Bispecific nucleic acid

Abstract

The present invention provides a bispecific nucleic acid molecule that includes an antisense strand that has a sequence that specifically binds CHST2 and also specifically binds CHST4.

Description

bispecific nucleic acids

[0001] The present invention relates to bispecific nucleic acid molecules, in particular nucleic acid molecules having dual specificity for sugar sulfotransferases CHST2 and CHST4, pharmaceutical compositions containing the same, and uses thereof.

[0002] Chronic inflammation is a persistent and often irreversible response to various external insults, resulting in tissue destruction, functional impairment, and progressive clinical symptoms in all organs throughout the body. Chronic inflammation is characterized by the infiltration of leukocytes, which control inflammation and immunity, and is thought to be a major cause of the exacerbation and persistence of functionally impaired lesions. Inflammation originates in capillaries, and the emergence of distinctive vascular endothelial cells at the site of inflammation has been observed and reported, particularly in chronic inflammation. Under physiological conditions, these cells are expressed only in lymphoid tissues, such as lymph nodes and the spleen, and are high endothelial venules (HEVs), which serve as the entry point for lymphocyte "homing" (the migration of lymphocytes from blood vessels to lymphoid tissues during lymphocyte recirculation). Since it has been reported in many chronic inflammatory and allergic diseases such as bronchial asthma, allergic rhinitis, contact dermatitis, rheumatoid arthritis, and inflammatory bowel disease, it is considered to be a phenomenon that is not specific to an organ or disease, but is induced in any tissue and is common to chronic inflammation. Therefore, it has been suggested that it is involved in the recruitment of leukocytes to infiltrated foci in chronic inflammatory and allergic diseases, the pathogenesis of chronic foci, functional disorders, and clinical symptoms, and it is expected to be applied to the development of new treatments (References 1-6).

[0003] The endothelial cells of HEV contain sialyl 6-sulfo Lewis X (S-sLe x ) is expressed, which is a ligand for L-selectin (CD62L) on the surface of leukocytes. For the purpose of treating chronic inflammation, it is necessary to bind L-selectin on the leukocyte side or S-sLe on the HEV side. xResearch and development has been conducted to attempt to suppress inflammatory cell infiltration by inhibiting L-selectin, but no inhibitors have yet been successfully applied in clinical practice. Antibodies such as MEL-14, which inhibit L-selectin on leukocytes, have been developed and shown in preclinical studies to inhibit the "rolling" function, an early stage of cell extravasation, and thus inhibit cell infiltration. Antibody drugs, administered via the systemic circulation (bloodstream), are particularly effective because they can directly affect circulating leukocytes rather than vascular endothelial cells distributed in tissues. However, because naive lymphocytes are the major cell population expressing L-selectin, inhibiting their circulation is thought to cause severe immune dysfunction. Systemically administered drugs, including antibody drugs, have safety issues, and approaches targeting HEV ligand molecules have been difficult to commercialize (References 1-8).

[0004] On the other hand, approaches that inhibit HEV molecules have the advantage of fewer safety concerns, especially in chronic inflammation, given the phenomenon of de novo HEV-like blood vessels appearing at the site of inflammation. The MECA-79 antibody has been identified as an antibody that recognizes HEV. This antibody recognizes peripheral node addression (PNAd), a sialomucin glycoprotein expressed in HEV, and is the most widely used antibody for detecting HEV by immunohistological staining (References 9-11). The antigenic determinant of the MECA-79 antibody is an O-linked glycan attached to the core protein (CD34, Podpcalyxin, GlyCAM-1, Endomucin, and Nepmucin have been identified so far), and contains an S-sLeu complex in the core 1 structure (N-acetylgalactosamine (GalNAc) residue + galactose (Gal) residue). xThe structure is an addition of the MECA-79 antibody (Figure 1). In non-clinical trials, the MECA-79 antibody has been shown to have an inhibitory effect on leukocyte infiltration and a therapeutic effect in an asthma model, suggesting the possibility of targeting PNAd for therapeutic purposes. However, as with many antibodies against glycans, the MECA-79 antibody is an IgM subclass, and although it is useful as a research tool and for diagnostics, it is not suitable for formulation or clinical use as a therapeutic agent. In addition, the S-sLe x Although antibodies that recognize the structure have been reported to be effective as research tools and in animal experimental models, the glycan structure and attachment state do not necessarily match between humans and animals, so there is a high possibility that the efficacy of the drug will be very heterogeneous. x Since the structure is also expressed on the surface of leukocytes, safety concerns remain, similar to those of antibodies against L-selectin, and it is thought that it is not suitable for clinical application as a pharmaceutical (References 1-17).

[0005] In response to this issue, the biosynthetic pathway of PNAd (L-selectin ligand glycan) has become clearer (Fig. 1). Effectively inhibiting any step in this process could potentially inhibit PNAd synthesis, particularly the de novo synthesis of PNAds that emerges during inflammation. PNAd synthesis involves four major steps: core 1 extension, core 2 branching, sulfation, sialylation, and fucosylation. Specific enzymes responsible for the addition of sugar chains at each step have been identified (underlined in Fig. 1). Previous studies have revealed that the sulfotransferase involved in sulfation, Carbohydrate sulfotransferase 2 (CHST2, also known as N-acetylglucosamine-6-sulfotransferase 1, GlcNAc6ST-1), is responsible for the 6-sulfation of GlcNAc residues on the core 2 branch, while CHST4 (also known as GlcNAc6ST-2) is responsible for the 6-sulfation of both core 1 extension and core 2 branch (References 9-11). Analysis of knockout mice for these genes revealed that simultaneous knockdown of both genes, but not just one of them, strongly suppressed MECA-79 expression and lymphocyte infiltration (References 1, 4, 5, 17).

[0006] In recent years, bispecific antibodies have been actively developed as bispecific inhibitors, and have already been put to clinical use. However, CHST2 and CHST4 are Golgi proteins, and extracellular administration of antibodies (anti-CHST2 and anti-CHST4 antibodies) via the bloodstream or other routes does not exert neutralizing activity, making antibody therapy impractical. Furthermore, the creation of small molecule inhibitors of glycosyltransferases has been extremely challenging. It has been impossible to obtain compounds with high specificity, even at IC50 levels comparable to those of small molecules typically used in clinical trials, making it difficult to advance them to the development stage as drug candidates. Given this situation, the inventors have pursued nucleic acid drug approaches, particularly those targeting nucleic acids (RNAs) using single-stranded RNA antisense oligonucleotides (ASOs) or double-stranded RNA siRNAs (Refs. 4, 5, 18, 19).

