Pharmaceutical composition for treating fibrotic disease

Abstract

A therapeutic agent for fibrotic diseases, which contains, as an active ingredient, a nucleic acid that inhibits the expression of the NDR2 gene.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating fibrotic diseases. Background Art

[0002] Fibrotic diseases are diseases that progress irreversibly due to the accumulation of excess collagen, which damages organ functions. For example, the skin, lungs, liver, pancreas, kidneys, and bone marrow are known to be affected organs. Although idiopathic pulmonary fibrosis (IPF), a type of pulmonary fibrosis, has a low prevalence rate of about 20 patients per 100,000 people, the average survival period after diagnosis is 2.5 to 5 years, and the course of the disease after treatment is poor, so it is designated as an intractable disease in Japan.

[0003] As IPF treatment drugs, Pirfenidone and Nintedanib have been approved and marketed by the Ministry of Health, Labor and Welfare of Japan. Although all drugs have shown effects on suppressing the decrease in vital capacity and prolonging the progression-free period, delaying the progression of the disease, they are not satisfactory as treatment effects, and the development of therapeutic drugs based on new mechanisms is expected.

[0004] On the other hand, the "NDR2 protein" that is the target of the present invention is a serine / threonine kinase that is one of the subtypes of NDR kinases (Nuclear Dbf2-related kinases) belonging to the AGC family. There are two subtypes of NDR kinases, NDR1 (also known as STK38) and NDR2 (also known as STK38L). NDR kinases are widely present in cells throughout the body and are known to be related to the morphogenesis of cells or tissues. On the other hand, it has been reported that no serious toxic results were observed in systemic knockout mice, especially with respect to NDR2 (non-patent literature 1). So far, there has been no report on the association of NDR2 with fibrotic diseases.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent document 1: International Publication No. 04 / 048576

[0008] Non-patent literature

[0009] Non-patent literature 1: Debora Schmitz-Rohmer, et al., "PLOS ONE", 2015, Vol. 10, e0136566 Summary of the invention

[0010] Technical problem to be solved by the invention

[0011] The object of the present invention is to provide a novel therapeutic agent for fibrotic diseases.

[0012] Technical solutions for solving technical problems

[0013] The inventors of the present invention focused on the fact that the expression level of the NDR2 gene was increased in LL29 cells, which are human IPF lung fibroblasts, by adding TGF-β1, a fibrosis-promoting factor, and conducted research. As a result, they found that the secretion of collagen, which is the main component of fibrosis, was suppressed by inhibiting the expression of the NDR2 gene, thereby completing the present invention.

[0014] That is, the present invention is as follows.

[0015] [1] A therapeutic agent for a fibrotic disease, comprising, as an active ingredient, a nucleic acid that inhibits the expression of an NDR2 gene.

[0016] [2] The therapeutic agent according to [1], wherein the fibrotic disease is pulmonary fibrosis, liver fibrosis or kidney fibrosis.

[0017] [3] The therapeutic agent according to [1] or [2], wherein the nucleic acid that inhibits NDR2 gene expression is an antisense nucleic acid.

[0018] [4] The therapeutic agent according to [1] or [2], wherein the nucleic acid that inhibits NDR2 gene expression is siRNA.

[0019] [5] A method for treating a fibrotic disease, comprising the step of administering a nucleic acid that inhibits NDR2 gene expression to a subject.

[0020] [6] A nucleic acid, wherein the nucleic acid is used in the treatment of fibrotic diseases and inhibits the expression of the NDR2 gene.

[0021] [7] Use of a nucleic acid that inhibits the expression of the NDR2 gene in the manufacture of a therapeutic agent for fibrotic diseases.

[0022] [8] The method, nucleic acid or use according to any one of [5] to [7], wherein the fibrotic disease is pulmonary fibrosis, liver fibrosis or kidney fibrosis.

