Tlr-inhibiting oligonucleotides and uses thereof

Abstract

Inhibitory oligonucleotides with reduced toxicity, partially phosphorothioated, strongly block NF-kB activation induced by TLR9 agonists and TLR7 / 8 agonists. Inhibitory oligonucleotides inhibit the production of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFa). Interferon (IFN) production by human PBMCs induced by TLR9 agonists is prevented by inhibitory oligonucleotides. These oligonucleotides can be used as therapeutic drugs to treat immune-mediated diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), sepsis, multiple organ dysfunction syndrome and inflammatory diseases mediated by inflammatory cytokines.

Description

Technical Field

[0001] This invention relates to oligonucleotides and therapeutic remedies using oligonucleotides to treat immune-mediated diseases. Immune-mediated diseases include autoimmune diseases, graft rejection, hypersensitivity reactions, diseases related to self-antigens, microbial overstimulation of the host immune system, Toll-like receptor (TLR)-mediated diseases, NF-κB-mediated diseases, interferon-mediated diseases, and inflammatory diseases mediated by inflammatory cytokines. Background Technology

[0002] The immune system protects the body from infections by bacteria, parasites, fungi, and viruses, and from the growth of tumor cells. Immunity can be divided into innate immunity and adaptive immunity. The innate immune response usually occurs immediately after infection, providing an early barrier against infectious diseases, while the adaptive immune response subsequently produces antigen-specific, long-term protective immunity.

[0003] However, sometimes unwanted immune responses occur, which can lead to immune-mediated conditions. These conditions include autoimmune diseases, graft rejection, hypersensitivity reactions, diseases associated with microbial overstimulation of the host's immune system, Toll-like receptor (TLR)-mediated diseases, interferon-mediated diseases, NF-κB-mediated diseases, and inflammatory cytokine-mediated inflammatory diseases. Autoimmune diseases are caused by adaptive immune responses, innate immune responses, or both against endogenous and / or exogenous antigens. Foreign substances derived from bacteria, parasites, fungi, or viruses may mimic their own proteins and stimulate the immune system to respond to its own cells and tissues, resulting in diseases including, but not limited to, systemic lupus erythematosus (SLE) and rheumatoid arthritis. Graft rejection is the result of organ or tissue transplantation caused by the recipient's (host's) immune response to the transplanted organ / tissue. When a subject receives a graft, including those for the kidney, pancreas, heart, lung, bone marrow, cornea, and skin, the subject is able to generate an immune response (rejection) against the graft. Hypersensitivity is an inappropriate immune response with detrimental effects, leading to significant tissue damage or even death. Hypersensitivity reactions are classified into four types (e.g., types I, II, III, and IV). Diseases associated with microbial overstimulation of the host's immune system are triggered by infections with viruses such as influenza virus and other microorganisms. In cases of influenza virus and Gram-negative bacterial infections, an excessive immune response to the invaders appears to be a fatal factor for patients. The response is characterized by the excessive production of cytokines. Studies of septic shock syndrome have shown that excessive / abnormal production of cytokines can lead to rapid death due to cytokine-mediated lethal shock (Slifka et al., J Mol Med. 2000, 78, 74-80). Post-Gram-negative septic shock is the leading cause of death in critically ill patients. Excessive cytokine production is known to promote sepsis characterized by cytokine-mediated lethal shock (Espat et al., J Surg Res, 1995, 59, 153-8). Multiple organ dysfunction syndrome (MODS) is a leading cause of morbidity and mortality in severe sepsis and shock. Cytokine-mediated lethal shock due to excessive host cytokine production is considered a major mechanism leading to MODS (Wang et al., Am J EmergMed, 2008, 26, 711-5). Toll-like receptor (TLR)-mediated diseases are symptoms caused by TLR activation.

[0004] TLRs are a family of receptors that recognize microbial-derived molecular structures (pathogen-associated molecular patterns, or PAMPs). Immune cells expressing TLRs are activated upon binding to PAMPs. TLRs recognize and are activated by a range of pathogen-derived products. Bacterial lipopolysaccharide (LPS) is recognized by TLR4; lipoteichoic acid and diacylated lipopeptides are recognized by the TLR2-TLR6 dimer; triacylated lipopeptides are recognized by the TLR2-TLR1 dimer; synthetic or viral or bacterial CpG-containing oligonucleotides (CpGODN) are recognized by TLR9; bacterial flagellin is recognized by TLR5; yeast glycans are recognized by the TLR2-TLR6 dimer; F protein from respiratory syncytial virus (RSV) is recognized by TLR4; viral-derived double-stranded RNA (dsRNA) and synthetic analogues of poly(I:C) dsRNA are recognized by TLR3; viral DNA is recognized by TLR9; single-stranded viral RNA (VSV and influenza virus) and synthetic guanosine analogues such as imidazoquinoline and imiquimod are recognized by TLR7 and TLR8 (Liew et al., Nature Reviews Immunology, 2005, 5, 446-458).

[0005] In recent years, TLR activation has been associated with the pathogenesis of several diseases, including sepsis, dilated cardiomyopathy, diabetes, experimental autoimmune encephalomyelitis, systemic lupus erythematosus, atherosclerosis, asthma, chronic obstructive pulmonary disease, and organ failure (Liew et al., Nature Review Immunology, 2005, 5, 446-458). Activation of TLR9 by autologous DNA plays an important role in the development of autoimmune diseases, such as psoriasis (Gilliet et al., Nat. Rev. Immunol. 2008, 8, 594-606), SLE (Christensen et al., Immunity, 2006, 25, 417-28; Barrat, et al., J Exp Med, 2005, 202, 1131-9; Wellmann, et al., Proc Natl Acad Sci USA, 2005, 102, 9258-63), and rheumatoid arthritis (Leadbetter et al., Nature, 2002, 416, 603-7; Boule et al., J Exp Med, 2004, 199, 1631-40). It also describes how TLR9 agonists activate innate and adaptive immune responses (Krieg, Nature Reviews Drug Discovery, 2006, 5, 471-484).

[0006] Oligonucleotides with the 5'-cctcctcctcctcctcctcct-3' nucleotide sequence have been reported to inhibit the proliferation of human peripheral blood mononuclear cells (PBMCs) and the production of interferon (IFN) induced by TLR9 agonists (US8030289B2). Oligonucleotides with the nucleotide sequence (CCT)nCm, where n is an integer from 2 to 50 and m is 0, 1, or 2, have been documented to possess TLR7, 8, and 9 inhibitory properties, inhibiting NF-κB-dependent immune responses and the production of inflammatory cytokines such as IFNα and tumor necrosis factor-α (TNFα) (EP2154144, WO2014 / 082254), suggesting the widespread use of oligonucleotides (CCT)nCm for the treatment of immune-mediated conditions.

[0007] Oligonucleotides with common phosphodiester bonds in the backbone have been reported to be susceptible to nucleases in vivo, such as serum exonucleases or intracellular endonucleases (Stein et al., Nuc Acid Res, 1988, 16, 3209-3221), leading to poor pharmacokinetic profiles. To address this issue, several strategies have been employed to modify the backbone with nuclease-resistant structures, including thiophosphorylation. However, thiophosphorylation is well-known for its potent hepatotoxicity and nephrotoxicity (Henry et al., Toxicol Pathol, 1999, 28, 95-100; Monteith et al., Toxicol Pathol, 1999, 27, 307-317), which hinders the use of thiophosphorylated oligonucleotides for the treatment of systemic diseases. Therefore, some experiments have been conducted to reduce toxicity, such as altering the nucleotide sequence (Hagedorn et al., Nuc Acid Ther, 2013, 23, 302-310; Burdick et al., Nuc Acid Res, 2014, 42, 4882-4891). However, with respect to the oligonucleotide described in this invention as CxTy(CCT)nCm, since the potency of the oligonucleotide depends entirely on its unique nucleotide sequence, the strategies described above for altering the nucleotide sequence cannot be performed.

[0008] Citation List

[0009] Patent documents

[0010] PTL 1: US8030289B2

[0011] PTL 2: EP2154144

[0012] PTL 3: WO2014 / 082254

[0013] PTL 4: JP5011520

[0014] Non-Patent Literature

[0015] NPL 1: Slifka et al., J Mol Med. 2000, 78, 74 - 80

[0016] NPL 2: Espat et al., J Surg Res, 1995, 59, 153 - 8

[0017] NPL 3: Wang et al., Am J Emerg Med, 2008, 26, 711 - 5

[0018] NPL 4: Liew et al., Nature Reviews Immunology, 2005, 5, 446 - 458

[0019] NPL 5: Gilliet et al., Nat.Rev.Immunol. 2008, 8, 594 - 606

[0020] NPL 6: Christensen et al., Immunity, 2006, 25, 417 - 28

[0021] NPL 7: Barrat, et al., J Exp Med, 2005, 202, 1131 - 9 [[ID=第二十七章]]

[0022] NPL 8: Wellmann, et al., Proc Natl Acad Sci USA, 2005, 102, 9258 - 63

[0023] NPL 9: Leadbetter et al., Nature, 2002, 416, 603 - 7

[0024] NPL 10: Boule et al., J Exp Med, 2004, 199, 1631 - 40

[0025] NPL 11: Krieg, Nature Reviews Drug Discovery, 2006, 5, 471 - 484

[0026] NPL 12: Stein et al., Nuc Acid Res, 1988, 16, 3209 - 3221

[0027] NPL 13: Henry et al.,Toxicol Pathol,1999,28,95-100

[0028] NPL 14: Monteith et al.,Toxicol Pathol,1999,27,307-317

[0029] NPL 15: Hagedorn et al.,Nuc Acid Ther,2013,23,302-310

[0030] NPL 16: Burdick et a l.,Nuc Acid Res,2014,42,4882-4891

[0031] NPL 17: Klinman, Nat. Rev. Immunol, 2004, 4, 249-258

[0032] NPL 18: Marshall et al.,Infect Immun,1998,66,1325-33

[0033] NPL 19: Peter et al.,Immunology,2008,123,118-28

[0034] NPL 20: The Gilmore Lab, Boston University Biology [Online, retrieved November 4, 2015] Retrieved from the following web:<URL:http: / / www.bu.edu / nf-kb / physiological-mediators / diseases / >

[0035] NPL 21: Tak et a l.,J Clin Invest,2001,107,7-11

[0036] NPL 22: Baldwin(Jr) et al., Annu Rev Immunol,1996,14,649-683

[0037] NPL 23: Miyazawa et al.,Am J Pathol 1998,152,793-803

[0038] NPL 24: Schottelius et al.,J Biol Chem 1999,274,31868-31874

[0039] NPL 25:Neurath et al.,Ann NY Acad Sci,1998,859,149-159

[0040] NPL 26:Liu et al.,Neurosci Bull,2012,28,131-44

[0041] NPL 27:Liu et al.,Itch:Mechanisms and Treatment,Carstens E,Akiyama T,editors,2014,CRC Press / Taylor & Francis

[0042] NPL 28:Sun et al.,Clin Immunol,2010,134,262-276

[0043] NPL 29:Zhang et al.,International Immunopharmacol,2012,446-453

[0044] NPL 30:Freier et al.,Nuc Acid Res,1997,25,4429-4443

[0045] NPL 31:Kuwahara et al.,Molecules,2010,15,5423-5444

[0046] NPL 32:Deleavey et al.,Chemistry&Biology,19,937-954

[0047] NPL 33:Mutisya et al.,Nucleic Acids Research,2014,42,6542-6551

[0048] NPL 34:Sharma et al.,Med.Chem.Commun.2014,5,1454-1471

[0049] NPL 35:Davidson,Engl J Med,2001,345,340-350

[0050] NPL 36:Delgado-Vega et al.,Arthritis Research & Therapy 2010,12 / S2

[0051] NPL 37: Goldsby et al., Immunology, Fifth Edition, 2003, W.H. FREEMAN AND COMPANY

[0052] NPL 38: The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A / H5, N Engl J Med, 2005, 353, 1374 - 85

[0053] NPL 39: Patel et al., Mol Cell Neurosci, 2014, 63, 38 - 48

[0054] NPL 40: Smith, Curr Opin Nehrol Hypertens, 2009, 18, 189

[0055] NPL 41: Gambuzza et al., J Neuroimmunol, 2011, 239, 1 - 12

[0056] NPL 42: Hatemi et al., Clin Exp Rheumatol, 2015, 33(6 Suppl 94), 3 - 14

[0057] NPL 43: Tang et al., Respirology, 2015, Oct 18. doi:10.1111 / resp.12657. [Electronic publication ahead of print]

[0058] NPL 44: Ito et al., Blood, 2006, 107, 2423 - 2431

[0059] NPL 45: Guidance for Industry,Estimating the Maximum Safe StartingDose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers,USDepartment of Health and Human Services Food and Drug Administrati on,Center for Drug Evaluation and Research(CDER),July 2005

[0060] NPL 46: Maeda et al.,Int J Hematol.,2005,81,148-54 Invention Overview

[0062] Technical issues

[0063] The present invention aims to provide safer pharmaceutical compositions and allow for increased dosing regimens of said pharmaceutical compositions, while increasing efficacy against targeted diseases.