[0007] Nucleic acid drugs (ASOs and siRNAs) exert their effects by specifically binding to specific base sequences in target mRNAs. They have emerged as highly specific new drug modalities and are rapidly being commercialized in clinical settings. Because they can target molecules that could not be targeted by conventional small molecule compounds or antibody drugs, they hold great promise for therapeutically targeting a wide range of disease-related molecules. The concept of designing a highly specific nucleic acid drug that simultaneously binds to two mRNAs with a single nucleic acid drug has traditionally been unheard of in the medical and biological community, as it violates the Watson-Crick principle (Ref. 20). However, as the popularity of bispecific antibodies demonstrates, there are many conditions in which targeting two distinct mRNAs may be more effective. Therefore, to target two or more distinct mRNAs, basic research has been conducted on the use of nucleic acid drug cocktails, such as the administration of two distinct siRNAs (Refs. 18, 19). Moving forward with clinical practical application of cocktails will not only entail increased costs due to the simple increase in the number of bases, but also a synergistic increase in development costs due to the increase in manufacturing processes and toxicity testing items, as well as an increased risk of discontinuation due to toxicity.Furthermore, since it is realistically difficult for the compounds to be taken up into the same cells at exactly the same time in the body, there is a high risk of instability and uncertainty in drug efficacy.

[0008] PCT / US93 / 11612PCT / US95 / 05463PCT / US2020 / 017879PCT / KR2021 / 003488

[0009] 1) Blanchard L, Girard JP. High endothelial venules (HEVs) in immunity, inflammation and cancer. Angiogenesis. 24:719-753. 2021. 2) Kogame T, Kabashima K, Egawa G. Putative Immunological Functions of Inducible Skin-Associated Lymphoid Tissue in the Context of Mucosa-Associated Lymphoid Tissue. Front Immunol. 12:733484. 2021. 3) Yoneyama H, Matsuno K, Zhang Y, Murai M, Itakura M, Ishikawa S, Hasegawa G, Naito M, Asakura H, Matsushima K. Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease. J Exp Med. 193: 35-49, 2001. 4) Kawashima H. ​​Roles of sulfated glycans in lymphocyte homing. Biol Pharm Bull. 29: 2343-2349, 2006. 5) Uchimura K. Cell surface sulfated glycans recognized by the "homing receptor" L-selectin. Seikagaku 85: 244-252, 2013. 6) van Zante A, Rosen SD. Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation. Biochem Soc Trans. 31: 313-3187, 2003. 7) Ley K.The role of selectins in inflammation and disease. Trends Mol Med. 9: 263-268, 2003. 8) Smith BAH, Bertozzi CR. The clinical impact of glycobiology: targeting selectins, Siglecs and mammalian glycans. Nat Rev Drug Discov. 20: 217-243, 2021. 9) Streeter PR, Rouse BT, Butcher EC. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J Cell Biol. 107: 1853-1862, 1998. 10)Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase. Cell. 105: 957-969, 2001. 11)Michie SA, Streeter PR, Bolt PA, Butcher EC, Picker LJ. The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing. Am J Pathol. 143: 1688-1698, 1993. 12)Lechleitner S, Kunstfeld R, Messeritsch-Fanta C, Wolff K, Petzelbauer P.Peripheral lymph node addressins are expressed on skin endothelial cells. J Invest Dermatol. 113: 410-414, 1999. 13)Yang J, Rosen SD, Bendele P, Hemmerich S. Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis. BMC Immunol. 7:12, 2006. 14)Rosen SD, Tsay D, Singer MS, Hemmerich S, Abraham WM. Therapeutic targeting of endothelial ligands for L-selectin (PNAd) in a sheep model of asthma. Am J Pathol. 166: 935-944, 2005. 15)Matsumura R, Hirakawa J, Sato K, Ikeda T, Nagai M, Fukuda M, Imai Y, Kawashima H. Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses. J Biol Chem. 290: 15313-15326, 2015. 16)Xiong W, Liu W, Nishida S, Komiyama D, Liu W, Hirakawa J, Kawashima H. Therapeutic Effects of an Anti-sialyl Lewis X Antibody in a Murine Model of Allergic Asthma. Int J Mol Sci. 22: 9961, 2021.17) Ohmichi Y, Hirakawa J, Imai Y, Fukuda M, Kawashima H. ​​Essential role of peripheral node addressin in lymphocyte homing to nasal-associated lymphoid tissues and allergic immune responses. J Exp Med. ;208: 1015-1025, 2011. 18) Friedrich M, Aigner A. Therapeutic siRNA: State-of-the-Art and Future Perspectives. BioDrugs. 36: 549-571, 2022. 19) Michael FM, Chandran P, Chandramohan K, Iyer K, Jayaraj K, Sundaramoorthy R, Venkatachalam S. Prospects of siRNA cocktails as tools for modifying multiple gene targets in the injured spinal cord. Exp Biol Med (Maywood). 244: 1096-1110, 2019. 20) Kondo J, Westhof E. Base pairs and pseudo pairs observed in RNA-ligand complexes. J Mol Recognit. 23: 241-252, 2010.

[0010] It has been difficult to inhibit the infiltration of leukocytes that form lesions, particularly chronic lesions, in various inflammatory or allergic diseases. However, an objective of the present invention is to provide a novel treatment method and therapeutic agent that controls the infiltration of leukocytes and improves lesions by inhibiting the HEV-like blood vessels that appear locally with inflammation.

[0011] The present inventors attempted to create a nucleic acid compound (bispecific nucleic acid) that simultaneously hybridizes to both human CHST2 mRNA and CHST4 mRNA, which are molecules responsible for the sulfation process in PNAd synthesis, in order to inhibit the synthesis of glycoprotein structures characteristic of HEV-like blood vessels. As a result, they found that by substituting artificial bases for the base sequence of the nucleic acid compound, rather than the original C (cytidylic acid), A (adenylic acid), U (uridylic acid), and G (guanylic acid), it was possible to design a nucleic acid compound that hybridizes to both CHST2 mRNA and CHST4 mRNA. Furthermore, because the sequences of CHST2 mRNA and CHST4 mRNA targeted by the nucleic acid compound of the present invention (the portions to which the nucleic acid compound binds) are identical in humans, mice, rats, monkeys, dogs, and cats, it was possible to design a nucleic acid compound without considering differences between these species, especially between mammalian species.