[0023] [9] The treatment method, nucleic acid or use according to any one of [5] to [8], wherein the nucleic acid that inhibits NDR2 gene expression is an antisense nucleic acid.

[0024]

[10] The treatment method, nucleic acid or use according to any one of [5] to [8], wherein the nucleic acid that inhibits NDR2 gene expression is siRNA.

[0025]

[11] The therapeutic agent, therapeutic method, nucleic acid or use according to [3] or [9], wherein the antisense nucleic acid that inhibits NDR2 gene expression contains 10 to 40 bases.

[0026]

[12] The therapeutic agent, therapeutic method, nucleic acid or use according to [3] or [9], wherein the antisense nucleic acid that inhibits NDR2 gene expression contains 12 to 21 bases.

[0027]

[13] The therapeutic agent, therapeutic method, nucleic acid or use according to [3] or [9], wherein artificial nucleic acids are introduced at both ends of the sequence of the antisense nucleic acid that inhibits NDR2 gene expression.

[0028]

[14] The therapeutic agent, therapeutic method, nucleic acid or use as described in [3] or [9], wherein the antisense nucleic acid that inhibits the expression of the NDR2 gene contains 12 to 21 bases and artificial nucleic acids are introduced at both ends of the sequence.

[0029]

[15] A therapeutic agent for fibrotic diseases as described in [1] or [2], wherein the nucleic acid that inhibits the expression of the NDR2 gene is a nucleic acid having a guide strand and a passenger strand, the guide strand and the passenger strand each contain 15 to 50 bases, and the guide strand contains an expression inhibitory sequence that is complementary to the NDR2 gene.

[0030]

[16] The therapeutic agent according to

[15] , wherein the guide strand and / or the passenger strand is a nucleic acid molecule having an overhanging end.

[0031]

[17] The therapeutic agent according to

[15] , wherein the guide strand and the passenger strand are nucleic acid molecules linked via a linker.

[0032]

[18] The treatment method, nucleic acid or use as described in [5] to [8], wherein the nucleic acid that inhibits the expression of the NDR2 gene is a nucleic acid molecule having a guide strand and a passenger strand, wherein the guide strand and the passenger strand are 15 to 50 bases respectively, and the expression inhibition sequence contained in the guide strand is a nucleic acid complementary to the NDR2 gene.

[0033]

[19] The method, nucleic acid or use as described in

[18] , wherein the guide strand and / or the passenger strand is a nucleic acid molecule with an overhang.

[0034]

[20] The method, nucleic acid or use according to

[18] , wherein the guide strand and the passenger strand are nucleic acid molecules linked via a linker.

[0035]

[21] The therapeutic agent, method of treatment, nucleic acid or use as described in [3], [5] to [9] and

[14] , wherein the antisense nucleic acid is encapsulated in a drug delivery carrier such as liposomes, poly(glycolic acid) copolymer (PLGA) microspheres, lipid microspheres, polymer micelles, gels or exosomes for administration.

[0036]

[22] A therapeutic agent for a fibrotic disease, comprising a low molecular weight compound having NDR2 inhibitory activity.

[0037]

[23] A method for treating a fibrotic disease, comprising the step of administering a low molecular weight compound having NDR2 inhibitory activity to a subject.

[0038]

[24] A low molecular weight compound, wherein the low molecular weight compound is used for treating fibrotic diseases and has NDR2 inhibitory activity.

[0039]

[25] Use of low molecular weight compounds having NDR2 inhibitory activity in the manufacture of therapeutic agents for fibrotic diseases.

[0040]

[26] The therapeutic agent, therapeutic method, low molecular weight compound or use as described in

[22] to

[25] , wherein the fibrotic disease is pulmonary fibrosis, liver fibrosis or kidney fibrosis.

