-terminal truncation of approximately 8 nt had little effect on the immunoreactivity of ODN60, as shown in Fig.23 Importantly, switching the first pair of adjacent nucleotides alone was sufficient to significantly reduce the immunoreactivity of ODN60, as shown in Fig. 22 These results indicate that the immunoreactivity of ODN60 is mainly determined by its 5′ end sequence and is sensitive to single nucleotide changes.

[0137] Next, nucleotides 1 to 7 at the 5′ end of ODN60 were systematically replaced with the other three types of nucleotides, and the resulting activity was evaluated by assessing cell viability and activation of TNF cytokine and JNK pathways. Fig.24 These results consistently showed that for the immunostimulatory activity of ODN60, the second and third nucleotides were highly restricted to C and G, respectively, and the fourth nucleotide T showed some tolerance to A, indicating that the CGT / A motif is unexpectedly required for the activation of immune responses not only by extracellular ssDNA but also by intracellular ssDNA. We then further analyzed the presence of the CGT / A motif in the 200 screened ODNs and found that the presence of the CGT and CGA (CGT / A) motifs determined the stimulation level of the ODN, as shown in the results. Fig.25 Any single nucleotide mutation in the CGT / A motif abolished the immunostimulatory activity of the top-ranked ODNs, as shown in Fig.26 Therefore, the presence of CGT / A is required for immunostimulatory ODN.

[0138] We found that in the screening experiment of Example 1, not all ODNs containing the CGT / A motif had strong immunostimulatory effects. Therefore, we speculate that the flanking sequence of the CGT / A motif cooperates with CGT / A to determine the immunostimulatory effect of ODN in cells. In order to determine the flanking sequence pattern that distinguishes the effectiveness of the CGT / A motif, we first analyzed the base composition of all ODNs and compared the CGT / A flanking motifs of the top-ranked (high activity) and middle-ranked (medium activity) ODNs. Fig. 27 As shown in Figure 2, since most of the bottom-ranked (low activity) CGT / A ODNs lacked downstream flanking sequences, they were excluded from this alignment analysis. We observed that the CGT / A flanking sequences in the top-ranked ODNs were G-rich, while the flanking sequences in the other ODNs were T-rich, as shown in Figure 2. Fig.28 We then synthesized ODN-H and ODN-L with a CGT / A motif surrounded by high- and medium-activity flanking sequences, respectively. As expected, Fig.29In the results shown, only ODN-H triggered TNF expression, while ODN-L did not. In addition, the substitution of G by T and the disruption of the G continuity downstream of the CGT / A motif impaired the immunostimulatory activity of ODN-H, as shown in Fig.30 This indicates that the highly active CGT / A motif requires the assistance of consecutive Gs downstream of CGT / A. This also explains why the CGT / A motifs in the bottom-ranked ODNs tend to be located within the last 10 nt of the 3′ end, as shown in Fig.28 .

[0139] To verify the universality of this sequence rule in predicting the potential immunostimulatory ability of single-stranded DNA, we analyzed the proportion of this immunostimulatory sequence in the genomes of multiple species. We found that the proportion of this motif in bacterial genomes is much higher than that in human genomes. Fig.31 As shown, this explains why bacterial ssDNA is more immunostimulatory than human ssDNA. Cas9 protein is a bacterial protein widely used in gene editing, so its coding sequence also contains a high proportion of immunostimulatory sequences. Compared with double-stranded DNA viruses, most single-stranded DNA viruses and retroviruses contain fewer immunostimulatory sequences, but the genome of adeno-associated virus is an exception. According to this motif rule, we tested the immunostimulatory ability of single-stranded DNA from bacteria, HIV and adeno-associated virus type 2 genomes respectively. At the same time, we also searched, merged and tested ODNs from other viral genomes. The test results are shown below. Fig.32 In summary, our results indicate that CGT / A with downstream consecutive Gs is a stimulatory motif for intracellular ssDNA, regardless of the species origin of the ssDNA.