[0064] This invention provides non-toxic oligonucleotides. The oligonucleotides of this invention comprise oligonucleotide segments having the formula CxTy(CCT)nCm, which are partially thiophosphorylated by substituting one or more non-bridging oxygen atoms with sulfur atoms in the internucleotide bonds, wherein n is an integer from 2 to 50, or preferably from 5 to 16, x represents an integer 0 or 1, y represents an integer 0 (only when x = 0) or 1 (x can be 0 or 1), and m is 0, 1, or 2.

[0065] In this invention, the inventors have identified certain oligonucleotides with partial thiophosphorylation that achieve reduced toxicity while maintaining stability in serum, enabling safer pharmaceutical composition formulations; at the same time, the safety profile of the oligonucleotides allows for increased dosing levels of each oligonucleotide, which will result in increased efficacy in treating targeted diseases in treated humans or non-human animals.

[0066] Brief description of the attached figures

[0067] [ Figure 1 ] Figure 1Figure a shows the stability of partially phosphorothiolated (CCT)12 oligonucleotides in serum-containing medium. The oligonucleotides were incubated in serum-supplemented medium, and the remaining oligonucleotides in the medium were quantified. As shown, CCT12PO with normal phosphodiester nucleotide bonds showed almost complete degradation after 1 day of incubation, while fully phosphorothiolated CCT12PS showed a longer duration, indicating that the oligonucleotides degrade rapidly without internucleotide modification. Meanwhile, partially phosphorothiolated CCT12-3 (see Examples section for details) showed comparable stability to CCT12PS, indicating that at least some level of internucleotide bond phosphorothiolation is sufficient to achieve stability against serum nucleases. Figure 1 b shows the stability of partially phosphorothiolated (TCC12) oligonucleotides in serum-containing medium. The results indicate that while the unphosphorothiolated oligonucleotide (TCC12PO) exhibited rapid degradation after 1 day of incubation, the partially phosphorothiolated oligonucleotide (TCC12-6) showed the same increased stability as the fully phosphorothiolated oligonucleotide (TCC12PS) with the same sequence.

[0068] [Figure 2] Figure 2 reveals the reduction in oligonucleotide toxicity through certain modes of decreased thiophosphorylation levels. Figure 2a This demonstrates that reducing thiophosphate esterification levels lowers (CCT). 12 The toxicity. As shown here, even though the oligonucleotide CCT12PS with the same sequence exhibits the same high toxicity as another fully thiophosphorylated oligonucleotide CCT8PS, CCT12-1 did not show significant weight loss. Figure 2b The comparison between oligonucleotides CCT12-2, CCT12-3, CCT12-4, and CCT12-5 is shown, which means that the reduction in toxicity is achieved not only by reducing the level of thiophosphorylation, but also by being related to the thiophosphorylation pattern. Figure 2c The reduction in toxicity through alteration of the thiophosphorylation pattern also applies to (TCC) repeats. TCC12-6 showed reduced toxicity, while TCC12-7, with the same level of thiophosphorylation, still exhibited significant toxicity. Figure 2d and 2e The study showed that even oligonucleotides exhibiting strong toxicity also resulted in an increase in ALT levels.

[0069] [Figure 3] Figure 3a The inhibitory effect of partially thiophosphate-esterified oligonucleotides on NF-κB transcriptional activity was demonstrated. Although CCT12-1, CCT12-2, and CCT12-3 showed reduced toxicity, they still retained inhibitory activity. Figure 3bc shows that the reduction in toxicity caused by changes in the thiophosphorylation mode does not affect the inhibitory activity of oligonucleotides, such as oligonucleotides CCT12-3 and TCC12-6.

[0070] [Figure 4] Figure 4a Figures 1, 2, and 3 show the inhibitory effects of the inhibitory oligonucleotides on TNFα and interleukin (IL)-6 production from CAL-1 cells stimulated with the TLR7 / 8 agonist gademod or the TLR9 agonist CpG2395. The altered thiophosphorylation pattern, which reduced toxicity, had no significant effect on the inhibitory effect against TLR7, 8, or TLR9 activation.

[0071] [ Figure 5 ] Figure 5 Figures a and b show plots depicting the inhibitory activity of the repressive oligonucleotides against IFNa production from human PBMCs stimulated with CpG2216. These figures (5a and 5b) also reveal that alterations in the thiophosphorylation pattern do not significantly affect the inhibitory activity of the oligonucleotides against levels of IFNa production induced by TLR9 agonists. Detailed Implementation

[0072] This invention provides pharmaceutical compositions containing one or more oligonucleotides, said oligonucleotide comprising an oligonucleotide having the formula CxTy(CCT)nCm, wherein n is an integer from 2 to 50, or preferably from 5 to 16, x represents an integer 0 or 1, y represents an integer 0 (only when x = 0) or 1 (x can be 0 or 1), and m is 0, 1, or 2. Examples of oligonucleotide sequences are as follows:

[0073] 5'-cctcctcctcctcctcct-3' (SEQ ID NO: 1).

[0074] 5'-cctcctcctcctcctcctcct-3' (SEQ ID NO: 2).

[0075] 5'-cctcctcctcctcctcctcctcct-3' (SEQ ID NO: 3).

[0076] 5'-cctcctcctcctcctcctcctcctc-3' (SEQ ID NO: 4).

[0077] 5'-cctcctcctcctcctcctcctcctcc-3' (SEQ ID NO: 5).

[0078] 5'-cctcctcctcctcctcctcctcctcct-3' (SEQ ID NO: 6).

[0079] 5'-cctcctcctcctcctcctcctcctcctc-3’(SEQ ID NO:7)。

[0080] 5'-cctcctcctcctcctcctcctcctcctcc-3’(SEQ ID NO:8)。

[0081] 5'-cctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO:9)。

[0082] 5'-cctcctcctcctcctcctcctcctcctcctc-3’(SEQ ID NO:10)。

[0083] 5'-cctcctcctcctcctcctcctcctcctcctcc-3’(SEQ ID NO:11)。

[0084] 5'-cctcctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO:12)。

[0085] 5'-cctcctcctcctcctcctcctcctcctcctcctc-3’(SEQ ID NO:13)。

[0086] 5'-cctcctcctcctcctcctcctcctcctcctcctcc-3’(SEQ ID NO:14)。

[0087] 5'-cctcctcctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO:15)。

[0088] 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO:16)。

[0089] 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO:17)。

[0090] 5'-tcctcctcctcctcctcc-3’(SEQ ID NO:18)。

[0091] 5'-tcctcctcctcctcctcctcctcctcc-3' (SEQ ID NO: 19).

[0092] 5'-tcctcctcctcctcctcctcctcctcctcctcctcc-3' (SEQ ID NO: 20).

[0093] The oligonucleotides of the present invention can be screened based on the arrangement of chemical modification patterns in the internucleotide bonds, based on the reduced toxicity compared to fully thiophosphorylated oligonucleotides having the same sequence; the chemical modification is optionally thiophosphorylation.

[0094] Preferably, the present invention provides oligodeoxynucleotides with partial thiophosphorylation, wherein S replaces non-bridging O atoms in a certain pattern, resulting in maintenance of oligonucleotide activity and in vivo stability, while minimizing toxicity when administered to animals. Examples of the oligodeoxynucleotides of the present invention with reduced toxicity comprise fully thiophosphorylated segments of CCTCCTCCT, CTCCTCCTC, or TCCTCCTCC, or truncated derivatives thereof, linked by normal phosphodiester bonds. The oligonucleotides include the following:

[0095] 5'-CCTCCTCCtCCTCCTCCt-3'(SEQ ID NO.:21),

[0096] 5'-CCTCCTCCtCCTCCTCCtCCTCCTCCt-3'(SEQ ID NO.:22),

[0097] 5'-CCTCCtCCTCCTCCtCCTCCTCCtCCt-3'(SEQ ID NO.:23),

[0098] 5'-CCtCCTCCTCCtCCTCCTCCtCCTCCt-3'(SEQ ID NO.:24),

[0099] 5'-CCTCCtCCTCCtCCTCCtCCTCCtCCTCCtCCTCCt-3' (SEQ ID NO.: 25)

[0100] 5'-CCTCCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCt-3' (SEQ ID NO.: 26),

[0101] 5'-CCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCtCCt-3' (SEQ ID NO.:27)

[0102] 5'-CCtCCTCCTCCtCCTCCTCCtCCTCCTCCtCCTCCt-3'(SEQ ID NO.:28),

[0103] 5'-CTCCTCCTcCTCCTCCTc-3'(SEQ ID NO.:29),

[0104] 5'-CTCCTCCTcCTCCTCCTcCTCCTCCTc-3'(SEQ ID NO.:30),

[0105] 5'-CTCCTcCTCCTCCTcCTCCTCCTcCTc-3'(SEQ ID NO.:31),

[0106] 5'-CTcCTCCTCCTcCTCCTCCTcCTCCTc-3'(SEQ ID NO.:32),

[0107] 5'-CTCCTCCTcCTCCTCCTcCTCCTCCTcCTCCTCCTc-3'(SEQ ID NO.:33),

[0108] 5'-CTCCTcCTCCTCCTcCTCCTCCTcCTCCTCCTcCTc-3'(SEQ ID NO.:34),

[0109] 5'-CTcCTCCTCCTcCTCCTCCTcCTCCTCCTcCTCCTc-3'(SEQ ID NO.:35),

[0110] 5'-TCCTCCTCcTCCTCCTCc-3'(SEQ ID NO.:36),

[0111] 5'-TCCTCCTCcTCCTCCTCcTCCTCCTCc-3'(SEQ ID NO.:37),

[0112] 5'-TCCTCcTCCTCCTCcTCCTCCTCcTCc-3'(SEQ ID NO.:38),

[0113] 5'-TCcTCCTCCTCcTCCTCCTCcTCCTCc-3'(SEQ ID NO.:39),

[0114] 5'-TCCTCCTCcTCCTCCTCcTCCTCCTCcTCCTCCTCc-3'(SEQ ID NO.:40),

[0115] 5'-TCCTCcTCCTCCTCcTCCTCCTCcTCCTCCTCcTCc-3' (SEQ ID NO.:41),

[0116] 5'-TCcTCCTCCTCcTCCTCCTCcTCCTCCTCcTCCTCc-3' (SEQ ID NO.:42),

[0117] In this context, uppercase letters indicate that the base is modified with thiophosphate at the 3' position in the phosphodiester nucleotide internucleotide bond, while lowercase letters indicate unmodified bases. The common structural features of oligonucleotides containing fully thiophosphate-modified segments of CCTCCTCCT, CTCCTCCTC, or TCCTCCTCC, or truncated derivatives thereof, allow for the reduction of the toxicity exhibited by fully thiophosphate-modified oligonucleotides having the structural formula CxTy(CCT)nCm, where n is an integer from 2 to 50, preferably from 5 to 16, x represents an integer 0 or 1, y represents an integer 0 (only when x = 0) or 1 (x can be 0 or 1), and m is 0, 1, or 2.