[0012] More specifically, the present invention provides the following [1] to

[10] . [1] A bispecific nucleic acid molecule comprising an antisense strand having a sequence that specifically binds to CHST2 and also specifically binds to CHST4. [2] The bispecific nucleic acid molecule of [1], which is an siRNA or an antisense oligonucleotide. [3] The bispecific nucleic acid molecule of [2], which is an siRNA having the sequence set forth in SEQ ID NOs: 5 and 6, 7 and 8, 9 and 10, 11 and 12, or 13 and 14, or an antisense oligonucleotide having any of SEQ ID NOs: 25 to 29. [4] The bispecific nucleic acid molecule of [2] or [3], which comprises an unnatural base. [5] The bispecific nucleic acid molecule of [4], which comprises a chemically modified unnatural base. [6] A pharmaceutical composition comprising the bispecific nucleic acid molecule of any of [1] to [5]. [7] The pharmaceutical composition of [6], for preventing or treating an inflammatory or allergic disease. [8] Use of the bispecific nucleic acid molecule of any one of [1] to [5] for the manufacture of a pharmaceutical composition for preventing or treating an inflammatory or allergic disease. [9] A method for preventing or treating an inflammatory or allergic disease in a patient, comprising administering the pharmaceutical composition of [6] or [7] to a patient in need of such treatment.

[0013] The bispecific nucleic acid molecules of the present invention can be artificially synthesized. The inventors have demonstrated that in human cells, the bispecific nucleic acid molecules of the present invention dose-dependently suppress the expression of CHST2 mRNA and CHST4 mRNA, and also suppress the expression of MECA-79, which recognizes the glycan structure of PNAd. Notably, although the bispecific nucleic acid molecules of the present invention contain unnatural bases, they were able to suppress dual targets without inducing nonspecific inflammatory responses such as interferon responses or TLR responses. Furthermore, in a mouse atopic dermatitis model, administration of the bispecific nucleic acid molecules of the present invention drastically reduced MECA-79-positive HEV-like vessels and significantly suppressed the associated inflammatory lymphocyte infiltration, demonstrating their biological activity.

[0014] The present invention provides a novel therapeutic approach to the formation of leukocyte-infiltrated lesions, which are a characteristic feature of various chronic inflammatory and allergic diseases and are the basis of their intractability, by safely inhibiting the localized appearance of HEV-like vessels associated with inflammation, thereby improving the lesions. Furthermore, while simultaneous inhibition of CHST2 and CHST4, which are molecules that synthesize HEV-like vessels, in the same cells is essential for inhibiting HEV-like vessel synthesis, the present nucleic acid compound is a bispecific nucleic acid that simultaneously inhibits CHST2 and CHST4, thereby providing a novel treatment that ameliorates inflammation by inhibiting the localized appearance of HEV-like vessels.

[0015] The synthesis process of the MECA-79 antibody antigenic determinant (sialomucin glycoprotein: PNAd) and glycosyltransferase (green) are shown. The MECA-79 epitope (PNAd) is an O-linked glycan attached to a core protein ("R" in the figure: CD34, Podpcalyxin, GlyCAM-1, Endomucin, and Nepmucin have been identified so far). The core 1 structure (N-acetylgalactosamine (GalNAc) residue + galactose (Gal) residue) is linked to S-sLe. xThis structure is a structure with the core 1 structure (boxed in the figure) added. The synthesis involves four major steps: elongation of the core 1 structure, branching of the core 2 structure, sulfation, sialylation, and fucosylation. Specific enzymes that add sugar chains at each step have been identified (underlined). The enzyme that elongates the core 1 structure (Core 1) by transferring N-acetylglucosamine (GlcNAc: square) is Core 1 b1-3GlucNacT, and the enzyme that produces the core 2 branch is Core 2 GlcNAcT (top center of the figure). In the next sulfation step (S: oval), CHST2 6-sulfates the GlcNAc residue on the core 2 branch, and CHST4 6-sulfates both the core 1 elongation and the core 2 branch (bottom left of the figure). The enzyme that adds galactose (Gal: circle) to sulfated N-acetylglucosamine (GlcNAc: square) is b4-GalT, and the enzyme that sialylates the galactose residue (sialic acid: diamond transfer) is STGal (bottom center of the figure). Finally, the enzyme undergoes fucosylation (fucose: triangle transfer) to form S-sLe xSynthesis of the structure is completed (bottom right). The enzymes involved in this step are FucT-IV and FucT-VII, with FucT-VII presumed to be more involved. This figure shows the knockdown effect of the target mRNA (CHST2 mRNA and CHST4 mRNA) of the dual-specific nucleic acids (10 nucleic acids, DS01 to DS10) for human CHST2 or CHST4. The stability of the binding complex between the antisense strand (single-stranded RNA, SS06 to SS10) of DS06 to 10 and human CHST2 mRNA is shown by the double-helix melting temperature (Tm). The stability of the binding complex between the antisense strand (single-stranded RNA, SS06 to SS10) of DS06 to 10 and human CHST4 mRNA is shown by the double-helix melting temperature (Tm). Figure 1 shows the dose-dependent knockdown effect of bispecific nucleic acids (DS06 and DS10) against human CHST2 and CHST4 on target mRNA (CHST2 mRNA and CHST4 mRNA). Control: Vehicle (saline) was used as a negative control, and PC: A cocktail of CHST2 siRNA and CHST4 siRNA was used as a positive control. Figure 2 shows the dose-dependent inhibitory effect of bispecific nucleic acids (DS06 and DS10) against human CHST2 and CHST4 on MECA-79 expression. Control: Vehicle (saline) was used as a negative control, and PC: A cocktail of CHST2 siRNA and CHST4 siRNA was used as a positive control. Figure 3 shows the effect of bispecific nucleic acids (DS06) against human CHST2 and CHST4 on the expression of interferon-responsive genes (IFITM1). Poly I:C is a positive control that induces interferon responses. Figure 4 shows the effect of bispecific nucleic acids (DS06) against human CHST2 and CHST4 on TLR responses. Control (positive control): Substance that induces each TLR response. The cumulative total amount of nucleic acid that penetrated the artificial skin over time using DS06 is shown. The effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis is shown.NC / Nga mice (7-week-old, male) were administered mite antigen to the ear pinna to induce dermatitis, and then DS-06-containing cream, a negative control cream, and tacrolimus ointment as a positive control were applied once daily for 20 days from days 11 to 30. The mice were sacrificed on day 31 and their skin lesions were evaluated. HE staining findings. Figure 1 shows the effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (HEV-like blood vessels). Figure 2 shows the effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (T lymphocytes). Figure 3 shows the effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (activated T lymphocytes). Figure 4 shows the effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (regulatory T lymphocytes). 1 shows the effects of a bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (IL13, IL-33). 2 shows the effects of a bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse allergic rhinitis (HEV-like vessels and T lymphocytes).