[0140] Although intracellularly active ODNs have certain sequence similarities with the A-class CpG ODNs sensed by TLR9, TLR9 cannot be activated by 5-methylated CpG ODNs, while ODNs with a phosphorothioate (PS) backbone enhance TLR9 activation. Fig.33As shown in the results, we found that the deoxyribose backbone of CGT-ODN can only be natural. Replacement with phosphorothioate (PS) backbone, 2'-O methylated backbone and ribonucleotide (RNA) backbone will destroy the immunostimulatory activity of intracellular ODNs, while the 5-methylated cytosine modification in the CGT / A motif has almost no effect on the efficacy of intracellular ODN. Therefore, the type of ODNs we discovered is not the same type of ODNs as the CpG ODNs recognized by TLR9. These elements define a class of intracellularly active ODNs: the CGT / A motif with natural phosphodiester (PO) linkage and methylation tolerance is poly G at the 3′ downstream, and the palindromic sequence is optional. For simplicity, we call ODNs with these elements CGT ODNs. Therefore, when delivered into cells, typical CpG-ODNs are not as active as ODN-H. At the same time, TLR9 deficiency does not affect the stimulatory activity of ODN-H, as shown in the results. Fig.34 These results further indicate that CGT-ODNs introduced into cells do not activate immune responses through TLR9, and further suggest the existence of a new intracellular receptor for CGT-ODNs.

[0141] Example 4 SLFN11 is essential for ODN-induced immune response

[0142] To identify potential receptors for intracellular ssDNA, we performed a genome-wide CRISPR-Cas9 screen. Fig.35 We focused on knocking out genes involved in ODN60-induced cell exhaustion. Interestingly, SLFN11, a member of the SLFN protein family, was the most significantly enriched in both single and three rounds of screening, while other identified DNA genes, such as TLR9, AIM2, and cGAS, were not highly enriched, as shown in the results. Fig.36 As shown. SLFN11 was previously identified as an interferon-stimulated gene (ISG) that plays a role in the repression of HIV translation and tumor responses to DNA damaging agents. To validate the role of SLFN11 in intracellular CGT-ODN-induced immune responses, we generated SLFN11 knockout clones from HEK293, 293A, and DU145 cells. Deletion of SLFN11 completely blocked all previously identified immune responses, including cytokine activation triggered by ODN60 and other top-ranked ODNs, decreased cell viability, and the results are shown in Figure 2. Fig.37 We were unable to use lentivirus to express SLFN11 - / - Stable re-expression of SLFN11 was established in HEK293 cells, because overexpression of SLFN11 from the vector strongly inhibited the production of lentivirus. - / -HEK293 cells responded to ODN60, although this re-expression of SLFN11 itself also partially triggered an immune response, as shown in Fig.38 In addition, virus-derived CGT / A ODN and bacterial ssDNA activated TNF and CXCL8 expression only in WT 293A cells but not in SLFN11- / -293A cells, as shown in Fig.32 , 39 As shown. Fig. 9 Consistently, only cells carrying SLFN11 expression responded to the top-ranked ODNs, as shown in Fig.40 Finally, in DU145 cells, SLFN11 was required for CGT-ODNs to activate IFNB1 and CXCL8, as shown in Fig.41 shown.

[0143] Example 5 SLFN11 is a sequence-specific ssDNA binding protein

[0144] To determine whether SLFN11 can bind and recognize ssDNA in the form of ODN, we developed a biotin pull-down complementary method called luminescent ODN immunoprecipitation (ODIP) to quantify the intracellular ODN binding activity of SLFNs. Fig.42 ODN60 was specifically immunoprecipitated with SLFN11, while ODN60rc was not, and the ODIP signal of ODN60 was dependent on SLFN11. Fig.43 Furthermore, ODN binding to SLFN11 is CGT motif-dependent, as any single mutation in the CGT motif of ODN60 reduced its binding to SLFN11 ( Fig.44 ), while the acquisition of the CGT motif in ODN60rc enhanced its binding to SLFN11 ( Fig.45 As expected, SLFN11 specifically bound to the top-ranked ODNs but not to the bottom-ranked ODNs ( Fig.44 ), indicating that high affinity for SLFN11 is necessary for the immunostimulatory activity of ODN.