[0118] The oligonucleotides of this invention strongly inhibit TLR9 activation. CpG-containing oligonucleotides (CpG ODNs) are known as TLR9 agonists (Klinman, Nat. Rev. Immunol, 2004, 4, 249-258). The oligonucleotides of this invention strongly inhibit cytokines stimulated by CpG ODNs, indicating that these oligonucleotides can be used as therapeutic agents for diseases associated with TLR9 overactivation or other diseases that can be treated by inhibiting TLR9 signaling. For example, since it has been reported that TLR9 activation promotes the development of psoriasis (Gilliet et al., Nat. Rev. Immunol., 2008, 8, 594-606), SLE (Barrat et al., J Exp Med, 2005, 202, 1131-9; Wellmann et al., Proc Natl Acad Sci USA, 2005, 102, 9258-63; Christensen et al., Immunity, 2006, 25, 417-28) and rheumatoid arthritis (Leadbetter et al., Nature, 2002, 416, 603-7; Boule et al., J Exp Med, 2004, 199, 1631-40), the oligonucleotides of the present invention can be used as therapeutic agents for psoriasis, SLE and rheumatoid arthritis by inhibiting TLR9 activation.

[0119] The oligonucleotides of the present invention strongly inhibit the production of IFN by human PBMCs induced by TLR9 agonists. Therefore, the present invention is capable of treating diseases associated with excessive IFN production or diseases treated by inhibiting IFN production. For example, as reported, since elevated IFN production promotes the development of SLE (Barrat et al., J Exp Med, 2005, 202, 1131-9; Wellmann et al., Proc Natl Acad Sci USA, 2005, 102, 9258-63), the oligonucleotides of the present invention can be used as therapeutic agents for SLE by inhibiting IFN production.

[0120] It has been demonstrated that injection of TLR9 agonists; CpG ODN and D-galactosamine (D-Gal) into mice elicited a strong immune response. The model mice died within 12 to 24 hours. Analysis of plasma cytokines revealed excessive production of pro-inflammatory cytokines such as TNFα (Marshall et al., Infect Immun, 1998, 66, 1325-33; Peter et al., Immunology, 2008, 123, 118-28). The oligonucleotides of this invention strongly inhibit TNFα production induced by TLR9 stimulation in mouse cells. Because cytokine-mediated lethal shock promotes septic shock (Slifka et al., J Mol Med, 2000, 78, 74-80; Espat et al., J Surg Res, 1995, 59, 153-8) and multiple organ dysfunction syndrome (MODS) (Wang et al., Am J Emerg Med, 2008, 26, 711-5), the oligonucleotides of the present invention can be used as therapeutic agents for sepsis and MOGS by rescuing the host from cytokine-mediated lethal shock.

[0121] The oligonucleotides of the present invention also strongly inhibit the production of cytokines induced by TLR7 or TLR8 agonists. The oligonucleotides of the present invention can be used as therapeutic agents for treating Toll-like receptor (TLR)-mediated diseases by inhibiting TLR7 or TLR8.

[0122] The oligonucleotides of this invention strongly inhibit TLR-induced NF-κB activation, indicating that they can be used as therapeutic agents for diseases associated with NF-κB activation. Since NF-κB activation has been reported to promote the development of various diseases (http: / / www.bu.edu / nf-kb / physiological-mediators / diseases / ; Tak et al., J Clin Invest, 2001, 107, 7-11), the oligonucleotides of this invention can be used as therapeutic agents for the symptoms occurring in these diseases by inhibiting NF-κB activation. In some cancers, excessive activation of NF-κB has been reported, and the inhibitory function of this invention can also be used to treat such cancers.

[0123] NF-κB is one of the most important regulators of pro-inflammatory gene expression. Activation of NF-κB plays a crucial role in inflammation through its ability to induce the transcription of pro-inflammatory cytokines (Baldwin(Jr) et al., Annu Rev Immunol, 1996, 14, 649-683). NF-κB has been shown to play a role in constitutive IL-6 production in synovial fibroblasts of rheumatoid arthritis (RA) (Miyazawa et al., Am J Pathol 1998, 152, 793-803). NF-κB is also involved in the activation of inflammatory genes in human monocytes induced by IL-1 or TNFα (Schottelius et al., J Biol Chem 1999, 274, 31868-31874). The number of NF-κB-positive cells is correlated with the severity of gastritis. Similarly, there is evidence of NF-κB activation in inflammatory bowel disease, with lamina propria macrophages exhibiting activated NF-κB (Neurath et al., Ann NY Acad Sci, 1998, 859, 149-159).

[0124] Ligand activation of TLRs induces the activation of transcription factors such as NF-κB and interferon response factors (IRF). These activated transcription factors further induce the production of inflammatory cytokines such as IL-6, IL-1, IL-8, TNFα, and IFN. The production of inflammatory cytokines can cause diseases such as, but not limited to, autoimmune diseases, allergic reactions, and hypersensitivity reactions; therefore, this invention is intended for the treatment of such inflammatory diseases.

[0125] Inflammatory cytokines such as TNFα, IFN, IL-1, IL-6, and IL-12 can also activate the production of downstream prostaglandins, which are involved in pain formation in disease conditions. TLRs have also been reported to be expressed in the CNS and PNS, which are involved in pain perception (Liu et al., Neurosci Bull, 2012, 28, 131-44; Liu et al., Itch: Mechanisms and Treatment, Carstens E, Akiyama T, editors, 2014, CRC Press / Taylor & Francis). Therefore, this invention is expected to be used to treat such pain symptoms caused by inflammatory conditions.

[0126] Since the function of the present invention can be derived from the above concepts, the present invention provides a therapeutic agent for treating immune-mediated diseases by administering the oligonucleotide of the present invention, alone or with a pharmaceutically acceptable carrier, to a subject; administration may be intravenous, parenteral, subcutaneous, intravenous, transdermal, sublingual, intranasal, transmucosal, pulmonary, oral, gastric, intestinal, rectal, vaginal, aerosol, intraocular, tracheal, rectal, spinal, intramuscular, intra-articular, intra-abdominal, intracardiac, intraosseous, intrasheath, intravitreal, inhaled, or local.

[0127] In another embodiment, the present invention provides a therapeutic agent for treating immune-mediated diseases by administering the oligonucleotide of the present invention alone or in combination with other active ingredients.

[0128] In another embodiment, the present invention provides a therapeutic agent for treating immune-mediated conditions by administering the oligonucleotides of the present invention in a delivery medium.

[0129] In another embodiment, the oligonucleotides of the present invention are modified with one or more polyethylene glycol (PEG) chains. PEGylated oligonucleotides can prolong in vivo circulation time by reducing renal clearance.

[0130] As described above, the present invention provides a method for modulating an individual's immune response, comprising administering to the individual an immunostimulatory compound sufficient to modulate the individual's immune response. The immunomodulation according to the method of the present invention can be performed on individuals, including those suffering from conditions associated with unwanted immune response activation.

[0131] In another embodiment, the present invention uses the oligonucleotides of the invention to provide therapeutic agents for treating immune-mediated conditions. Immune-mediated conditions include autoimmune diseases, graft rejection, hypersensitivity reactions, diseases related to self-antigens, microbial overstimulation of the host immune system, and Toll-like receptor (TLR)-mediated diseases.

[0132] In another embodiment, the present invention uses the oligonucleotides of the invention to provide therapeutics for Toll-like receptor (TLR)-mediated diseases, TLR9-mediated diseases, TLR7 and / or 8-mediated diseases, hypersensitivity reactions, diseases associated with microbial overstimulation of the host immune system, graft rejection, interferon-mediated diseases, and inflammatory cytokine-mediated inflammatory diseases by inhibiting TLR activation and inflammatory cytokine production induced by TLR antagonists such as viruses or autoantigens in patients with autoimmune diseases.

[0133] In another embodiment, the present invention provides a method for modulating a TLR9-dependent immune response in an individual to an amount sufficient to prevent the production of TLR9-dependent cytokines, comprising co-administering an immunostimulatory compound to the individual.

[0134] In another embodiment, the present invention provides a method for modulating a TLR7 and / or TLR8-dependent immune response in an individual to an amount sufficient to inhibit the production of TLR7 and / or TLR8-dependent cytokines, comprising co-administering an immunostimulatory compound to the individual.

[0135] In another embodiment, the present invention provides a therapeutic agent for treating immune-mediated diseases by using the oligonucleotides of the present invention to inhibit the production of pro-inflammatory cytokines to rescue subjects from diseases such as inflammation or cytokine-mediated lethal shock.

[0136] In another embodiment, the present invention provides a method for modulating an NF-κB-dependent immune response in an individual to an amount sufficient to prevent the production of NF-κB-dependent cytokines, comprising co-administering an immunostimulatory compound to the individual.

[0137] In another embodiment, the present invention provides a therapeutic agent for treating immune-mediated conditions by using the oligonucleotides of the present invention to inhibit NF-κB activation induced by TLR stimulation.

[0138] In another embodiment, the present invention primarily inhibits TLR activation and associated downstream signaling, and exhibits various effects on a broad spectrum of important biological systems, such as NF-κB transcriptional activity, IFN and inflammatory cytokine secretion. Therefore, pharmaceutical compositions comprising the oligonucleotides of the present invention can be used to combat a variety of diseases, including but not limited to autoimmune diseases, inflammation, cancer, tumors, and allergic reactions, which are caused by abnormalities in the aforementioned systems.

[0139] The oligonucleotides of the present invention may contain chemical modifications because the inhibitory effect on the TLR-containing targets shown in the present invention depends on the sequence of the oligonucleotide described as CxTy(CCT)nCm, where n represents an integer from 2 to 50, or preferably 5 to 16, x represents an integer 0 or 1, y represents an integer 0 (only when x = 0) or 1 (x can be 0 or 1), and m represents an integer of 0, 1, or 2 (Sun et al., Clin Immunol, 2010, 134, 262-276; Zhang et al., International Immunopharmacol, 2012, 446-453; EP2154144, WO2014 / 082254) and chemical modifications to improve the drug profile commonly used by those skilled in the art.

[0140] The oligonucleotides of this invention are partially phosphorothiolated at the internucleotide bonds, where non-bridging Os are replaced by S. As previously reported, oligonucleotides are unstable in vivo due to rapid degradation that can be caused by nucleases present in serum. Therefore, numerous attempts have been made to improve the stability of oligonucleotides, thereby allowing for longer half-lives in vivo, such as chemical modifications or the use of drug delivery tools to prevent attack by serum nucleases. Among such chemical modifications, phosphorothiolation of internucleotides has been utilized from the early stages of nucleic acid drug development. However, the toxicity of this phosphorothiolation is also well-known, posing a barrier to the development of nucleic acid drugs. Meanwhile, the oligonucleotides of this invention successfully reduce hepatotoxicity without the major challenges of activity and stability by reducing phosphorothiolate bonds with a specific pattern.

[0141] In one embodiment, the oligonucleotides of the present invention, in addition to the thiophosphorylation as described above, also contain various chemical modifications corresponding to those chemical residues in the phosphodiester internucleic bonds, in the sugars and / or nucleobases of the native nucleic acids (DNA or RNA).