[0016] The present invention will be described in detail below.

[0017] The present invention relates to bispecific nucleic acid molecules comprising an antisense strand having a sequence that specifically binds to CHST2 and also specifically binds to CHST4.

[0018] The CHST2 gene and CSHT4 gene of the present invention are not particularly limited, but are typically derived from animals, more preferably from mammals, and most preferably from humans. The CHST2 of the present invention is also known as GlcNAc6ST-1 (N-acetylglucosamine 6-O sulfotransferase-1), and the CHST4 of the present invention is also known as GlcNAc6ST-2 (N-acetylglucosamine 6-O sulfotransferase-2).

[0019] The sequences of CHST2 (GlcNAc6ST1) and CHST4 (GlcNAc6ST-2) of the present invention can be obtained, for example, based on accession numbers NM_004267.4 and NM_001166395.1. For example, the nucleotide sequence of the CHST2 gene of the present invention is set forth in SEQ ID NO: 1, and the amino acid sequence encoded by the gene is set forth in SEQ ID NO: 2. The nucleotide sequence of the CHST4 gene is set forth in SEQ ID NO: 3, and the amino acid sequence encoded by the gene is set forth in SEQ ID NO: 4. Proteins consisting of amino acid sequences other than those listed above are also included in the CHST2 protein and CHST4 protein of the present invention, as long as they share a high identity, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, with the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and have the functions of the above proteins. Furthermore, the CHST2 protein or CHST4 protein of the present invention may be, for example, a protein consisting of an amino acid sequence in which one or more amino acids have been added, deleted, substituted, or inserted in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and the number of amino acids that have changed may usually be 30 amino acids or less, preferably 10 amino acids or less, more preferably 5 amino acids or less, and most preferably 3 amino acids or less.

[0020] The CHST2 gene or CHST4 gene of the present invention includes, for example, endogenous genes in other organisms corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 (e.g., homologues of the above human genes). Furthermore, endogenous DNA in other organisms corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 generally has a high identity (homology) to the DNA set forth in SEQ ID NO: 1 or SEQ ID NO: 3, respectively. High identity preferably means a homology of 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, or even 96%, 97%, 98%, or 99% or more). This homology can be determined using the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). Furthermore, when isolated from a living organism, the DNA is believed to hybridize under stringent conditions with the DNA shown in SEQ ID NO: 1 or SEQ ID NO: 3. Here, "stringent conditions" include, for example, "2xSSC, 0.1% SDS, 50°C," "2xSSC, 0.1% SDS, 42°C," and "1xSSC, 0.1% SDS, 37°C," and, as more stringent conditions, "2xSSC, 0.1% SDS, 65°C," "0.5xSSC, 0.1% SDS, 42°C," and "0.2xSSC, 0.1% SDS, 65°C."

[0021] The bispecific nucleic acid molecules of the present invention may be, for example, but are not limited to, siRNA, antisense oligonucleotide, miRNA, or decoy.

[0022] In the siRNA of the present invention, not all nucleotides need to be ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides, as long as the molecule itself has the function of suppressing the expression of the CHST2 gene and the CHST4 gene. This "corresponding" refers to the same base type (adenine, guanine, cytosine, thymine (uracil)) although the sugar moiety structure is different. For example, a deoxyribonucleotide corresponding to a ribonucleotide containing adenine refers to a deoxyribonucleotide containing adenine. Furthermore, the term "multiple" is not particularly limited, but preferably refers to a small number of about 2 to 5.

[0023] Those skilled in the art can prepare the siRNA of the present invention using a commercially available nucleic acid synthesizer. Alternatively, the desired RNA can be synthesized using a general synthesis service.

[0024] In this specification, "an siRNA that suppresses the expression of the CHST2 gene" can also be expressed as "CHST2 siRNA," and "an siRNA that suppresses the expression of the CHST4 gene" can also be expressed as "CHST4 siRNA."

[0025] The bispecific nucleic acid molecules of the present invention can be, but are not limited to, siRNA or antisense oligonucleotides, preferably having any of the following sequences:

[0026] siRNA (DS01 to DS05): In one embodiment, the bispecific nucleic acid molecule of the present invention may be an siRNA consisting of the antisense strand and the sense strand shown in the following SEQ ID NOS: 5 to 24. The double-underlined bases in the antisense strand are mismatched base sites (sites that are not identical between CHST2 and CHST4), and include, for example, positions 3, 7, 11, 12, 13, 24, and 27 of SEQ ID NOS: 5; positions 5, 8, 14, 16, 24, 26, and 27 of SEQ ID NOS: 7; positions 9, 12, 18, and 20 of SEQ ID NOS: 9; positions 8, 11, 17, 19, and 27 of SEQ ID NOS: 11; positions 7, 10, 16, 18, and 26 of SEQ ID NOS: 13; and positions 10, 16, 18, and 26 of SEQ ID NOS: 14. One or more positions selected from the group consisting of positions 3, 7, 11, 12, 13, 24, and 27 of sequence number 15, positions 5, 8, 14, 16, 24, 26, and 27 of SEQ ID NO: 17, positions 9, 12, 18, and 20 of SEQ ID NO: 19, positions 8, 11, 17, 19, and 27 of SEQ ID NO: 21, and positions 7, 10, 16, 18, and 26 of SEQ ID NO: 23 may be modified, for example, but not limited to, 2'-OMe, 2'-MOE, or LNA modified. DNA is represented in bold. DS01 to DS05 have natural bases (G, A, U, and C).