[0145] SLFN11 belongs to subgroup III of the SLFN family, and its members contain three predicted domains: the N-terminal AAA domain, the SWADL domain, and the C-terminal helicase domain, as shown in the schematic diagram. Fig.46 Truncation analysis showed that the C-terminal fragment of SLFN11 containing residues 349 to 901 was capable of binding to ssDNA, and any of the indicated deletions between residues 349 to 901 reduced the ssDNA binding activity of SLFN11 ( Fig.47The direct ssDNA binding activity of SLFN11 was also determined using purified GST-fused and His-fused C-terminal SLFN11 ( Fig.48 ). Notably, in SLFN11- / - HEK293 cells, only full-length SLFN11 was able to restore responsiveness to ODN60 ( Fig.49 This suggests that the C-terminal helicase domain is responsible for ssDNA binding, whereas the N-terminal domain is required for signaling of the presence of intracellular ssDNA. In summary, the requirement for CGT-DNA-induced immune responses and the specific binding activity to sequences containing CGT / A motifs suggest that SLFN11 is an intracellular CGT-DNA immune sensor.

[0146] Compared with other SLFN subgroups, only SLFN in subgroup III contains the entire C-terminal ssDNA binding domain, which is conserved from elephant nose fish to humans ( Fig.50 ). In addition, subgroup III SLFNs showed similar evolutionary distribution among taxa with dsDNA sensors ( Fig.51 ), indicating that full-length subgroup III SLFNs are under potential selection pressure. However, among all subgroup III SLFNs expressed in humans ( Fig.52 ), only SLFN11 had detectable ssDNA binding activity ( Fig.53 ).

[0147] Example 6 SLFN11 colocalizes and binds to CGT-ODNs in the cytoplasm

[0148] Upon ODN60 stimulation, SLFN11 was exported from the nucleus and accumulated in puncta that colocalized with ODN60 in the cytoplasm. This was in contrast to the diffuse distribution of SLFN11 in the nucleus observed under basal conditions ( Fig.54 When we analyzed cytoplasmic and nuclear extracts by immunoblotting, we found that the distribution of endogenous SLFN11 was differentially sensitive to different extraction protocols ( Fig.55 To further investigate this observation, we compartmentalized cells into the nucleus, perinuclear, and cytosol and found that SLFN11 was mainly stable in the perinuclear fraction under basal conditions but translocated to the cytoplasm upon ODN60 stimulation ( Fig.56 Consistent with the immunofluorescence results, biotin pull-down experiments and EMSAs showed that ODNs-bound SLFN11 was mainly distributed in the cytoplasm ( Fig.57 Finally, we confirmed the colocalization between SLFN11 and the top-ranked ODNs in the cytoplasm of DU145 cells ( Fig.58 ).

[0149] Example 7 SLFN9 (SLFN11 homolog) is essential for CGT-ODN-induced immune response in mice

[0150] Since mice do not have SLFN11, to further investigate the role of SLFNs in the response to CGT-ODNs in vivo, we screened for a mouse SLFN that could functionally complement human SLFN11. Among all mouse subgroup III SLFNs that were highly homologous to human SLFN11, only SLFN9 was able to complement SLFN11. - / - Binds to ssDNA and restores ssDNA presence signaling in HEK293 cells ( Fig.59 Therefore, we established Slfn9 knockout mice ( Fig.60 ). - / - Initial evaluation of the mice indicated that they were healthy and fertile in our colony under specific pathogen-free conditions.

[0151] We first analyzed Slfn9 in primary cells after stimulation with CGT-ODNs. Deficiency of Slfn9 in primary bone marrow-derived macrophages (BMDMs) blocked activation of Ifnb1, Il6, and Cxcl2 by ODN-H and HIV6441 (a CGT ODN from HIV), but not by poly(dA:dT), poly(I:C), or LPS ( Fig.61 ), indicating that Slfn9 is specifically required for the intracellular CGT-ODNs-induced response. HIV6441 was found to be more active than ODN-H in mouse cells; therefore, we used HIV6441 to express the intracellular ODN for the following mouse experiments. To explore the role of SLFN9 and TLR9 in the response to different types of ODN, we delivered HIV6441 and all three types of typical CpG ODNs, ODN1585 (class A), ODN1826 (class B), and ODN2395 (class C), into primary mouse fibroblasts (MAFs), lung fibroblasts (LFs), primary BMDMs, and plasmacytoid dendritic cells (pDCs), respectively.