[0142] Chemical modifications of oligonucleotides containing deoxyribonucleotides or ribonucleotides for pharmaceutical purposes have been extensively studied to date (Freier et al., Nuc Acid Res, 1997, 25, 4429-4443; Kuwahara et al., Molecules, 2010, 15, 5423-5444; Deleavey et al., Chemistry & Biology, 19, 937-954; Mutisya et al., Nucleic Acids Research, 2014, 42, 6542-6551; Sharma et al., Med. Chem. Commun. 2014, 5, 1454-1471).

[0143] Modifications to internucleotide bonds include, but are not limited to, thiophosphates, dithiophosphates, phosphonoacetate, methylphosphonate, methylthiophosphate, methyl phosphate, ethyl phosphate, ethylthiophosphate, boron phosphate, boron thiophosphate, methylborane phosphate, methylborane thiophosphate, methylborane phosphonate, phosphoramide, aminophosphate, diaminophosphate, aminothiophosphate, diaminothiophosphate, aminosulfonate, dimethylene sulfone, sulfonic acid, sulfide, sulfonate, methyleneimine, oxalyl, thioacetamide, methyl acetal, thiomethyl acetal, carboxylic acid ester, formamide, amide, triazolyl, imino and their derivatives.

[0144] Modifications to the sugar moiety include, but are not limited to, (a) introducing an electronegative atom / substituent at the 2'-position of the sugar moiety, (b) forming a bicyclic structure in the sugar moiety by fusing an additional ring, (c) modifications to the pentose ring structure, and (d) introducing a spirocyclic structure into the carbon atom of the sugar ring (Sharma et al., Med. Chem. Commun. 2014, 5, 1454-1471). Modifications to the sugar moiety also include converting nucleic acids into nucleic acid analogs, such as morpholino, peptide nucleic acids (PNA), locked / bridged nucleic acids (LNA, BNA), cyclohexenyl nucleic acids (HNA), and glycol nucleic acids and / or threononucleotides (TNA).

[0145] Modifications to the nucleobases include, but are not limited to: (a) modified pyrimidine bases, such as 5-propynyl U, 2-thioT, and N3-thioethylT, and (b) general modified bases, such as 3-nitropyrrole, imidazole-4-carboxamide, and 5-nitroindole. It may also include modifications to cytosine nucleoside bases to form cytosine analogs.

[0146] The above-mentioned chemical modifications in this invention can occur alone, or they can occur simultaneously with other modifications or with natural nucleic acids.

[0147] The oligonucleotides constituting the present invention can be obtained from existing nucleic acid sources (e.g., genomes or cDNA), but are preferably synthetic. The oligonucleotides of the present invention can be synthesized using various commercially available automated nucleic acid synthesizers. These oligonucleotides are referred to as synthetic oligonucleotides.

[0148] The oligonucleotides of the present invention can be single-stranded, double-stranded, or hybrids of single-stranded and double-stranded. They are also capable of forming cyclic structures linked at the 5' and 3' ends of a single molecule; more than one oligonucleotide of the present invention can be linked at the 5' or 3' ends by covalent bonding or by unique linkers to form tandemly linked elongated structures or multivalent structures. Linkers are organic chemical compounds commonly used by those skilled in the art, including but not limited to glycerol, (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis, cis-1,3,5-cyclohexanetriol, cis-trans-1,3,5-cyclohexanetriol, 1,3,5-tris-(2-hydroxyethyl)isocyanurate, tetraethylene glycol, and hexaethylene glycol, and may also be composed of amino acid nucleotides and / or derivatives thereof. The oligonucleotides in this invention preferably have a fully thiophosphorylated (CCT)3, (CTC)3, or (TCC)3 motif or a truncated form thereof linked by phosphodiester bonds in the core structure, which may be spaced in the middle by one or more nucleotides by alteration or insertion of nucleotides, but need to contain 2 to 50, or preferably 5 to 16, repeats of CCT, CTC, or TCC in a single molecule.

[0149] In one embodiment, the oligonucleotides of the present invention can be used in combination with each other, in combination with other oligonucleotides having similar functional mechanisms, or in combination with other organic compounds such as (poly)peptides / antibodies or nucleic acids / oligonucleotides, or inorganic compounds commonly used to improve or modify the physical properties of pharmaceuticals, such as cytotoxic agents. Such combinations may or may not be formed through covalent bonding.

[0150] “Oligonucleotide”: An oligonucleotide is a molecule consisting of multiple nucleotides (i.e., sugars (e.g., deoxyribose) linked to a phosphate ester group and an exchangeable organic base, where the organic base is a substituted pyrimidine (Py) (e.g., cytosine (C), thymine (T)) or a substituted purine (Pu) (e.g., adenine (A) or guanine (G)). As used herein, the term oligonucleotide primarily refers to oligodeoxyribonucleotides (ODNs), but is not limited to such terms if they function similarly.

[0151] "Chemical Modification": The oligonucleotides disclosed in this invention may include various chemical modifications in phosphodiester nucleoside bridges, ribosomes, and / or native nucleoside bases (cytosine and thymine). Modifications can occur during or after oligonucleotide synthesis. During synthesis, the modified bases can be incorporated internally or terminally. After synthesis, modifications can be made using active groups (via amino modifiers, via 3' or 5' hydroxyl groups, or via phosphate groups). Examples of chemical modifications are known to those skilled in the art. Oligonucleotides according to the invention may have one or more modifications, wherein each modification is located at a specific phosphodiester nucleoside bridge and / or a specific ribosome and / or a specific native nucleoside base position compared to an oligonucleotide of the same sequence composed of native DNA. Chemical modifications include "backbone modifications" of the oligonucleotides of the invention. As used herein, the modified backbone of the oligonucleotides of the invention includes, but is not limited to, a "thiophosphate backbone," which refers to a stable sugar-phosphate backbone of a nucleic acid molecule in which non-bridging phosphate ester oxygens are replaced by sulfur. Other backbone modifications refer to modifications using nonionic DNA analogs such as alkyl- and aryl-phosphonates (where the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiesters, and alkyl phosphotriesters (where the charged oxygen moiety is alkylated). Chemical modifications also include base substitutions of the oligonucleotides of the present invention. Substituted bases include, but are not limited to, cytosine, thymine, and other structurally related naturally occurring or non-naturally occurring nucleobases. Chemical modifications of the oligonucleotides of the present invention also include modifications to the bases of the oligonucleotides. Modified bases are chemically different from naturally occurring bases commonly found in DNA, such as T, C, G, and A, but they share a common basic chemical structure with these natural bases. The oligonucleotides of the present invention can be modified by using cytidine derivatives and / or thymidine derivatives. The term "cytidine derivative" refers to cytidine-like nucleotides (excluding cytidine), and the term "thymidine derivative" refers to thymidine-like nucleotides (excluding thymidine). Additionally, the oligonucleotides of the present invention can be chemically modified by attaching a diol, such as tetraethylene glycol or hexaethylene glycol, to either or both ends of the oligonucleotide.

[0152] "Immune-mediated disorders": Immune-mediated disorders are diseases caused by unwanted immune responses in a subject. Disorders include autoimmune diseases, graft rejection, hypersensitivity reactions, and diseases associated with microbial overstimulation of the host's immune system. Immune-mediated disorders can also be classified based on the physiological pathways affected by the disease, such as diseases related to TLR activation (TLR-mediated diseases, TLR9-mediated diseases, TLR7 and / or 8-mediated diseases), NF-κB-mediated diseases, interferon-mediated diseases, or inflammatory cytokine-mediated diseases. As described above, the oligonucleotides disclosed in this invention can be used as therapeutic agents for treating immune-mediated disorders.

[0153] "Immune response": The response of immune system cells, such as B cells, T cells, natural killer cells, dendritic cells, neutrophils, and macrophages, to stimuli. Responses include innate immune responses and adaptive (specific or acquired) immune responses. Adaptive (specific or acquired) immune responses include humoral immune responses and cellular immune responses.

[0154] "Prevention or treatment of immune-mediated diseases": As used in this article, prevention refers to stopping the full development of immune-mediated diseases in subjects; treatment refers to therapeutic interventions in subjects to improve the signs or symptoms of immune-mediated diseases, stop their progression, or eliminate the pathological condition.

[0155] "Subject": As used herein, a subject refers to a human or non-human vertebrate. Non-human vertebrates include non-human primates, livestock, and companion animals. The oligonucleotides of the present invention can be administered to prevent and / or treat immune-mediated conditions in subjects.

[0156] "Autoimmune Diseases": The term "autoimmune disease" refers to a disease caused by the disruption of self-tolerance, resulting in an adverse immune response to self-antigens and mediating cellular and tissue damage. Autoimmune diseases are often characterized by their involvement of a single organ or cell type, or multiple organs or tissue systems. Autoimmune diseases are also known as "collagen," "collagenous vessel," or "connective tissue" diseases. Autoimmune diseases are often associated with hypersensitivity reactions. The oligonucleotides of this invention can be used to treat and / or prevent various types of autoimmune diseases. Specifically, non-limiting examples of autoimmune diseases include systemic lupus erythematosus, insulin-dependent (type I) diabetes mellitus, inflammatory arthritis, rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, chronic invasive hepatitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, autoimmune atrophic gastritis with pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, Behçet's disease, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, polymyositis dermatomyositis, discoid lupus, sympathetic ophthalmia, basic mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barré syndrome, idiopathic pulmonary fibrosis, and idiopathic pulmonary fibrosis. Thrombocytopenic purpura, IgA nephropathy, juvenile arthritis, systemic sclerosis, polyarteritis nodosa, polychondritis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, hyperimmune globulin E syndrome, progressive systemic sclerosis, psoriasis, Leter syndrome, sarcoidosis, stiff-person syndrome, uveitis, vasculitis, vitiligo, Hashimoto's thyroiditis, pulmonary hemorrhage and nephritis, pernicious anemia, Addison's disease, Sjögren's syndrome, myasthenia gravis, Graves' disease, allergic encephalomyelitis, glomerulonephritis, microscopic polyangiitis, Wechsler's granulomatosis, autoimmune thyroid disease, juvenile idiopathic arthritis, giant cell arteritis, ulcerative colitis, Crohn's disease, etc. (Davidson, Engl) J Med, 2001, 345, 340-350; Delgado-Vega et al., Arthritis Research & Therapy 2010, 12 / S2). DNA or RNA released from DNA- or RNA-containing microorganisms can stimulate the production of autoantibodies specifically against their own DNA- or RNA-containing complexes, thus leading to many of the aforementioned autoimmune diseases, including but not limited to SLE.

[0157] "Graft rejection": Graft rejection is an immune-mediated condition caused by organ or tissue transplantation; transplantation means transferring a graft from a donor to a recipient. A graft is a living cell, tissue, or organ transplanted from a donor to a recipient. An autologous graft is a graft in which one's own tissue is transferred from one location to another; a syngeneic graft is a graft between identical twins; an allogeneic graft is a graft between genetically different members of the same species; and a xenogeneic graft is a graft between members of different species. When a subject is a recipient of an allogeneic or xenograft, the body can produce an immune response against the donor tissue. In such cases, it is clearly necessary to suppress the immune response to avoid graft rejection (Goldsby et al., Immunology, Fifth Edition, 2003, WHFREEMAN AND COMPANY). The oligonucleotides of the present invention are useful in preventing graft rejection. Examples of grafts are the heart, kidney, liver, medullary bone, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, small intestine, pancreatic islet cells, etc. In some cases, the recipient may be an animal as defined in the “subject” section of the present invention.