[0027]

[0028] siRNA with Unnatural Bases (DS06 to DS10) In one embodiment, the siRNA in the bispecific nucleic acid molecule of the present invention may contain unnatural bases. In particular, as shown in DS06 to DS10, the double-underlined bases in the antisense strand are mismatched base sites (sites that are not identical between CHST2 and CHST4). For example, unnatural bases may be present at one or more N positions selected from the group consisting of positions 3, 7, 11, 12, 13, 24, and 27 of SEQ ID NO: 15; positions 5, 8, 14, 16, 24, 26, and 27 of SEQ ID NO: 17; positions 9, 12, 18, and 20 of SEQ ID NO: 19; positions 8, 11, 17, 19, and 27 of SEQ ID NO: 21; and positions 7, 10, 16, 18, and 26 of SEQ ID NO: 23. Examples of unnatural bases include 2,6-diaminopurine and ribavirin, which may be modified with 2'-OMe, 2'-MOE, or LNA, but are not limited thereto.

[0029]

[0030] Antisense oligonucleotides (SS01 to SS05): In one embodiment, the bispecific nucleic acid molecule of the present invention may be an antisense oligonucleotide consisting only of the antisense strand shown in SEQ ID NOs: 25 to 34 below. As with SEQ ID NOs: 25 to 34, the double-underlined bases in the antisense strand are mismatched base sites (sites that are not identical between CHST2 and CHST4), such as positions 3, 7, 11, 12, 13, 24, and 27 of SEQ ID NO: 25; positions 5, 8, 14, 16, 24, 26, and 27 of SEQ ID NO: 26; positions 9, 12, 18, and 20 of SEQ ID NO: 27; positions 8, 11, 17, 19, and 27 of SEQ ID NO: 28; and positions 7, 10, and 16 of SEQ ID NO: 29. , 18, 26, positions 3, 7, 11, 12, 13, 24, 27 of SEQ ID NO: 30, positions 5, 8, 14, 16, 24, 26, 27 of SEQ ID NO: 31, positions 9, 12, 18, 20 of SEQ ID NO: 32, positions 8, 11, 17, 19, 27 of SEQ ID NO: 33, and positions 7, 10, 16, 18, 26 of SEQ ID NO: 34 may be modified, for example, but not limited to, with 2'-OMe, 2'-MOE, or LNA. SS01 to SS05 have natural bases.

[0031]

[0032] Antisense Oligonucleotides Containing Unnatural Bases (SS06 to SS10) In one embodiment, the antisense oligonucleotides in the bispecific nucleic acid molecules of the present invention may contain unnatural bases. SS06 to SS10 are single-stranded nucleic acid sequences present only in the antisense strand. In particular, as shown in SS06 to SS10, the double-underlined bases in the antisense strand represent mismatched base sites (sites not identical between CHST2 and CHST4). For example, one or more N positions selected from the group consisting of positions 3, 7, 11, 12, 13, 24, and 27 of SEQ ID NO: 30; positions 5, 8, 14, 16, 24, 26, and 27 of SEQ ID NO: 31; positions 9, 12, 18, and 20 of SEQ ID NO: 32; positions 8, 11, 17, 19, and 27 of SEQ ID NO: 33; and positions 7, 10, 16, 18, and 26 of SEQ ID NO: 34 may be substituted with unnatural bases, or even all of the selected N positions may be substituted with unnatural bases. Examples of unnatural bases include 2,6-diaminopurine and ribavirin. Regardless of whether they are unnatural bases or not, one or more to all of the bases may be modified with 2'-OMe, 2'-MOE, or LNA, but are not limited thereto.

[0033]

[0034] In one aspect of the invention, in the bispecific nucleic acid molecule of the present invention, bases in the antisense strand, particularly bases at positions that do not match between CHST2 and CHST4, may be substituted with artificial bases rather than natural bases. The artificial base may be any artificial base that is suitable for pharmaceutical use, preferably a universal base such as 2,6-diaminopurine, ribavirin, or inosine. Regardless of whether the base is a natural base or an artificial base, one or more to all of the bases may be modified with 2'-OMe, 2'-MOE, or LNA, but this is not limitative.

[0035] In one aspect of the invention, the bispecific nucleic acid molecule of the present invention may have a chemical modification at the unnatural base in the antisense strand. The chemical modification may be any chemical modification suitable for pharmaceutical use, including phosphonate modifications including methylphosphonate (MP), phosphorodithioate (PS2), methoxypropyl-phosphonate (MPO), 5'-phosphorothioate (5'-PS), (S)-5'-C-methyl having phosphoate, peptide nucleic acid (PNA), phosphorothioate (PS, Rp isomer), phosphorothioate (PS, Sp isomer), 5'-(E)-vinylphosphole (5'-(E)-VP), and 5'-methylphosphonate (5'-MP); pseudouridine (Ψ), 2'-thiouridine (s2U), N6'-methyladenosine (m ６ A), base modifications including N-ethylpiperidine 7'-EAA triazole modified adenine, N-ethylpiperidine 6'-triazole modified adenine, 6'-phenylpyrrolo-cytosine (PhpC), 5'-methylcytidine (m5C), 5'-fluoro-2'-deoxyuridine, 2'4'-difluorotoluylribonucleoside (rF) and 5'-nitroindole; and 2'-O-methyl (2'-OMe), 2'-deoxy-2'-fluoro (2'F), The ribose modifications may be selected from the group consisting of 2'-methyl-4-pyridinefluoro (2'-O-CH2Py(4)), PMO, 2'-O-methoxyethyl (2'-O-MOE), tricyclo-DNA (tcDNA), locked nucleic acid (LNA), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, glycol nucleic acid (GNA), unlocked nucleic acid (UNA) and (S)-cEt-BNA, preferably 2'-OMe.