[0152] TLR9 has been reported to play a role mainly in pDCs and B cells. Consistent with this, all types of ODN stimulated IFNα production in WT and Slfn9- / - pDCs, but not in Tlr9- / - pDCs, suggesting that when Tlr9 is highly expressed, transfected ODN can also activate Tlr9 in endosomes due to endocytosis. However, only knockdown of Slfn9, but not Tlr9, blocked HIV6441- and ODN1585-induced Ifnb1 expression in MAFs, LFs, and BMDMs ( Fig.62). Therefore, the intracellular stimulatory activities of typical CpG ODNs and CGT ODNs are both dependent on SLFN9, and SLNF9 functions in a wider range of tissue types than TLR9.

[0153] We stimulated WT and Slfn9 by intracellular delivery of ODN using a lipid nanoparticle (LNP) system. - / - mice, extending our findings to primary mouse cells, a system also used for the SARS-CoV-2 mRNA vaccine. Since intravenous administration of LNPs resulted primarily in the accumulation of nucleotides in the liver ( Fig.63 A) We evaluated the role of Slfn9 in the liver response to ODNs. HIV6441 activated the expression of Ifnb1, Tnf, Il6, Cxcl2, and Cxcl10 in the liver, as well as the STAT1, JNK, and NFκB pathways ( Fig.63 B, C). Slfn9 deficiency almost completely abolished HIV6441-induced responses. Similarly, Slfn9 was also required for HIV6441-induced cytokine expression in adult mouse liver ( Fig.63 D), except Ifnb1. Consistent with the morphological characteristics of cell death in human cells in Example 1, HIV6441 even triggered histological necrosis and acute hepatitis in the liver ( Fig.63 E, F), which is also SLFN9-dependent. Of note, lethal toxic shock induced by CpG-ODNs has only been reported in D-galactosamine-sensitized mice, and only CpG-DNA injection into mice has been reported to be nontoxic, even at fairly high doses. However, we found that intravenous injection of 60 µg LNP-packaged HIV6441 was sufficient to cause death in WT mice ( Fig.63 G). On the contrary, all Slfn9 - / - In addition, we packaged the single-stranded DNA genome of adeno-associated virus type 2 (AAV2) into LNP and injected it intravenously into mice, which also stimulated the SLFN9-dependent immune response ( Fig.63 H, I). Taken together, these in vivo results confirm previous cellular studies showing that SLFN11 and SLFN9 are essential for intracellular ODN-induced immune responses in humans and mice, respectively.

[0154] Previous basic and clinical studies have shown the intratumoral monotherapy activity of classical CpG-ODNs, which are generally regarded as TLR9 agonists. We speculated that CGT ODN should also have a good tumor therapeutic effect by activating SLFN9. To clarify the role of SLFN9 and TLR9 in ODN anti-tumor monotherapy, we generated SLFN9- / - and TLR9 - / - B16-F10 cells, although WT B16-F10 cells barely expressed TLR9 ( Fig.64 ). We found that Slfn9 - / - cells, but not Tlr9 - / - , were completely resistant to the ODN-induced decrease in cell viability and increase in cytokine activation ( Fig.65 To further confirm these findings, WT and Slfn9 - / - B16-F10 cells were subcutaneously transplanted into mice ( Fig.63 g). Consistently, intratumoral delivery of HIV6441 significantly reduced the growth of WT tumors but had no effect on Slfn9 - / - There was no effect on tumor growth ( Fig.63 h, i), indicating that Slfn9 is a key target for ODN monotherapy in tumor cells.