[0158] "Hypersensitivity reaction": Hypersensitivity reactions are conditions that cause tissue damage due to humoral or cellular-mediated reactions to endogenous or exogenous antigens, and have been classified into four categories. Type I hypersensitivity reactions (commonly referred to as anaphylactic, immediate, atopic, reaginic, IgE-mediated, or allergic reactions) are typically caused by the release of eosinophil chemokines (ECF) formed after pharmacologically active substances such as histamine, slow-reacting substances of anaphylaxis (SRS-A), and IgE-sensitized basophils and mast cells come into contact with specific exogenous antigens. Type I hypersensitivity reactions include, but are not limited to, allergic extrinsic asthma, seasonal allergic rhinitis, and systemic allergies. Type II hypersensitivity reactions (also known as cytotoxic, cytolytic complement-dependent, or cell-stimulating hypersensitivity reactions) occur when antibodies react with antigenic components of cells or tissue elements, or with antigens or haptens that have become tightly bound to cells or tissues. Type II hypersensitivity reactions include, but are not limited to, autoimmune hemolytic anemia, fetal erythroblastosis, and pulmonary hemorrhage with nephritis. Type III hypersensitivity reactions (also known as toxic complex, soluble complex, or immune complex hypersensitivity reactions) result from acute inflammatory reactions accompanying the deposition of soluble circulating antigen-antibody complexes in blood vessels or tissues. Type III hypersensitivity reactions include, but are not limited to, Artes' reaction, serum sickness, systemic lupus erythematosus, and certain types of glomerulonephritis. Type IV hypersensitivity reactions (commonly referred to as cellular, cell-mediated, delayed, or tuberculin-induced hypersensitivity reactions) result from sensitized T lymphocytes generated by contact with specific antigens. Type IV hypersensitivity reactions include, but are not limited to, contact dermatitis and allogeneic graft rejection (Goldsby et al., Immunology, Fifth Edition, 2003, WHFREEMAN AND COMPANY).

[0159] "Diseases Associated with Microbial Overstimulation of the Host Immune System": Microbial invasion, if severe, can sometimes trigger a systemic inflammatory response in subjects, leading to diseases associated with microbial overstimulation of the host immune system. For example, events in the development of diseases such as influenza A (H5N1) or bacterial infections include significantly elevated blood levels of TNFα, IL-1, IL-6, IL-12, IFNα, IFNβ, IFNγ, the chemokine IFN-induced protein 10, monocyte chemoattractant protein 1, IL-8, IL-1β, and monocyte chemoattractant protein 1. Such responses can lead to cytokine-mediated lethal shock, contributing in part to sepsis, ARDS, and multiple organ failure observed in many patients (The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A / H5, N Engl J Med, 2005, 353, 1374-85). A significant increase in blood cytokine levels following microbial infection is termed hypercytokinemia or a cytokine storm. This study indicates that patients infected with avian influenza or SARS require immunosuppressive drugs in addition to antiviral medications to suppress the elevated cytokine levels. Therefore, the oligonucleotides of this invention can be used to treat and / or prevent diseases associated with microbial stimulation of the host immune system in subjects. Disease-causing microorganisms include, but are not limited to, viruses, bacteria, fungi, parasites, and pathogens of spongiform encephalopathy. Viruses causing diseases associated with microbial overstimulation of the host immune system include SARS. Pathogens of CoV, influenza virus, avian influenza virus, HIV-1, poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus, equine encephalitis virus, rubella virus, dengue virus, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, Hantavirus, bungalow virus, sandfly virus, Nairo virus, hemorrhagic fever virus; reovirus, circovirus and rotavirus, hepatitis B virus, parvovirus, papillomavirus, polyomavirus, adenovirus, herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus, smallpox virus, vaccinia virus, poxvirus, African swine fever virus, spongiform encephalopathy, hepatitis D virus, hepatitis C virus, foot-and-mouth disease virus and avian influenza virus.Bacteria that can cause diseases related to microbial overstimulation of the host's immune system include: Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, certain genera of mycobacteria (such as Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium Kansas, Mycobacterium Gordonnae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Group A streptococci, Group B streptococci, streptococci, Enterococcus faecalis, Streptococcus bovis, streptococci (certain genera of anaerobic bacteria), Streptococcus pneumoniae, certain genera of pathogenic Karnofsky Performancey, certain genera of Enterococci, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, certain genera of Corynebacterium, Erysipelothrix rhusiopathiae, and Clostridium perfringens. Perfringers, Clostridium tetani, Enterobacter aerogeytes, Klebsiella pneumoniae, Pasteurella multocida, certain genera of Bacteroides, Fusobacterium nucleatum, Streptococcus moniliforme, Treponema pallidum, Treponema pallidum, Leptospira, and Actinobacter isatis. Fungi that can cause diseases related to microbial overstimulation of the host's immune system include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Parasites that can cause diseases related to microbial overstimulation of the host's immune system include Plasmodium falciparum and Toxoplasma gondii.

[0160] "Toll-like receptor (TLR) mediated diseases": Toll-like receptor (TLR) mediated diseases refer to immune-mediated conditions associated with the activation of TLR family members. These diseases include, but are not limited to, sepsis associated with lipopolysaccharide (LPS)-induced TLR4 activation, dilated cardiomyopathy associated with TLR2, 3, 4, and 9 activation, diabetes associated with TLR2, 3, 4, and 9 activation, experimental autoimmune encephalomyelitis associated with TLR3 activation, age-related macular degeneration associated with TLR3 activation (Patelet et al., Mol Cell Neurosci, 2014, 63, 38-48), systemic lupus erythematosus associated with TLR9 activation, atherosclerosis associated with TLR4 activation, asthma associated with LPS-induced TLR4 activation, chronic obstructive pulmonary disease associated with TLR4 activation, acute exacerbation syndrome (EAE) associated with TLR4 activation, and organ failure associated with TLR4 activation (Liew et al., Nature Review Immunology, 2005, 5, 446-458). CpG-containing DNA (TLR9 ​​agonists) derived from nucleic acid-containing infectious agents can be identified from SLE serum, which induces an effective immune response dominated by IFNa secretion, a process believed to promote SLE development. The oligonucleotides of this invention can be used to treat and / or prevent Toll-like receptor (TLR)-mediated diseases in subjects, including but not limited to SLE.

[0161] "TLR9-mediated diseases": The term "TLR-9-mediated diseases" refers to diseases involving uncontrolled TLR9 expression and / or activation of TLR9 signaling that are considered to be the cause of symptoms, and such diseases include, but are not limited to, systemic lupus erythematosus, IgA nephropathy, periodontitis, sclerosing cholangitis, multiple sclerosis, inflammatory bowel disease, graft-versus-host disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), fulminant type 1 diabetes mellitus (FT1D), end-stage renal disease, acute pancreatitis (AP), granulomatous disease with polyangiitis (GPA), xerophthalmia, Wescott-Aldrich syndrome, idiopathic pulmonary fibrosis, cystic fibrosis, chronic sinusitis, rheumatoid arthritis (RA), Sjögren's syndrome, pain, preterm birth, and preeclampsia.

[0162] "TLR7 and / or 8 mediated diseases": The term "TLR7 and / or 8 mediated diseases" refers to diseases involving uncontrolled overexpression of TLR7 and / or 8 and / or excessive activation of TLR7 and / or 8 signaling. These phenomena are considered good targets for treatment of diseases, including but not limited to systemic lupus erythematosus, lupus nephritis (Smith, Curr Opin Nehrol Hypertens, 2009, 18, 189), multiple sclerosis (Gambuzza et al., J Neuroimmunol, 2011, 239, 1-12), type 1 diabetes mellitus, rheumatoid arthritis (RA), Sjögren's syndrome, Behçet's syndrome (Hatemi et al., Clin Exp Rheumatol, 2015, 33(6Suppl 94), 3-14), and asthma (Tang et al., Respirology, 2015, Oct 18. doi:10.1111 / resp.12657. [Electronic publication prior to print edition]).

[0163] "NF-κB-mediated diseases": The term "NF-κB-mediated diseases" refers to diseases involving uncontrolled overactivation of NF-κB and affecting the expression of downstream genes. These include, but are not limited to, rheumatoid arthritis, gastritis and inflammatory bowel disease, allergies, headaches, pain, complex regional pain syndromes, cardiac hypertrophy, muscular dystrophy, muscle atrophy, catabolic diseases, type I diabetes, type II diabetes, fetal growth restriction, hypercholesterolemia, atherosclerosis, heart disease, chronic heart failure, ischemia / reperfusion, stroke, cerebral aneurysm, angina pectoris, lung diseases, cystic fibrosis, acid lung injury, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), hyaline membrane disease, nephropathy, glomerular diseases, alcoholic liver disease, leptospirosis nephropathy, intestinal diseases, peritoneal endometriosis, sinusitis, anhidrotic ectodermal dysplasia-ID, Behçet's disease, pigmentary disorders, tuberculosis, and asthma. Arthritis, Crohn's disease, eye allergies, glaucoma, appendicitis, Paget's disease, pancreatitis, periodontitis, endometriosis, inflammatory lung disease, sepsis, sleep apnea, antiphospholipid syndrome, lupus, lupus nephritis, chronic disease syndrome, familial Mediterranean fever, hereditary periodic fever syndrome, Parkinson's disease, multiple sclerosis, rheumatic diseases, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cataracts, chronic inflammatory demyelinating polyneuropathy, Helicobacter pylori-associated gastritis, systemic inflammatory response syndrome, and cancer (http: / / www.bu.edu / nf-kb / physiological-mediators / diseases / ; Tak et al., J Clin Invest, 2001, 107, 7-11).

[0164] "Interferon-mediated diseases": These are caused by excessive interferon production and / or overactivation of interferon downstream genes, including but not limited to systemic lupus erythematosus, psoriasis, Sjögren's syndrome, rheumatoid arthritis (RA), scleroderma, inflammatory arthritis, type 1 diabetes mellitus, inflammatory bowel disease, multiple sclerosis, Graves' disease, microscopic polyangiitis, Wechsler's granulomatosis, autoimmune thyroid disease, juvenile idiopathic arthritis, giant cell arteritis, ulcerative colitis, and Crohn's disease (Delgado-Vega et al., Arthritis Research & Therapy, 2010, 12 / S2).

[0165] "Inflammatory cytokine-mediated inflammatory diseases": These are induced by inflammatory cytokines such as TNFα, IFNα, IL-1, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), and / or IL-12, which excessively activate the immune system through downstream signaling pathways of these cytokines. Inflammatory cytokines can also activate the production of downstream prostaglandins, which are involved in pain formation during disease conditions. Diseases induced by these inflammatory cytokines exhibit inflammatory responses marked by signs such as swelling, redness, fever, pain, and loss of function. Chronic inflammation can also lead to fibrosis associated with wound healing responses, which can interfere with the function of normal tissues. Fibrosis includes, but is not limited to, pulmonary fibrosis, cirrhosis, cardiac fibrosis, and cystic fibrosis. Chronic inflammation can also cause granulomas, which are uncontrolled inflammatory masses composed of activated macrophages (epithelial-like cells) (Goldsby et al., Immunology, Fifth Edition, 2003, WHFREEMAN AND COMPANY).