[0036] In one aspect of the invention, the bispecific nucleic acid molecules of the present invention may be modified to enhance drug delivery, and examples thereof include, but are not limited to, drug conjugates including antibody-siRNA conjugates, polymer-siRNA conjugates, GalNAc-siRNA conjugates, and cholesterol-siRNA conjugates; lipid-based nanocarriers including liposomes, stealth liposomes, solid-lipid nanoparticles, stable nucleic acid-lipid particles, and lipid nanoparticles; polymeric nanocarriers including nanoparticles based on degradable or non-differentiable polymers (chitosan, cyclodextrin, poly(ethyleneimine), poly(L-lysine), poly(beta-amino-ester)), dendrimers (poly(propyleneimine), poly(amidoamine)); and inorganic nanocarriers including silica nanoparticles and metal / metal oxide nanoparticles (Au, FexOy); and other modifications including carbon-based nanoparticles, hydrogels, quantum dots, and natural ECVs (exosomes, larger ECVs).

[0037] The bispecific nucleic acid molecule of the present invention suppresses the formation of inflammatory cell infiltration lesions in inflammatory or allergic diseases as a single agent. Therefore, the present invention provides pharmaceutical compositions for preventing or treating inflammatory or allergic diseases, which comprise as active ingredients bispecific nucleic acid molecules against CHST2 and CHST4.

[0038] The present invention provides methods for preventing or treating inflammation or allergic diseases, comprising the step of administering bispecific nucleic acid molecules against CHST2 and CHST4; pharmaceutical compositions comprising bispecific nucleic acid molecules against CHST2 and CHST4 for use in the prevention or treatment of inflammation or allergic diseases; use of bispecific nucleic acid molecules against CHST2 and CHST4 in the production of pharmaceutical compositions for the prevention or treatment of inflammation or allergic diseases; and methods for producing therapeutic agents for inflammation or allergic diseases, comprising the step of using (formulating and / or mixing with a pharmaceutically or physiologically acceptable carrier) bispecific nucleic acid molecules against CHST2 and CHST4.

[0039] The inflammatory or allergic diseases to be treated or prevented in the present invention are inflammatory or allergic diseases associated with the appearance of HEV-like blood vessels at the site of inflammation, for which the bispecific nucleic acid molecules of the present invention exert a therapeutic effect, particularly chronic inflammatory or allergic diseases, and include Guillain-Barré syndrome, myasthenia gravis, inflammatory myopathy, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, autoimmune pancreatitis, Takayasu's arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, chronic disc disease, and other conditions. These conditions include, but are not limited to, lupus erythematosus, localized scleroderma, pemphigus, pustular psoriasis, plaque psoriasis, epidermolysis bullosa supervenes, autoimmune optic neuropathy, rheumatoid arthritis, arthritis, spondyloarthritis, systemic lupus erythematosus, scleroderma (systemic sclerosis), Sjogren's syndrome, polymyositis, dermatomyositis, vasculitis syndrome, mixed connective tissue disease, bronchial asthma, bronchiectasis, interstitial pneumonia, pulmonary fibrosis, allergic rhinitis, chronic sinusitis, allergic conjunctivitis, chronic thyroiditis, transplant rejection, Warthin's tumor, benign prostatic hyperplasia, cutaneous pseudolymphoma, contact dermatitis, Th2-dominant disease or type 2 inflammation, and atopic dermatitis. Note that "treatment" in the present invention is not necessarily limited to cases where a complete therapeutic effect is obtained, and may also include cases where a partial effect is obtained.

[0040] The pharmaceutical composition for treating inflammatory or allergic diseases of the present invention can be mixed with a pharmaceutically or physiologically acceptable carrier, excipient, diluent, or the like and administered orally or parenterally. Oral preparations can be in the form of granules, powders, tablets, capsules, solutions, emulsions, suspensions, or the like. Parenteral preparations can be selected from injections and infusions. Injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, intracranial injections, and intranasal injections. Formulation techniques for preparing the above dosage forms so as to contain the pharmaceutical composition of the present invention as the main ingredient are known.

[0041] For example, tablets for oral administration can be produced by adding excipients, disintegrants, binders, lubricants, etc. to the pharmaceutical composition for restoring tissue integrity of the present invention, mixing the mixture, and compressing and shaping the mixture. Commonly used excipients include lactose, starch, mannitol, etc. Commonly used disintegrants include calcium carbonate and calcium carboxymethylcellulose. Commonly used binders include gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone. Commonly known lubricants include talc and magnesium stearate.

[0042] The tablets containing the pharmaceutical composition for treating inflammatory or allergic diseases of the present invention can be coated with a known coating agent for masking or to prepare an enteric-coated formulation. Examples of coating agents that can be used include ethyl cellulose and polyoxyethylene glycol.

[0043] Injectable preparations can be obtained by dissolving or dispersing the pharmaceutical composition for restoring tissue integrity, which is the main ingredient, in a suitable dispersant or in a dispersion medium. Depending on the dispersion medium selected, either an aqueous or oily solvent formulation can be used. For aqueous solvents, distilled water, physiological saline, or Ringer's solution can be used as the dispersion medium. For oily solvents, various vegetable oils or propylene glycol can be used as the dispersion medium. Preservatives such as parabens can also be added as needed. Known isotonicity agents such as sodium chloride and glucose can also be added to the injectable preparation. Furthermore, soothing agents such as benzalkonium chloride and procaine hydrochloride can also be added.

[0044] When the pharmaceutical composition for preventing or treating inflammatory or allergic diseases of the present invention is used as a gene therapy agent, the pharmaceutical composition for preventing or treating inflammatory or allergic diseases of the present invention can be directly administered by injection, or a vector having a nucleic acid incorporated therein can be administered. Examples of the vector include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, and lentivirus vectors, and these viral vectors can be used to enable efficient administration.

[0045] Alternatively, the pharmaceutical composition of the present invention for preventing or treating inflammatory or allergic diseases can be incorporated into phospholipid vesicles such as liposomes, and the vesicles can be administered. The vesicles containing siRNA can be introduced into specific cells by lipofection. The resulting cells can then be administered systemically, for example, intravenously or intraarterially.

[0046] The present invention also provides a method for ameliorating lesions in a subject, comprising the step of administering to an individual (e.g., a patient) or the inflamed tissue thereof a pharmaceutical composition for preventing or treating inflammatory or allergic diseases of the present invention. The subject of the present invention is not particularly limited as long as it is an organism capable of developing inflammatory or allergic lesion changes, but is preferably a mammal, including a human, dog, or cat. Administration to a subject can be carried out by methods known to those skilled in the art, such as oral administration, application to the skin, spraying, intradermal or subcutaneous administration, or intravenous injection. Systemic administration or direct local administration into inflamed tissue is possible. Furthermore, commercially available gene transfer kits can be used to introduce the bispecific nucleic acid molecules of the present invention into target cells, tissues, or organs.