[0041] Effects of the Invention

[0042] The present invention can provide a novel therapeutic agent for fibrotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a diagram showing the sequence of the mRNA from the 5' end to the 2400th base of the human NDR2 gene and the target sites of each antisense nucleic acid and siRNA described in the present disclosure. In the diagram, for example, ASO#4 indicates that the underlined sequence is the target sequence of the antisense nucleic acid shown in sequence number 4. In addition, for example, siRNA#23 indicates that the underlined sequence is the target sequence of the siRNA containing the guide strand shown in sequence number 23. In addition, U (uracil) on the mRNA sequence is marked with T (thymine). In addition, these marks are Figure 2 to Figure 4 It is the same in.

[0044] Figure 2 This is a diagram showing the base sequence from the 5' end to position 2401 and thereafter of mRNA derived from the human NDR2 gene and the target sites of each antisense nucleic acid and siRNA described in the present disclosure.

[0045] Figure 3This is a diagram showing the sequence from the 5' end to the 2400th base of the mRNA of the mouse NDR2 gene and the target site of each antisense nucleic acid and siRNA described in the present disclosure.

[0046] Figure 4 This is a diagram showing the base sequence from the 5' end to position 2401 and thereafter of the mRNA of the mouse NDR2 gene and the target sites of each antisense nucleic acid and siRNA described in the present disclosure.

[0047] Figure 5 This is a graph showing the transcription level of the NDR2 gene when each antisense nucleic acid was introduced into human IPF lung fibroblasts.

[0048] Figure 6 This is a graph showing the secretion amount of collagen when NDR2 is knocked down in human IPF lung fibroblasts using various antisense nucleic acids.

[0049] Figure 7 This is a graph showing the transcription level of the NDR2 gene when antisense nucleic acid at different concentrations was introduced into human IPF lung fibroblasts.

[0050] Figure 8 This is a graph showing the amount of collagen secreted when NDR2 is knocked down in human IPF lung fibroblasts using antisense nucleic acids at different concentrations.

[0051] Fig. 9 This is a graph showing the transcription level of the NDR2 gene when each siRNA was introduced into human IPF lung fibroblasts.

[0052] Fig.10 This is a graph showing the transcription level of the NDR2 gene when ON-TARGET plus pool siRNA was introduced as siRNA into human IPF lung fibroblasts.

[0053] Fig.11 This is a graph showing the transcription level of the NDR2 gene when Silencer Select siRNA was introduced as siRNA into human IPF lung fibroblasts.

[0054] Fig.12 This is a graph showing the secretion amount of collagen when NDR2 is knocked down in human IPF lung fibroblasts using various siRNAs.

[0055] Fig.13 This is a graph showing the amount of collagen secretion when NDR2 is knocked down in human IPF lung fibroblasts using ON-TARGET plus pool siRNA.

[0056] Fig.14This is a graph showing the amount of collagen secretion when NDR2 is knocked down using Silencer Select siRNA as siRNA in human IPF lung fibroblasts.

[0057] Fig.15 This is a graph showing the transcription level of the NDR2 gene when each antisense nucleic acid was introduced into human hepatic stellate cells.

[0058] Fig.16 This is a graph showing the secretion amount of collagen when NDR2 is knocked down in human hepatic stellate cells using various antisense nucleic acids.

[0059] Fig.17 This is a graph showing the transcription level of the NDR2 gene when each siRNA was introduced into human hepatic stellate cells.

[0060] Fig.18 This is a graph showing the transcription level of the NDR2 gene when Silencer Select siRNA was introduced as siRNA into human hepatic stellate cells.

[0061] Fig.19 This is a graph showing the secretion amount of collagen when NDR2 is knocked down in human hepatic stellate cells using various siRNAs.

[0062] Fig. 20 This is a graph showing the secretion amount of collagen when NDR2 is knocked down using Silencer Select siRNA as siRNA in human hepatic stellate cells.

[0063] Fig.21 This is a graph showing the transcription level of the NDR2 gene when ON-TARGET plus pool siRNA was introduced as siRNA into human renal fibroblasts.