[0166] “CpG ODN”: TLR9 agonists have been documented to activate innate and adaptive immune responses (Krieg, Nature Reviews Drug Discovery, 2006, 5, 471-484). CpG-containing oligonucleotides (CpG ODNs) are TLR9 agonists (Klinman, Nat. Rev. Immunol, 2004, 4, 249-258). Based on functional characteristics, CpG ODNs are classified into three types (Ito et al., Blood, 2006, 107, 2423-2431). Type A CpG ODNs activate human plasmacytoid dendritic cells (pDCs) to produce large amounts of type I IFN (IFNa / b) and strongly activate natural killer cells (NK cells). Type B CpG ODNs primarily activate B cells, leading to their proliferation and antibody secretion. Type C CpG ODNs possess the activities of both type A and type B CpG ODNs. As TLR9 agonists, CpG ODNs such as CpG 2216, CpG 2006, or CpG 2395 can be endocytosed into the cell compartments they are exposed to and activate TLR9. In pDCs, TLR9 activation initiates a rapid innate immune response, characterized by the secretion of pro-inflammatory cytokines (IL-6, TNFα), type I IFN, and IFN-induced chemokines. Innate immune cells, including natural killer (NK) cells, monocytes, and neutrophils, are secondary activated by pDCs via IFN-dependent and IFN-independent pathways. TLR9-activated B cells exhibit significantly increased sensitivity to antigenic stimulation and efficiently differentiate into antibody-secreting cells, thus promoting adaptive immune responses, particularly humoral immune responses. TLR9-activated pDCs secrete IFNa, which drives their migration and aggregation to lymph nodes and other secondary lymphoid tissues. These pDCs activate naive and memory T cells, assist in the cross-presentation of soluble protein antigens to CD8+ cytotoxic T lymphocytes (CTLs), and promote a strong TH1-biased CD4 and CD8 T cell response. Based on these findings, it is evident that agents antagonizing CpG ODN activity can be used to treat or prevent immune-mediated diseases by suppressing both innate and adaptive immune responses.

[0167] "Pharmaceutically acceptable carrier": A pharmaceutically acceptable carrier refers to one or more solid or liquid fillers, diluents, or encapsulating substances suitable for administering the oligonucleotides of the present invention to a subject. The carrier can be organic, inorganic, natural, or synthetic. Carriers include any and all solutions, diluents, solvents, dispersion media, liposomes, emulsions, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, as well as any other carriers suitable for administering the oligonucleotides of the present invention, and whose uses are well known in the art. The pharmaceutically acceptable carrier is selected depending on the specific mode of oligonucleotide administration. Parenteral preparations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, hydrated dextran, glycerol, etc., as a medium. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0168] "Therapeutic effective amount": This refers to the administration of a therapeutically effective amount of the oligonucleotides of the present invention to a subject for the treatment or prevention of an immune-mediated condition. One or more oligonucleotides in a "therapeutic effective amount" refer to a sufficient quantity of oligonucleotides to achieve the desired outcome of treating or preventing the immune-mediated condition in the subject. The oligonucleotides of the present invention can be utilized in pure form or in a pharmaceutically acceptable carrier. Alternatively, the oligonucleotides can be administered as a pharmaceutical composition. "Amount" in this invention refers to dosage. Dosage can be determined using standard techniques well known to those skilled in the art and can vary depending on factors including, but not limited to, the size of the subject and / or the severity of overall health or disease symptoms. The introduction of the oligonucleotides of the present invention can be performed as a single treatment or as part of a series of treatments. The dosage range for administration of the oligonucleotides of the present invention to a subject is from about 1 μg (microgram) to 10 g per administration. Preferably, the dosage range is from 0.1 mg to 5 g. More preferably, the dosage range is from 0.3 mg to 3 g. Most preferably, the dosage range is from 1 mg to 1 g. More preferred dosages can be adjusted by those skilled in the art, for example by an attending physician, within appropriate medical judgment to provide optimal therapeutic effects.

[0169] "Route of Administration": For clinical applications, the oligonucleotides of the present invention can be administered alone or formulated into pharmaceutical compositions via any suitable route of administration to effectively achieve the desired therapeutic outcome. The "route" of administration of the oligonucleotides of the present invention should refer to intravenous, parenteral, and topical application or inhalation. Intravenous routes of administration of the oligonucleotides of the present invention include oral, gastric, intestinal, and rectal routes. Parenteral routes include subcutaneous, intravenous, transdermal, sublingual, intranasal, transmucosal, pulmonary, vaginal, aerosol, intraocular, intratracheal, rectal, intraspinal, intramuscular, intra-articular, intraperitoneal, intracardiac, intraosseous, intrasheathal, intravitreal, inhalation, or topical application. Topical administration routes of the oligonucleotides of the present invention refer to external application of the oligonucleotides to the epidermis, mouth and ears, eyes, and nose.

[0170] "Pharmaceutical Composition": A pharmaceutical composition refers to a composition comprising a therapeutically effective amount of the oligonucleotides of the present invention and a pharmaceutically acceptable carrier, with or without. The pharmaceutical composition may comprise one or more oligonucleotides of the present invention. Compositions include, but are not limited to, aqueous or saline solutions, granules, aerosols, pills, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, and other pharmaceutical compositions suitable for various drug delivery systems. The composition may be administered parenterally, orally, rectally, vaginally, intraperitoneally, topically (in the form of powders, ointments, gels, drops, or transdermal patches), buccally, or as an oral or nasal spray. In all cases, the composition must be sterile and stable under preparation and storage conditions and protected against microbial contamination. Pharmaceutical compositions of the present invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersants, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injections or dispersions prior to use. The oligonucleotides of the present invention can be resuspended in an aqueous carrier, such as in an isotonic buffer solution with a pH of about 3.0 to about 8.0, preferably about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. The buffer solution includes sodium citrate-citric acid and sodium phosphate-phosphate, as well as sodium acetate-acetic acid buffer. For oral administration, the composition will be formulated with an edible carrier into powders, tablets, pills, lozenges, capsules, liquids, gels, syrups, pastes, suspensions, etc. For solid compositions, conventional non-toxic solid carriers may include pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. For buccal administration, the composition will be a conventionally prepared tablet or lozenge. For inhalation, the composition will be from pressurized packaging, aerosol sprays, or dry powder, and may be selected by those skilled in the art. In some cases, to prolong the effect of the oligonucleotides of the present invention, the oligonucleotides of the present invention are also suitable for administration via a sustained-release system. The oligonucleotides of the present invention are used in the form of liquid suspensions of poorly water-soluble crystals or amorphous materials to slow the release of the oligonucleotides. Alternatively, delayed release of parenteral oligonucleotide drug forms can be achieved by dissolving or suspending the oligonucleotides in a hydrophobic material (e.g., an acceptable oily medium). Injectable reservoir forms can be prepared by encapsulating the oligonucleotides in liposomes or microemulsions or other biodegradable semi-permeable polymer matrices such as polylactide-polyglycolic acid, poly(orthoester), and poly(anhydride).

[0171] "Active Ingredient": The oligonucleotides of the present invention can be used alone, in combination, in pharmaceutically acceptable carriers, or in combination with one or more other active ingredients. The administration of the oligonucleotides and other active ingredients of the present invention can be performed sequentially or simultaneously. Active ingredients include nonsteroidal anti-inflammatory agents, steroids, nonspecific immunosuppressants, biological response modifiers, chemical compounds, small molecules, nucleic acids, and TLR antagonists. Active ingredients also refer to agents that inhibit immune activation by antagonizing chemokines, by inducing the production of regulatory T cells (CD4+CD25+ T cells), by inhibiting complement, matrix metalloproteinases, and nitric oxide synthase, by blocking co-stimulatory factors, and by inhibiting signal transduction cascades in immune cells. Nonsteroidal anti-inflammatory agents include, but are not limited to, diclofenac, difluniform, etodoxacin, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprazin, piroxicam, sulindac, tohnetin, celecoxib, and rofecoxib. Steroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, and triamcinolone. Nonspecific immunosuppressants refer to agents used to prevent the development of immune-mediated diseases. Nonspecific immunosuppressants include, but are not limited to, cyclophosphamide, cyclosporine, methotrexate, steroids, FK506, tacrolimus, mycophenolic acid, and sirolimus. Biological response modulators include, but are not limited to, the following small molecules, recombinant proteins, or monoclonal antibody-targeting molecules:

[0172] i) Targeting IL-1: Anakinra and Linasep / Arcalyst

[0173] ii) Targeting TNFα signaling: etanercept / Enbrel, infliximab / Remicade, and golimumab / Simponi

[0174] iii) Targeting IL-6: Tocilizumab / Actemra, Sylvant / Sirukumab, and Olokizumab

[0175] iv) Targeting IFNa: sifalumab and rontalizumab

[0176] v) Targeting BAFF: belimumab / Benlysita, blisimod, and acecipaspirin

[0177] vi) Targeting IL-17: Secukinumab / Cosentyx, brodalumab / Lumicef, and ixekizumab / Talz

[0178] vii) Targeting IL-23 or IL-12 / 23: ustekinumab / Stelara, guselkumab, briakinumab and tildrakizumab

[0179] viii) Targeting JAK (Janus kinase): Ruxotinib / Jakafi and Tofacitinib / Xeljanz

[0180] The agents also include interferon beta-1a, IL-10, and TGFβ or their derivatives. They also include monoclonal antibodies / recombinant proteins for eliminating certain immune cells, such as B cells (rituximab / Rituxan, Eptratuzumab)) and proteins that inhibit lymphocyte activation, such as abatacept / Orencia, nastatin / Tysabri, and dacroizumab / Zimbryta.

[0181] "Delivery medium": The oligonucleotides of the present invention can be administered in a delivery medium, or in combination with a delivery medium, or in a form linked to a delivery medium. Delivery media include, but are not limited to, steroids (e.g., cholesterol), helices, milk bodies, ISCOM; lipids (e.g., cationic lipids, anionic lipids), liposomes; ethylene glycol (PEG); live bacterial vectors (e.g., Salmonella, Escherichia coli, BCG, Shigella, Lactobacillus), live viral vectors (e.g., vaccinia virus, adenovirus, herpes simplex virus), virions, virus-like particles, microspheres, nucleic acid vaccines, polymers (e.g., carboxymethyl cellulose, chitosan), polymer rings, and targeting agents that recognize target cells by specific receptors.

[0182] "Polyglycolization": Polyglycolization is the process of covalently linking a poly(ethylene glycol) polymer chain to another molecule (usually a drug or therapeutic protein). Polyglycolization is typically achieved by incubating a reactive derivative of PEG with the target agent. PEGylating agents can "mask" the agent from the host immune system and increase the hydrodynamic dimensions of the agent, thereby prolonging its circulation time. The oligonucleotides of this invention can be PEGylated.

[0183] Unless otherwise defined, all terms in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless the context otherwise indicates, the singular terms “a,” “an,” and “the” include plural referents. Similarly, unless the context otherwise indicates, the word “or” is intended to include “and.” In this specification, the term “few” refers to a number from 2 to 3. In this specification, the term “several” refers to a number from 2 to 6. In case of conflict, this application (including the interpretation of terms) shall prevail. Furthermore, the materials, methods, and examples are merely exemplary and not intended to be limiting. Treatment (treat, treating, or treatment) should have the same meaning without grammatical implications. Similarly, prevention (prevent, preventing, or prevention) should have the same meaning without grammatical implications.

[0184] Example

[0185] The invention will be described in more detail in the following embodiments. However, the invention is not limited to these embodiments. Unless otherwise stated, experiments using commercially available kits and reagents in these embodiments are performed according to the appended protocols. Those skilled in the art will understand that the oligonucleotides of the present invention can be readily applied to the treatment of immune-mediated conditions. The invention will now be demonstrated through the following non-limiting examples.