[0047] The pharmaceutical composition of the present invention for preventing or treating inflammatory or allergic diseases is administered to a mammalian subject, including a human, in a required amount (effective amount) within a range of dosages considered safe. The dosage of the pharmaceutical composition of the present invention for preventing or treating inflammatory or allergic diseases can ultimately be appropriately determined by the judgment of a person skilled in the art (a physician or veterinarian) taking into consideration the type of dosage form, the method of administration, the age and weight of the subject, and the subject's symptoms, etc. For example, the dosage for local administration of the bispecific nucleic acid molecule of the present invention varies depending on the age, sex, symptoms, route of administration, number of administrations, and dosage form, but is, for example, approximately 1 pM to 10,000 nM per day, and is administered as a single dose, at weekly to monthly intervals (e.g., weekly, biweekly, or monthly intervals), or daily.

[0048] All prior art documents cited in this specification are hereby incorporated by reference.

[0049] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0050] Example 1: Target mRNA (CHST2 mRNA and CHST4 mRNA) knockdown effect of bispecific nucleic acids (10 nucleic acids, DS01-DS10; BioSpring) for human CHST2 and CHST4: A cell line (dual transfectant) co-expressing human CHST2 and CHST4 was created and cultured in vitro. The CHST2 / CHST4 mRNA expression suppression effect of 10 synthetic double-stranded RNAs (dsRNAs, nucleic acid compound numbers: DS01-DS10) at a standardized concentration of 50 nM was examined by quantitative RT-PCR. All 10 designed and synthesized dsRNAs simultaneously suppressed both CHST2 and CHST4 mRNA. Control: Vehicle (physiological saline) was used as a negative control, and PC: a cocktail of CHST2 siRNA and CHST4 siRNA was used as a positive control. The results are shown in Figure 2.

[0051] Example 2: Binding affinity of bispecific nucleic acids (5 strands, DS06 to DS10) to human CHST2: The stability of the binding complex between the antisense strands of the dsRNAs DS06 to DS10 (single-stranded RNAs, SS06 to SS10) used in Example 1 and human CHST2 mRNA was analyzed by measuring the duplex melting temperature (Tm). The results showed that all SSs (antisense strands of the bispecific nucleic acids) showed strong affinity, with SS06 having the strongest binding affinity to human CHST2 mRNA. The results are shown in Figure 3.

[0052] Example 3: Binding affinity of bispecific nucleic acids (5 strands, DS06 to DS10) to human CHST4: The stability of the binding complex between the antisense strands (single-stranded RNAs, SS06 to SS10) of the dsRNAs, DS06 to DS10, used in Example 1 and human CHST4 mRNA was analyzed by measuring the duplex melting temperature (Tm). The results showed that all SSs (antisense strands of the bispecific nucleic acids) showed strong affinity, with SS06 having the strongest binding affinity to human CHST4 mRNA. The results are shown in Figure 4.

[0053] Example 4: Dose-dependent knockdown of target mRNAs (CHST2 mRNA and CHST4 mRNA) by dual-specific nucleic acids (DS06 and DS10) for human CHST2 and CHST4: Cell lines co-expressing human CHST2 and CHST4 (dual transfectants) were generated and cultured in vitro using the same method as in Example 1. The dose-dependent inhibitory effect of DS06 or DS10 on CHST2 / CHST4 mRNA expression was examined by quantitative RT-PCR. As a result, both DS06 and DS10 simultaneously inhibited both CHST2 and CHST4 mRNA at concentrations as low as 50 nM. Control: Vehicle (physiological saline) was used as a negative control, and PC: a cocktail of CHST2 siRNA and CHST4 siRNA was used as a positive control. The results are shown in Figure 5.

[0054] Example 5: Dose-dependent suppression of MECA-79 expression by dual-specific nucleic acids (DS06 and DS10) against human CHST2 and CHST4: Cell lines simultaneously expressing human CHST2 and CHST4 (dual transfectants) were generated and cultured in vitro using the same method as in Example 1. The dose-dependent suppression of MECA-79 expression by DS06 or DS10 against the MECA-79 epitope was examined using a flow cytometer (FACS). Both DS06 and DS10 demonstrated greater suppression of MECA-79 expression than the positive control, but DS06 demonstrated a more pronounced dose-dependent suppression. Control: Vehicle (physiological saline) was used as a negative control, and PC: a cocktail of CHST2 siRNA and CHST4 siRNA was used as a positive control. The results are shown in Figure 6.

[0055] Example 6: Effect of a bispecific nucleic acid (DS06) for human CHST2 and CHST4 on interferon-responsive gene (IFITM1) expression: A cell line (dual transfectant) co-expressing human CHST2 / CHST4 was created and cultured in vitro using the same method as in Example 1. Quantitative RT-PCR was used to examine whether DS06 increases the expression of the interferon-responsive gene IFITM1 mRNA, i.e., whether it may induce a nonspecific interferon response. As a result, DS06 did not induce an increase in IFITM1 mRNA expression even at the maximum dose of 500 nM. On the other hand, Poly I:C was used as a positive control for inducing an interferon response and indeed induced a strong interferon response in this experimental system. The results for DS06 are shown in Figure 7.

[0056] Example 7: Effect of bispecific nucleic acids (DS06 and DS10) against human CHST2 and CHST4 on TLR responses: To determine whether DS06 induces nonspecific inflammation mediated by TLRs (Toll-like receptors), we analyzed its effect on responses to human TLRs 2, 3, 4, 5, 7, 8, and 9. As a result, DS06 did not induce the above TLR responses even at a high dose of 1 μM. Control: Substances that induce each TLR response were used as positive controls. The results are shown in Figure 8.

[0057] Example 8: Skin penetration test of DS06 using artificial skin: A base composition (cream) containing DS-06 was prepared and applied to the surface of artificial skin (Strat M) using a Franz cell. The cumulative total amount of nucleic acid that penetrated over time (0, 0.5, 1, 2, 4, 6, 12, 18, and 24 hours) was measured by HPLC up to 24 hours. As shown in Figure 9, DS06 demonstrated excellent permeability. The results are shown in Figure 9.