[0064] Fig. 22 This is a graph showing the amount of collagen secreted when NDR2 was knocked down in human renal fibroblasts using ON-TARGET plus pool siRNA.

[0065] Fig.23 This is a graph showing the transcription level of the NDR2 gene when the antisense nucleic acid was introduced into mouse lung fibroblasts.

[0066] Fig.24 This is a graph showing the secretion amount of procollagen when NDR2 is knocked down in mouse lung fibroblasts using antisense nucleic acid.

[0067] Fig.25 This is a graph showing the transcription level of the NDR2 gene when each siRNA was introduced into mouse hepatic stellate cells.

[0068] Fig.26 This is a graph showing the secretion amount of collagen when NDR2 is knocked down in mouse hepatic stellate cells using various siRNAs.

[0069] Fig. 27 This is a graph showing the transcription level of NDR2 gene in the lung of normal mice when antisense nucleic acid for suppressing NDR2 gene expression was orally administered to normal mice.

[0070] Fig.28 This is a graph showing the transcription level of NDR2 gene in mouse liver when antisense nucleic acid for suppressing NDR2 gene expression was subcutaneously or intravenously administered to normal mice.

[0071] Fig.29 This is a graph showing the transcription level of the NDR2 gene in the liver of normal mice when siRNA that inhibits the expression of the NDR2 gene was administered via the tail vein.

[0072] Fig.30 This is a graph showing the transcription level of the NDR1 gene in the liver of normal mice when siRNA that inhibits the expression of the NDR2 gene was administered via the tail vein.

[0073] Fig.31 This is a graph showing the results of Western blotting of NDR2 protein and CYP2E1 protein in the liver of normal mice when siRNA that inhibits NDR2 gene expression was administered via the tail vein.

[0074] Fig.32 This is a graph showing the level of NDR2 protein corrected by vinculin in the liver of normal mice when siRNA that inhibits NDR2 gene expression was administered via the tail vein.

[0075] Fig.33 This is a graph showing the level of CYP2E1 protein corrected by vinculin in the liver of normal mice when siRNA that inhibits NDR2 gene expression was administered via the tail vein.

[0076] Fig.34 This is a graph showing the transcription level of NDR2 gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0077] Fig.35This is a graph showing the transcription level of CYR61 gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0078] Fig.36 This is a graph showing the transcription level of CTGF gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0079] Fig.37 This is a graph showing the transcription level of TNF gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0080] Fig.38 This is a graph showing the transcription level of CCL2 gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0081] Fig.39 This is a graph showing the transcription level of COL1A1 gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0082] Fig.40 This is a graph showing the transcription level of TGF-β1 gene in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0083] Fig.41 The results of Western blotting show the αSMA protein in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein.

[0084] Fig.42 This is a graph showing the level of αSMA protein corrected by vinculin in the liver of mice when siRNA that inhibits NDR2 gene expression was administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice via tail vein. DETAILED DESCRIPTION

[0085] Unless otherwise specified, the terms used in this specification are used in the sense commonly used in the technical field. In addition, in the present invention, "number of bases" refers to, for example, "length" and may also be referred to as "base length". In the present invention, the numerical range of the number of bases, for example, discloses all positive integers belonging to the range, and as a specific example, the description of "1 to 4 bases" means that all "1, 2, 3, 4 bases" are disclosed.

[0086] The present invention provides a therapeutic agent for fibrotic diseases comprising a nucleic acid that inhibits the expression of the NDR2 gene as an active ingredient. The present invention will be described in detail below.

[0087] (1) Nucleic acid that inhibits NDR2 gene expression

[0088] The NDR2 protein is a serine / threonine kinase that is one of the subtypes of NDR kinases (Nuclear Dbf2-related kinases) belonging to the AGC family.