[0186] The TLR9-stimulating oligonucleotides used in the following examples are CpG2395 (5'-TCGTCGTTTTCGGCGCGCGCGCCg-3', SEQ ID NO.:43) and CpG2216 (5'-GGgggacgatcgtcGGGGGg-3', SEQ ID NO.:44), both of which are single-stranded DNA and synthesized at Hokkaido System Science Co. Ltd. (Sapporo, Japan). In the oligonucleotides, uppercase letters indicate phosphorylated thiophosphate nucleotides, while lowercase letters indicate nucleotides with a phosphodiester bond at their 3' end. TLR7 or 8 stimulation is achieved by adding Invivogen. All oligonucleotides of the present invention used in this example are also single-stranded DNA and synthesized at Hokkaido System Science Co. Ltd. (Sapporo, Japan). The oligonucleotides used in the examples are described as CxTy(CCT)nCm, where n is an integer from 2 to 50 or preferably from 5 to 16, x represents an integer 0 or 1, y represents an integer 0 (only when x = 0) or 1 (x can be 0 or 1), and m is 0, 1, or 2, and the internucleotide bonds are fully or partially phosphorylated. The sample oligonucleotides described in the examples are as follows:

[0187] CCT8PS (23 thiophosphate bonds): 5'-CCTCCTCCTCCTCCTCCTCCTCCt-3' (SEQ ID NO.:45),

[0188] CCT12PO (0 thiophosphate bonds):

[0189] 5'-cctcctcctcctcctcctcctcctcctcctcctcct-3'(SEQ ID NO.:15)

[0190] CCT12PS (35 thiophosphate bonds):

[0191] 5'-CCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCt-3' (SEQ ID NO.:46)

[0192] CCT12-1 (30 thiophosphate bonds):

[0193] 5'-CCTCCtCCTCCtCCTCCtCCTCCtCCTCCtCCTCCt-3' (SEQ ID NO.: 25)

[0194] CCT12-2 (32 thiophosphate bonds):

[0195] 5'-CCTCCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCt-3' (SEQ ID NO.: 26)

[0196] CCT12-3 (31 thiophosphate bonds):

[0197] 5'-CCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCtCCt-3' (SEQ ID NO.:27)

[0198] CCT12-4 (32 thiophosphate bonds):

[0199] 5'-CCTCCtCCTCTCCTCCtCCTCCTCCTCCtCCTCCt-3' (SEQ ID NO.:47)

[0200] CCT12-5 (31 thiophosphate bonds):

[0201] 5'-CCtCCTCCtCCTCCTCCTCCtCCTCCTCCtCCtCCt-3' (SEQ ID NO.:48),

[0202] TCC12PO (0 thiophosphate bonds):

[0203] 5'-tcctcctcctcctcctcctcctcctcctcctcctcc-3'(SEQ ID NO:20)

[0204] TCC12PS (35 thiophosphate bonds):

[0205] 5'-TCCTCCTCTCCTCCTCCTCCTCCTCCTCCTCCTCc-3' (SEQ ID NO: 49)

[0206] TCC12-6 (32 thiophosphate bonds):

[0207] 5'-TCCTCCTCcTCCTCCTCcTCCTCCTCcTCCTCCTCc-3' (SEQ ID NO.:40),

[0208] TCC12-7 (32 thiophosphate bonds):

[0209] 5'-TCCTCcTCCTCCTCCTCcTCCTCCTCCTCcTCCTCc-3' (SEQ ID NO.:50),

[0210] TCC6-1 (16 thiophosphate bonds): 5'-TCCTCCTCcTCCTCCTCc-3' (SEQ ID NO.:36),

[0211] In this context, uppercase letters indicate that the base is modified by thiophosphorylation at the 3' position within the phosphodiester nucleotide internucleotide bond, while lowercase letters indicate that the base is unmodified. All reagents used to manipulate oligonucleotides in the following examples are pyrogen-free.

[0212] Example 1

[0213] Compared to oligonucleotides with normal phosphodiester bonds or thiophosphate-esterified oligonucleotides, partially thiophosphate-esterified oligonucleotides exhibit higher stability.

[0214] <Experimental Methods>

[0215] Oligonucleotides (10 ng / µL) were incubated in distilled water (DW) with 20% (v / v) human serum (Cosmo Bio, Cat. 12181201) at 37°C. After 1 day of incubation, the solution containing the oligonucleotides was collected and the concentration of remaining undegraded oligonucleotides was examined. The concentration of oligonucleotides in the solution was determined by hybridization assay using a complementary oligonucleotide of (CCT)12 or (TCC)12 as a probe, which detects only oligonucleotides having the full-length oligonucleotide sequence (CCT)12 or (TCC)12.

[0216] <Experimental Results>

[0217] Figure 1 This indicates the percentage of intact oligonucleotides remaining after one day of incubation in serum-containing medium; this percentage is expressed as a percentage (%) compared to the oligonucleotide concentration at the start of the study. Figure 1 As shown in figure a, CCT12PO with normal phosphodiester internucleotide bonds showed almost complete degradation after 1 day of incubation, while fully thiophosphate-modified CCT12PS showed a considerably longer degradation duration, indicating that oligonucleotides degrade rapidly without internucleotide modifications. Figure 1 As shown in a, the non-thiophosphorylated oligonucleotide CCT12PO exhibited rapid degradation in the presence of serum in aqueous solution, while the fully or partially thiophosphorylated oligonucleotides (CCT12PS and CCT12-3) remained stable for more than 24 hours. Figure 1 b shows the stability of the partially phosphorothiolated (TCC)12 oligonucleotide. The results indicate that while the unphosphorothiolated oligonucleotide (TCC12PO) exhibited rapid degradation after 1 day of incubation, the partially phosphorothiolated oligonucleotide (TCC12-6) showed increased stability compared to the fully phosphorothiolated oligonucleotide (TCC12PS) with the same sequence.

[0218] Example 2

[0219] In vivo toxicity testing of oligonucleotides

[0220] <Experimental Methods>

[0221] Oligonucleotides were dissolved in saline at a concentration of 2 mg / ml and subcutaneously injected into mice (BALB / c) once daily at a dose of 100 μL / 10 g body weight for 7 consecutive days to achieve a dose of 20 mg / kg (mpk). Mouse body weight was examined to determine if the oligonucleotides showed any effect on body weight. Figures 2a to 2c In the graph, the X-axis represents the number of days since the first administration (i.e., day 0 is the first day of administration; day 6 is the last day of administration). The Y-axis displays the average body weight (3 animals per group), with the percentage set to 100% at the start of the study.

[0222] On the second day after the last administration (day 7), blood collected from mice was subjected to biochemical analysis to observe the effect on markers of liver damage, such as alanine aminotransferase (ALT). Figure 2d and 2e The concentration of blood ALT is displayed on the Y-axis. ALT is commonly measured clinically as part of the diagnostic assessment of hepatocellular injury to determine liver health. Typically, 10-40 IU / L is the standard reference range for any mammal, such as humans, cats, dogs, mice, rats, etc. The range of ALT can vary depending on factors such as the subject's age, sex, and race, and even in humans; therefore, the toxicity of the target compound is usually assessed by comparison with reference values ​​obtained from subjects with similar backgrounds. In this application, the determination of ALT was outsourced to the Nagahama Life Sciences Laboratory of Oriental Yeast Co., Ltd., and measurements were performed using the laboratory test kit L-type ALT J2 (Wako Pure Chemicals) according to the manufacturer's standard protocol. If the measured ALT value significantly exceeds that of animals given saline ( Figure 2d and 2e If the saline solution is used, the subject is diagnosed with liver toxicity.

[0223] <Experimental Results>

[0224] like Figure 2a As shown, mice given saline showed no weight loss, while mice given CCT8PS or CCT12PS showed significant weight loss, accompanied by the death of one animal in each administration group due to severe toxicity. Meanwhile, partially phosphorothiolated oligonucleotides exhibited variable behavior among them (from...). Figure 2a up to c), so that for CCT12-4 ( Figure 2b ), CCT12-5 and TCC12-7 ( Figure 2c The high toxicity is demonstrated by a sharp drop in body weight, while for CCT12-2 and CCT12-3... Figure 2b ), TCC6-1 and TCC12-6 ( Figure 2c No significant toxicity was observed. In the group of mice administered TCC12-7, one mouse died during administration, demonstrating strong toxicity. Interestingly, even oligonucleotides with the same thiophosphorylation ratios, such as CCT12-2 and CCT12-4, CCT12-3 and CCT12-5, and TCC12-6 and TCC12-7, showed differences in toxicity.

[0225] The mice underwent biochemical analysis one day after the last administration (day 7). Figure 2d and 2eMice with toxic oligonucleotides, such as fully phosphorothiolated CCT12PS or partially phosphorothiolated CCT12-4 or TCC12-7, showed a sharp increase in ALT. Meanwhile, some partially phosphorothiolated oligonucleotides (e.g., CCT12-1, CCT12-2, CCT12-3) also showed a sharp increase in ALT. Figure 2d ) and TCC12-6 ( Figure 2e The administration of the drug did not show an increase in ALT, indicating a lack of liver damage as in the saline control animals.

[0226] In summary, the results showed that toxicity was independent of the proportion or number of thiophosphate bonds in each molecule; among oligonucleotides with 12 CCT repeats of 32 thiophosphate bonds, CCT12-2 showed no toxicity while CCT12-4 showed strong toxicity; among oligonucleotides with 12 CCT repeats of 31 thiophosphate bonds, CCT12-3 showed no toxicity while CCT12-5 showed strong toxicity; and among oligonucleotides with 12 TCC repeats of 32 thiophosphate bonds, TCC12-6 showed no toxicity while TCC12-7 showed strong toxicity.

[0227] The common feature of all oligonucleotides with reduced toxicity is the structure of a fully thiophosphorylated segment of 3 CCTs or TCCs or a truncated segment linked by a phosphodiester bond, while toxic oligonucleotides typically have at least 4 fully thiophosphorylated segments of 4 CCTs or TCCs.

[0228] Furthermore, when calculated using dose conversion according to FDA guidelines based on body surface area, the 20 mg / kg dose examined in this embodiment can be considered equivalent to 1.62 mg / kg in humans (Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, USDapartment of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005), where a single dose is approximately 100 mg (at a body weight of 60 kg). Therefore, this study demonstrates that when the 100 mg oligonucleotide of the present invention is administered to humans, continuous daily administration of the 100 mg oligonucleotide of the present invention for at least one week is sufficiently tolerable, and also indicates that even higher doses may be permissible. The dosing schedule and dosage levels will be adjusted by a physician or other person skilled in the art.

[0229] Example 3

[0230] Inhibition of NF-κB transcriptional activity

[0231] <Experimental Methods>

[0232] A CAL-1 / NF-κB-GFP cell line was established to monitor the activity of NF-κB transcription factors in cell-based assays (WO2014 / 082254). A vector encoding a GFP reporter gene driven by a shared NF-κB transcriptional response element was transfected into a human plasma cell-like dendritic (DC) cell line, CAL-1 (human pDC cell line, Maeda et al., Int J Hematol., 2005, 81, 148-54, JP5011520). Transfected cells were further selected with antibiotics. Then, CAL-1 / NF-κB-GFP cells (1 × 10⁻⁶ cells) were... 5Cells were seeded in 96-well flat-bottom plates (Costar) and cultured with or without TLR agonists and oligonucleotides of the present invention. Cells were cultured at 37°C in a humidified incubator with 5% CO2 for 4 hours (TLR7 / 8) or 6 hours (TLR9). Changes in NF-κB transcriptional activity were indicated by the proportion of GFP-positive cells detected by flow cytometry (FACSCalibur, BD Bioscience Co., Ltd.) from cells set to 100% inhibitor-free medium. Figure 3a (Oligonucleotides, 0.1 μM) and 2 μg / ml Figure 3b and 3c Activation of downstream signaling in TLR7 / 8 was achieved by using (oligonucleotide, 0.33 μM) gademot and activation of downstream signaling in TLR9 was achieved by adding 1 μM of CpG2395.

[0233] <Experimental Results>

[0234] exist Figure 3a In the diagram, the horizontal axis represents the proportion of luminescence driven by active NF-κB compared to samples shown in cells treated solely with culture medium as 100% (culture medium). For example... Figure 3a As shown, partially phosphorothiolated oligonucleotides (CCT12-1, 2, 3, and 4) exhibited similar levels of repressive activity to the fully phosphorothiolated oligonucleotide CCT12-PS. Therefore, the oligonucleotides shown here do not show changes in repressive activity against NF-κB transcriptional activity regardless of the decreased level of phosphorothiolation at the internucleotide bonds. Figure 3b and Figure 3c Furthermore, it was shown that changes in the thiophosphorylation pattern did not affect the inhibitory effect, as revealed by the fact that oligonucleotides with the same thiophosphorylation ratio (CCT12-3 and CCT12-5; TCC12-6 and TCC12-7) did not show significant differences in inhibitory effect on TLR7 / 8 or TLR9 activation.