[0155] Example 8 CGT ODN and pathogenic ssDNA such as bacteria or AAV lead to reduced tRNA cleavage

[0156] After stimulating 293A cells with CGT ODN, the RNA of the cells was extracted and analyzed by PAGE gel electrophoresis staining, and it was found that the abundance of type II tRNA was significantly reduced ( Fig.66 A), and the amount of CGT ODN and the degree of tRNA degradation have a dose-effect relationship ( Fig.66 B). In HUVEC cells, CGT ODN can also lead to tRNA cleavage. In order to verify whether the reduction of type II tRNA caused by CGT ODN is dependent on the tRNA cleavage activity of SLFN11, we mutated the tRNA cleavage active site E209 of endogenous SLFN11 in HEK293 cells in situ to 209A, so that the endogenous SLFN11 expressed in HEK293 lost its tRNA cleavage ability. When we stimulated HEK293 cells with CGT ODN, only WT cells and E209A heterozygous cells (one allele expressed WT SLFN11 and the other allele expressed E209A SLFN11) were able to respond to CGT ODN stimulation to produce tRNA cleavage, while SLFN11 knockout cells and cells expressing only E209A SLFN11 completely resisted tRNA cleavage caused by CGT ODN stimulation ( Fig.66 CD). And E209A SLFN11 also cannot mediate downstream cell death ( Fig.66 EF) and cytokine expression ( Fig.66G), indicating that the tRNA cleavage activity of SLFN11 is necessary for inducing downstream innate immune responses. In addition to CGT ODN, we also verified that ssDNA derived from bacteria, AAV, and HIV genomes can cause SLFN11-dependent tRNA degradation ( Fig.66 H), and similarly, the tRNA cleavage activity of SLFN11 is also necessary for cell death caused by these pathogenic ssDNA ( Fig.66 I). HeLa cells hardly express endogenous SLFN11 and are insensitive to CGT ODN stimulation. Therefore, we artificially constructed cell lines that stably express exogenous wild-type SLFN11 and cleavage-active mutant SLFN11 (E209A, E214A, E209 / 214A) in HeLa cells. When HeLa cells expressing wild-type SLFN11 were stimulated by CGT ODN, we could see obvious tRNA degradation ( Fig.66 J), while HeLa cells expressing SLFN11 with lost tRNA cleavage activity could not respond to CGT ODN stimulation. Similar to the result of endogenous SLFN11 tRNA active point mutation in HEK293 cells, when exogenous SLFN11 was expressed in HeLa cells, it responded to CGT ODN stimulation and led to downstream cell death ( Fig.66 K) and inflammatory response ( Fig.66 L) also requires the tRNA cleavage activity of SLFN11. Combined with the data in our work that SLFN11 can directly recognize and bind to CGT ODN, we identified SLFN11 as an RNase activated by ssDNA. This is also the first ssDNA-activated RNA nuclease discovered in eukaryotic cells. It directly indicates that type II tRNA levels can be used as a clinical detection and diagnostic indicator for ssDNA-induced immune responses.

[0157] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A CGT-ODN nucleotide sequence, characterized in that: The CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-15.

2. The CGT-ODN nucleotide sequence according to claim 1, characterized in that The CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-9.

3. The CGT-ODN nucleotide sequence according to claim 1, characterized in that: The CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1, 3, 4, 6, and 8.

4. The CGT-ODN nucleotide sequence according to claim 1, characterized in that The CGT-ODN nucleotide sequence may be of natural origin or artificially synthesized.

5. The CGT-ODN nucleotide sequence according to claim 1, characterized in that: The CGT-ODN nucleotide sequence has a CGT motif, a CGA motif, and a CGT+CGA motif.

6. A carrier, characterized in that The vector comprises the CGT-ODN nucleotide sequence according to any one of claims 1 to 5.

7. The carrier according to claim 6, characterized in that The vector includes a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a piggyBac vector or a Sleeping Beauty transposase vector.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the CGT-ODN nucleotide sequence according to any one of claims 1 to 5, the carrier according to claim 6 or 7, and a pharmaceutically acceptable adjuvant.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable adjuvants include adjuvants required for intravenous drip, subcutaneous injection, intravenous push injection, intravitreal injection, intramuscular injection and nucleic acid delivery system.