[0089] The NDR2 gene as a target of the present invention is a gene from mammals, preferably a gene from humans or mice, and more preferably a gene from humans. For example, the mRNA sequence of one isoform of the human NDR2 gene (NCBI GenBank accession number: NM_015000.4) is shown in Figure 1-2 (SEQ ID NO: 1). For example, the mRNA sequence of one isoform of the mouse NDR2 gene (NCBI GenBank accession number: NM_172734.3) is shown in Figure 3-4 (Sequence number 2).

[0090] The mechanism of suppressing the expression of the NDR2 gene by the nucleic acid molecule is not particularly limited, as long as the expression can be reduced. The suppression of the expression of the NDR2 gene can be confirmed by a reduction in the amount of transcription products generated from the NDR2 gene, a reduction in the amount of translation products generated from the NDR2 gene, or a reduction in the activity of the above-mentioned translation products.

[0091] Examples of the nucleic acid that inhibits the expression of the NDR2 gene include antisense nucleic acid of NDR2 mRNA, siRNA, etc. The nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid.

[0092] Antisense nucleic acid and siRNA can be easily obtained based on the nucleotide sequence of the above-mentioned NDR2 gene as long as they are skilled in the art. Preferably, it is a nucleic acid prepared based on the NDR2mRNA sequence recorded in sequence number 1 (from humans) or sequence number 2 (from mice), and more preferably a nucleic acid prepared based on the NDR2mRNA sequence recorded in sequence number 1 (from humans). For example, the above-mentioned nucleic acid can be prepared based on the translation region or 3' non-translation region of the mRNA sequence of sequence number 1. Specifically, the above-mentioned nucleic acid can be prepared based on the DNA sequence of base numbers 59 to 1450 from the 5' end of sequence number 1 or the DNA sequence of base numbers 1451 to 4957. In addition, for example, the above-mentioned nucleic acid can be prepared based on the translation region or 3' non-translation region of the mRNA sequence of sequence number 2. Specifically, the above-mentioned nucleic acid can be prepared based on the DNA sequence of base numbers 130 to 1524 or base numbers 1525 to 4617 from the 5' end of sequence number 2. Alternatively, the nucleic acid can be prepared based on, for example, the precursor of the mRNA of SEQ ID NO: 1 (NCBI GenBank accession number: NC_000012.12REGION: 27244286..27325959) or the precursor of the mRNA of SEQ ID NO: 2 (NC_000072.7REGION: 146626428..146680312).

[0093] (2) Antisense Nucleic Acids

[0094] Antisense nucleic acids are oligonucleotides (antisense DNA and / or antisense RNA) that form double strands with target mRNA in a sequence-specific manner and perform functional inhibition such as splicing inhibition and translation inhibition. The effect is exerted by introducing antisense nucleic acids against the entire length or a portion of the target mRNA into cells.

[0095] Examples of the mechanism of inhibition of expression of antisense nucleic acid include:

[0096] 1) The region from the 5' cap of mRNA to about 25 bases downstream of the start codon is the steric hindrance of the translation initiation complex as the target sequence

[0097] 2) Single-stranded DNA complementary to the target mRNA or pre-mRNA (mRNA precursor), which is degraded by RNase H

[0098] 3) Splicing inhibition (mRNA maturation inhibition) targeting the boundary region between exons and introns of pre-mRNA. The mechanism is not particularly limited as long as the expression of the NDR2 gene is inhibited.

[0099] As the sequence contained in the antisense nucleic acid involved in the present embodiment, it is preferred to select a sequence specific to the NDR2 gene. For example, it can be cited that the sequence is complementary to the NDR2 gene and not complementary to the NDR1 gene (RefSeq database: NM_001305102.2 and / or NM_007271.4), or one or more (e.g., 1 to 3) bases in the region are non-complementary (mismatched) to the NDR1 gene. Increasing the (mismatched) bases (e.g., 4 or more, preferably 5 to 7) that are complementary to the NDR2 gene and non-complementary to the NDR1 gene is useful in avoiding off-target effects.