[0235] Example 4

[0236] Inhibition of cytokine production by oligonucleotides in CAL-1 cells

[0237] <Experimental Methods>

[0238] CAL-1 cells (at 1×10⁻⁶) 5 / well inoculation) with TLR agonist (gadermotide as a TLR7 / 8 agonist, 3ug / ml for Figure 4a Or 2ug / ml for Figure 4bAlternatively, CpG2395 (1 μM) as a TLR9 agonist was incubated at 37°C for 24 hours with or without an inhibitory oligonucleotide, and the supernatant was collected by centrifugation. Cytokines such as TNFα or IL-6 were then detected by ELISA targeting the concentration in the culture medium. ELISA was performed using the following kits according to the manufacturer's protocol: Human TNFα DuoSet ELISA (R&D Systems, Cat. Dy210), Human IL-6 DuoSet ELISA (R&D Systems, Cat. Dy206).

[0239] Figure 4a , 4b 4c depicts the levels of TNFα or IL-6 production as shown by the proportion of samples without inhibitory oligonucleotides, with the concentration of cytokines in the culture medium (containing samples without inhibitory oligonucleotides) set at 100%. Figure 4a In this study, oligonucleotides CCT12PS, CCT12-1, CCT12-2, CCT12-3, and CCT12-4 were used at 0.1 μM for both TLR7 / 8 and TLR9 agonists. Figure 4b and 4c In this study, oligonucleotides CCT12-3, CCT12-5, CCT12-6, and CCT12-7 were used at 0.037 μM for TLR7 / 8 agonists or at 0.011 μM for TLR9 agonists.

[0240] <Experimental Results>

[0241] like Figure 4a As shown, all oligonucleotides, including partially thiophosphorylated oligonucleotides (CCT12-1, 2, 3 and 4), exhibited similar levels of inhibition in cytokine production induced by TLR7 / 8 or TLR9, indicating that partial thiophosphorylation does not affect the inhibition of cytokine production. Figure 4b The results showed that when comparing between CCT12-3 and CCT12-5 or TCC12-6 and TCC12-7, the inhibitory effects of gademod or CpG2395 on TNFα and IL-6 production were not affected by changes in thiophosphorylation levels.

[0242] Example 5

[0243] Inhibitory effect of oligonucleotides on IFNa in human PBMCs

[0244] <Experimental Methods>

[0245] Human PBMCs collected from two healthy volunteer donors were incubated with the TLR9 agonist CpG2216 at 1 μM at 37°C for 24 hours with or without the inhibitory oligonucleotide, and IFNa in the supernatant was detected by ELISA.

[0246] According to the manufacturer's instructions, ELISA analysis of IFNa concentration was performed using the Human IFNa Module Set (Bender MedSystems, Cat.BMS216MST).

[0247] exist Figure 5 In a (0.03 μM oligonucleotide) and b (0.011 μM oligonucleotide), the IFNa level in each sample is shown as a proportion of IFNa produced using a TLR agonist but without an inhibitory oligonucleotide, and the average of the results from the two donors is plotted on the Y-axis.

[0248] <Experimental Results>

[0249] like Figure 5 As shown in figure a, IFNa production induced by TLR9 agonists was inhibited by any inhibitory oligonucleotide, whether toxic or non-toxic, revealing that a reduction in thiophosphorylation does not affect the inhibitory effect of oligonucleotides, even those with the same level of thiophosphorylation. The t-test results support no significant difference between CCT12PS and each partially thiophosphorylated oligonucleotide (i.e., CCT12-1, CCT12-2, CCT12-3, or CCT12-4; with p-values ​​of 0.3501, 0.5108, 0.3096, and 0.2922, respectively).

[0250] exist Figure 5 In c, the inhibition of IFNa production by TCC12 substances with different thiophosphorylation patterns was compared, and it was found that the thiophosphorylation pattern did not affect the activity of oligonucleotides, which was supported by the p-value calculated by the t-test. There was no significant difference between the results of TCC12-6 and TCC12-7 (p-value = 0.4541).

[0251] In summary, this invention provides oligonucleotides that are more stable yet less toxic than those of previous inventions for treating immune-mediated conditions. Interestingly, the oligonucleotides of this invention with low toxicity typically have a structure containing fully thiophosphorylated segments with three CCT or TCC repeats and / or truncated segments linked by phosphodiester bonds (e.g., CCT12-1, CCT12-2, CCT12-3, TCC12-6, and TCC6-1), while the oligonucleotides with high toxicity have larger (more than three) fully thiophosphorylated segments (e.g., CCT12-4, CCT12-5, TCC12-7, including CCT12PS and TCC12PS).

[0252] Therefore, compared with the prior art, the oligonucleotides in this application are effective in treating immune-mediated diseases because oligonucleotides have lower toxicity, thereby making it possible to increase the dosage regimen of pharmaceutical compositions containing said oligonucleotides. sequence list <110> SBI Biotechnology Co., Ltd. <120> TLR-inhibiting oligonucleotides and their uses <130> W7560-00 <150> JP 2015-255008 <151> 2015-12-25 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> (CCT)6 inhibitory oligonucleotide <400> 1 cctcctcctc ctcctcct 18 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> (CCT)7 inhibitory oligonucleotide <400> 2 cctcctcctc ctcctcctcc t 21 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> (CCT)8 inhibitory oligonucleotide <400> 3 cctcctcctc ctcctcctcc tcct 24 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> (CCT)8C inhibitory olionucleotide <400> 4 cctcctcctc ctcctcctcc tcctc 25 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> (CCT)8CC inhibitory oligonucleotide <400> 5 cctcctcctc ctcctcctcc tcctcc 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> (CCT)9 inhibitory oligonucleotide <400> 6 cctcctcctc ctcctcctcc tcctcct 27 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <400> 7 cctcctcctc ctcctcctcc tcctcctc 28 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <400> 8 cctcctcctc ctcctcctcc tcctcctcc 29 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> (CCT)10 inhibitory oligonucleotide <400> 9 cctcctcctc ctcctcctcc tcctcctcct 30 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> (CCT)10C inhibitory oligonucleotide <400> 10 cctcctcctc ctcctcctcc tcctcctcctc 31 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> (CCT)10CC inhibitory oligonucleotide <400> 11 cctcctcctc ctcctcctcc tcctcctcctcc 32 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> (CCT)11 inhibitory oligonucleotide <400> 12 cctcctcctc ctcctcctcc tcctcctcctcct 33 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> (CCT)11C inhibitory oligonucleotide <400> 13 cctcctcctc ctcctcctcc tcctcctcctcctc 34 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> (CCT)11CC inhibitory oligonucleotide <400> 14 cctcctcctc ctcctcctcc tcctcctcctcctcc 35 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> (CCT)12 inhibitory oligonucleotide <400> 15 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> (CCT)14 inhibitory oligonucleotide <400> 16 cctcctcctc ctcctcctcc tcctcctcctcctcctcctc ct 42 <210> 17 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> (CCT)16 inhibitory oligonucleotide <400> 17 cctcctcctc ctcctcctcc tcctcctcctcctcctcctc ctcctcct 48 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> (TCC)6 inhibitory oligonucleotide <400> 18 tcctcctcct cctcctcc 18 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> (TCC)9 inhibitory oligonucleotide <400> 19 tcctcctcct cctcctcctc ctcctcc 27 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> (TCC)12 inhibitory oligonucleotide <400> 20 tcctcctcct cctcctcctc ctcctcctcctcctcc 36 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <400> 21 cctcctcctc ctcctcct 18 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 22 cctcctcctc ctcctcctcc tcctcct 27 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 23 cctcctcctc ctcctcctcc tcctcct 27 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 24 cctcctcctc ctcctcctcc tcctcct 27 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 25 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (28)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 26 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonculeotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(32) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (34)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 27 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 28 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <400> 29 ctcctcctcc tcctcctc 18 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 30 ctcctcctcc tcctcctcct cctcctc 27 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 31 ctcctcctcc tcctcctcct cctcctc 27 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 32 ctcctcctcc tcctcctcct cctcctc 27 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (28)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 33 ctcctcctcc tcctcctcct cctcctcctcctcctc 36 <210> 34 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(32) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (34)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 34 ctcctcctcc tcctcctcct cctcctcctcctcctc 36 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 35 ctcctcctcc tcctcctcct cctcctcctcctcctc 36 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <400> 36 tcctcctcct cctcctcc 18 <210> 37 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 37 tcctcctcct cctcctcctc ctcctcc 27 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 38 tcctcctcct cctcctcctc ctcctcc 27 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <400> 39 tcctcctcct cctcctcctc ctcctcc 27 <210> 40 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(26) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (28)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 40 tcctcctcct cctcctcctc ctcctcctcctcctcc 36 <210> 41 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(14) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (16)..(23) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (25)..(32) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (34)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 41 tcctcctcct cctcctcctc ctcctcctcctcctcc 36 <210> 42 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(11) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (13)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 42 tcctcctcct cctcctcctc ctcctcctcctcctcc 36 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CpG2395 TLR9 stimulatory oligonucleotide <220> <221> modified_base <222> (1)..(21) <223> phosphorothioate-modified at 3' internucleotide bond <400> 43 tcgtcgtttt cggcgcgcgc cg 22 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpG2216 stimulatory oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (15)..(19) <223> phosphorothioate-modified at 3' internucleotide bond <400> 44 gggggacgat cgtcgggggg 20 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory oligonucleotide <220> <221> modified_base <222> (1)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <400> 45 cctcctcctc ctcctcct 18 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 46 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 47 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 47 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 48 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (4)..(8) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (10)..(20) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (22)..(32) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (34)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 48 cctcctcctc ctcctcctcc tcctcctcctcctcct 36 <210> 49 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 49 tcctcctcct cctcctcctc ctcctcctcctcctcc 36 <210> 50 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Inhibitory Oligonucleotide <220> <221> modified_base <222> (1)..(5) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (7)..(17) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (19)..(29) <223> phosphorothioate-modified at 3' internucleotide bond <220> <221> modified_base <222> (31)..(35) <223> phosphorothioate-modified at 3' internucleotide bond <400> 50 tcctcctcct cctcctcctc ctcctcctcctcctcc 36

Claims

1. A partially phosphorothiolated oligonucleotide, selected from the group consisting of: The oligonucleotide represented by 5'-CCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCtCCTCCTCCt-3' (SEQ ID NO:27) The oligonucleotide represented by 5'-TCCTCCTCcTCCTCCTCc-3' (SEQ ID NO:36), and The oligonucleotide shown in SEQ ID NO:40 is 5'-TCCTCCTCcTCCTCCTCcTCCTCCTCcTCCTCCTCc-3'. The uppercase letters indicate nucleotides modified with thiophosphate at the 3' position in the phosphodiester nucleotide internucleotide bond, while the lowercase letters indicate unmodified nucleotides.

2. The oligonucleotide of claim 1, wherein the oligonucleotide constitutes part of another DNA molecule, plasmid, or viral vector.

3. The oligonucleotide of claim 1, wherein the oligonucleotide comprises chemical modifications other than thiophosphorylation.

4. The oligonucleotide of claim 1, wherein the oligonucleotide is polyethylene glycolated.

5. The oligonucleotide of any one of claims 1 to 4 for in vitro use in inhibiting interferon production induced by Toll-like receptor (TLR) agonists.

6. The oligonucleotide of any one of claims 1 to 4 for in vitro use in inhibiting the production of pro-inflammatory cytokines induced by Toll-like receptor (TLR) agonists.

Images