[0058] Example 9: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on atopic dermatitis in mice: Based on a previous report (Sasakawa T, Higashi Y, Sakuma S, et al. Atopic dermatitis-like skin lesions induced by topical application of mite antigens in NC / Nga mice. Int Arch Allergy Immunol. 126: 239-247, 2001), NC / Nga mice (7-week-old, male) were administered mite antigens to the ear to induce dermatitis. Then, for 20 days from days 11 to 30, DS-06-containing cream, a negative control cream, and tacrolimus ointment as a positive control were applied once daily. The mice were sacrificed on day 31, and skin lesions were evaluated. The results are shown in HE staining. Compared to the control cream group, the DS06 cream group showed a significant decrease in inflammatory cell infiltration accompanied by thickening of the dermis and deeper. The results are shown in Figure 10.

[0059] Example 10: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (HEV-like blood vessels): In the test of Example 9, the extent of HEV-like blood vessel appearance was quantified as MECA-79-positive area (%) by immunostaining with MECA-79 antibody. While MECA-79-positive blood vessels significantly increased in the dermatitis model, application of DS06 cream significantly reduced the MECA-79-positive area compared to the control group. The results are shown in Figure 11.

[0060] Example 11: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on atopic dermatitis in mice (T lymphocytes): In the test of Example 9, the degree of lymphocyte infiltration into the dermis was quantified as CD4-positive area (%) by immunostaining with anti-CD4 antibody. + T cells significantly increased, but application of DS06 cream significantly increased CD4 + The T cell-positive area was significantly reduced compared to the control group. The results are shown in Figure 12.

[0061] Example 12: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (activated T lymphocytes): In the test of Example 9, the degree of activated lymphocyte infiltration into the dermis was quantified as CD69-positive area (%) by immunostaining with anti-CD69 antibody, which is also a marker for resident memory T cells. + DS06 cream application significantly increased the number of CD69 cells. + The cell-positive area was significantly reduced compared to the control group. The results are shown in Figure 13.

[0062] Example 13: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (regulatory T lymphocytes): In the test of Example 9, the degree of regulatory T cell (Treg) infiltration into the dermis was quantified as FoxP3-positive area (%) by immunostaining with anti-FoxP3 antibody, a marker for Treg. + Tregs showed a tendency to decrease, but application of DS06 cream increased FoxP3 + The Treg-positive area tended to increase significantly compared to the control group, maintaining the same level as normal mice. Meanwhile, tacrolimus ointment, used as a positive control, also tended to decrease Tregs, but DS06 cream significantly maintained Treg infiltration compared to the tacrolimus group. The results are shown in Figure 14.

[0063] Example 14: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on mouse atopic dermatitis (IL-13, IL-33): In the test of Example 9, RNA was purified from skin tissue, and the level of Type 2 cytokine expression was quantified by RT-PCR. The results are shown here. IL-13 and IL-33 expression was increased in the dermatitis model, but application of DS06 cream tended to significantly decrease IL-13 and IL-33 expression compared to the control group. The results are shown in Figure 15.

[0064] Example 15: Effect of bispecific nucleic acid (DS06) against human CHST2 and CHST4 on allergic rhinitis in mice (HEV-like vessels and T lymphocytes): Based on a previous report (Ohmichi Y, Hirakawa J, Imai Y, et al. Essential role of peripheral node addressin in lymphocyte homing to nasal-associated lymphoid tissues and allergic immune responses. J Exp Med. 208: 1015-25, 2011), C57BL / 6J mice (6-week-old, male) were intraperitoneally administered a mixture of ovalbumin (OVA) and cholera toxin (CT) three times (days 0, 7, and 14) at weekly intervals to induce allergic rhinitis. From days 8 to 19, DS06 solution or saline (as a negative control) was administered intranasally five times per week (total of 10 administrations). Mice were sacrificed on day 21, and nasal tissue lesions were evaluated. The degree of HEV-like vessel appearance in nasal-associated lymphoid tissue (NALT) was quantified as MECA-79-positive area (%) by immunostaining with MECA-79 antibody in the same manner as in Example 10. In the rhinitis model, nasal administration of DS06 significantly reduced the MECA-79-positive area compared to the control group. Furthermore, the degree of lymphocyte infiltration was quantified as CD4-positive area (%) by immunostaining with anti-CD4 antibody in the same manner as in Example 11. In the rhinitis model, nasal administration of DS06 significantly reduced the CD4-positive area. + The T cell-positive area was significantly reduced compared to the control group. The results are shown in Figure 16.

[0065] The bispecific nucleic acid molecules of the present invention against the glycosyl sulfotransferases CHST2 and CHST4 have been shown to inhibit the expression of HEV-like vessels, which induce the recruitment of leukocytes to infiltrated foci in inflammatory or allergic diseases, and in particular to alleviate the pathogenesis and functional impairment of chronic lesions. These results demonstrate that the bispecific nucleic acid molecules of the present invention are useful as pharmaceutical compositions for preventing or treating inflammatory or allergic diseases.

Claims

1. A bispecific nucleic acid molecule comprising an antisense strand having a sequence that specifically binds to CHST2 and also specifically binds to CHST4.

2. The bispecific nucleic acid molecule of claim 1, which is an siRNA or an antisense oligonucleotide.

3. The bispecific nucleic acid molecule of claim 2, which is an siRNA having the sequence shown in SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, or SEQ ID NOs: 13 and 14, or an antisense oligonucleotide having any of SEQ ID NOs: 25 to 29.

4. A bispecific nucleic acid molecule according to claim 2 or 3, which comprises an artificial base.

5. The bispecific nucleic acid molecule of claim 4, which comprises a chemically modified artificial base.

6. A pharmaceutical composition comprising a bispecific nucleic acid molecule according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6 for preventing or treating an inflammatory or allergic disease.

8. Use of a bispecific nucleic acid molecule according to any one of claims 1 to 5 for the manufacture of a pharmaceutical composition for preventing or treating an inflammatory or allergic disease.

9. A method for preventing or treating an inflammatory or allergic disease in a patient, comprising administering a pharmaceutical composition according to claim 6 or 7 to a patient in need of such treatment.