[0100] The number of bases contained in the antisense nucleic acid involved in this embodiment is not particularly limited as long as it exerts the effect of inhibiting the expression of the NDR2 gene, and can be, for example, 10 to 40 bases, 10 to 35 bases, 12 to 30 bases, 13 to 30 bases, or 13 to 21 bases.

[0101] Antisense nucleic acids that inhibit the gene expression of NDR2 can be obtained based on the cDNA sequence information of mature mRNA of NDR2 according to an antisense nucleic acid sequence design system such as PFRED (Pfizer RNAi Enumeration and Design).

[0102] The antisense nucleic acid preferably contains a modified nucleotide residue from the viewpoints of binding stability with RNA (Tm value, etc.), mismatch sequence recognition ability, nuclease resistance, RNase H activity, and the like.

[0103] Specific examples of the antisense nucleic acid that inhibits the expression of the NDR2 gene include the following single-stranded nucleic acid molecules.

[0104] [a] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 3

[0105] [b] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 4

[0106] [c] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 5

[0107] [d] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 6

[0108] [e] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 7

[0109] [f] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 8

[0110] [g] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 9

[0111] [h] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 10

[0112] [i] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 11

[0113] [j] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 12

[0114] [k] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 13

[0115] [1] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 14

[0116] [m] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 15

[0117] [n] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 16

[0118] [o] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 17

[0119] [p] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 18

[0120] [q] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 19

[0121] [r] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 20

[0122] [s] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 21

[0123] [t] A nucleic acid comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 22

[0124] The sequence identity of the sequence contained in the antisense nucleic acid to the sequence of sequence numbering 3 to 22 can be 90% or more, 92% or more, 95% or more, or 98% or more, or 100%. The antisense nucleic acid of [a] to [t] can be added with various modifications within the range of not losing the effect of inhibiting the expression of the NDR2 gene. Various modifications can exist in the sequence of sequence numbering 3 to 22, or can exist outside the sequence. The details of the modified nucleotides, etc. will be described later.

[0125] The antisense nucleic acid for inhibiting the gene expression of NDR2 can be easily obtained by a known method based on the nucleotide sequence of the NDR2 gene. For example, it can be appropriately designed based on the mRNA sequence information of the NDR2 gene such as the sequence of the NDR2 mRNA shown in sequence number 1 or sequence number 2, and synthesized by a known method. The above-mentioned synthesis method can include, for example, a synthesis method by a genetic engineering method, a chemical synthesis method, etc. The genetic engineering method can include, for example, an in vitro transcription synthesis method, a method using a vector, a method using a PCR box, etc. The above-mentioned vector is not particularly limited, and non-viral vectors such as plasmids, viral vectors, etc. can be listed. The above-mentioned chemical synthesis method is not particularly limited, and for example, the phosphoramidite method and the H-phosphonate method can be listed. The above-mentioned chemical synthesis method can use, for example, a commercially available automatic nucleic acid synthesizer. In addition, the gene information of NDR2 can also be provided to manufacturers (such as Qiagen, Hokkaido System Science CO, Ltd.), and the sequence of the Gapmer type antisense with a cross-linked nucleic acid introduced at both ends can be designed to obtain it.

[0126] (3) siRNA

[0127] The following describes siRNA (small interfering RNA), which is one of the nucleic acids that inhibit the expression of the NDR2 gene.