10. Use of the CGT-ODN nucleotide sequence, the vector according to claim 6 or 7, or the pharmaceutical composition according to claim 8 or 9 in regulating cytokine expression, wherein the use does not include diagnostic or therapeutic purposes; The CGT-ODN nucleotide sequence is the CGT-ODN nucleotide sequence according to any one of claims 1 to 5, wherein the nucleotide sequence is unmethylated, or, The CGT-ODN nucleotide sequence is a methylated CGT-ODN nucleotide sequence, as shown below: 5’-(X) n CGT / A(X) m (G) a (X) b (G) c (X) d -3’, in, X is any non-methylated nucleotide base, n represents any integer value ≥0, m represents any integer value ≥0, a represents any integer value ≥0, b represents any integer value ≥0, c represents any integer value ≥0, d represents any integer value ≥0, C is 5-methylcytosine, G represents guanine, T represents thymine, A represents adenine, and at least one of a and c is an integer ≥1.

11. The use according to claim 10, characterized in that: The cytokines include cytokines of JNK, p38 and NFκB signaling pathways.

12. The use according to claim 10, characterized in that: The cytokines include TNF, IFNB1, CXCL8, IL-11, IL-12A, IL-32, CCL2, CCL4, ANKRD1, TNFAIP3, JUN, FOS, FOSL1, FOSL2, NFKB1, NFKB2, CASP3, and MAP3K14.

13. The use according to claim 10, characterized in that: At least one of a and c has a value greater than or equal to 2.

14. The use according to claim 10, characterized in that: The flanking sequence of the methylated CGT-ODN nucleotide sequence is rich in consecutive G nucleotides.

15. The use according to claim 10, characterized in that The methylated CGT-ODN nucleotide sequence has phosphodiester linkages.

16. The use according to claim 10, characterized in that: The methylated CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-15.

17. The use according to claim 10, characterized in that: The methylated CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1-9.

18. The use according to claim 10, characterized in that: The methylated CGT-ODN nucleotide sequence is shown in any one of SEQ ID NOs: 1, 3, 4, 6, and 8.

19. The use according to claim 10, characterized in that: The methylated CGT-ODN nucleotide sequence may be of natural origin or artificially synthesized.

20. The use according to claim 10, characterized in that The methylated CGT-ODN nucleotide sequence has a CGT motif, a CGA motif, and a CGT+CGA motif.

21. Use of the CGT-ODN nucleotide sequence in any one of claims 10 to 20 and the vector in claim 6 or 7 in the preparation of a pharmaceutical composition for treating or preventing infectious diseases, allergic diseases, immunodeficiency diseases, and tumors.

22. Use of the CGT-ODN nucleotide sequence in any one of claims 10 to 20 and the vector according to claim 6 or 7 in the preparation of a vaccine.

23. The use according to claim 22, characterized in that The vaccines include subunit vaccines and viral vector vaccines.

24. The use according to claim 23, characterized in that The vectors in the viral vector vaccine self-assemble into virus-like particles.

25. The use according to claim 22, characterized in that The vector includes a self-inactivating vector.

26. The use according to claim 23, characterized in that The virus is selected from the group consisting of a lentivirus, an influenza virus, a hepatitis virus, an alphavirus, a filovirus, an adenovirus, an adeno-associated virus and / or a flavivirus.

27. Use of the CGT-ODN nucleotide sequence in any one of claims 10 to 20, or the vector in claim 6 or 7 in antibody production.

28. Use of the CGT-ODN nucleotide sequence in any one of claims 10 to 20, or the vector according to claim 6 or 7, in regulating the degree of tRNA degradation, wherein the use does not include the purpose of diagnosis or treatment.

29. The use according to claim 28, characterized in that The tRNA includes type II tRNA.

30. A method for preparing CGT-ODN related antibodies, characterized in that: The method comprises using the CGT-ODN nucleotide sequence in the application according to any one of claims 10 to 20 and the vector according to claim 6 or 7 to immunize non-human animals to obtain antibodies.

31. The method according to claim 30, characterized in that The non-human animal is a mammal.

32. The method according to claim 31, characterized in that The mammals include mice, rats, guinea pigs, rabbits, sheep, cows, horses, and non-human primates.

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