[0128] siRNA is a nucleic acid molecule consisting of a guide strand (antisense strand) paired with a target gene and a passenger strand (sense strand) that forms a double strand with the guide strand. After siRNA enters the complex called RNA-inducing silencing complex (RISC) with Argonaute (AGO) protein as the core in the cell, the sense strand is decomposed by AGO and the guide strand remains in RISC. The seed region of the guide strand (the 7-base region from the 5' end of the guide strand to 2-8 bases) is considered to play an important role in identifying the target sequence. From the purpose of avoiding off-target effects, a seed region specific to the target gene can be selected. For example, with respect to the seed region of the nucleic acid as the active ingredient of the present invention, it is also preferred to select a sequence specific to NDR2 mature mRNA (RefSeq database: NM_015000.4 and / or NM_172734.3). For example, a nucleic acid that is complementary to NDR2 mature mRNA and not complementary to NDR1 mature mRNA (RefSeq database: NM_001305102.2 and / or NM_007271.4) or a sequence in which one or more bases (e.g., 1 to 3) in the region are non-complementary to NDR2 mature mRNA (mismatch) can be selected as a seed region. In addition, with respect to the full-length sequence, for example, increasing the number of (mismatch) bases (e.g., 4 or more, preferably 5 to 7) that are complementary to NDR2 mature mRNA and non-complementary to NDR1 mature mRNA is also useful in avoiding off-target effects. The number of bases of the expression inhibition sequence contained in the guide chain is, for example, 15 to 30 bases, preferably 19 to 25 bases, more preferably 19 to 23 bases, further preferably 21, 22, 23 bases, and particularly preferably 23 bases.

[0129] The expression inhibitory sequence may further have an additional sequence on the 3' side to form an overhang. The number of bases of the additional sequence is, for example, 1 to 11 bases, preferably 1 to 4 bases. The additional sequence may be a ribonucleotide residue or a deoxyribonucleotide residue.

[0130] The number of bases of the guide strand is, for example, 15 to 50 bases, preferably 17 to 30 bases, more preferably 19 to 25 bases, further preferably 19 to 23 bases, further preferably 21, 22, or 23 bases, and particularly preferably 23 bases.

[0131] The number of bases of the passenger strand is, for example, 15 to 50 bases, preferably 17 to 30 bases, more preferably 19 to 25 bases, further preferably 19 to 23 bases, further preferably 21, 22, or 23 bases, and particularly preferably 21 bases.

[0132] The region of the passenger chain that shows complementarity with the guide chain is, for example, 15 to 50 bases, preferably 17 to 30 bases, more preferably 19 to 25 bases, and even more preferably 19 to 23 bases. The above region may further have an additional sequence on the 3' side. The number of bases of the above additional sequence is, for example, 1 to 11 bases, preferably 1 to 4 bases, and the additional sequence may be a ribonucleotide residue or a deoxyribonucleotide residue. The region of the passenger chain that shows complementarity with respect to the guide chain may be, for example, complementary, or 1 or several bases may be non-complementary, but preferably complementary. The above 1 base or several bases are, for example, 1 to 3 bases, preferably 1 base or 2 bases.

[0133] siRNA that inhibits NDR2 gene expression can be obtained based on the cDNA sequence information of NDR2 mature mRNA, for example, according to siRNA sequence design systems such as siSNIPER (registered trademark), siDirect (registered trademark) for drug development and diagnostic research, and PFRED.

[0134] The NDR2 siRNA is preferably an siRNA that specifically acts on NDR2, and examples thereof include the following double-stranded nucleic acids.

[0135] (a) a double-stranded nucleic acid molecule formed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 23 and a passenger strand (sense strand) complementary to the guide strand

[0136] (b) a double-stranded nucleic acid molecule composed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 24 and a passenger strand (sense strand) complementary to the guide strand

[0137] (c) a double-stranded nucleic acid molecule formed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 25 and a passenger strand (sense strand) complementary to the guide strand

[0138] (d) a double-stranded nucleic acid molecule formed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 26 and a passenger strand (sense strand) complementary to the guide strand

[0139] (e) a double-stranded nucleic acid molecule formed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 27 and a passenger strand (sense strand) complementary to the guide strand

[0140] (f) a double-stranded nucleic acid molecule formed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 28 and a passenger strand (sense strand) complementary to the guide strand

[0141] (g) a double-stranded nucleic acid molecule composed of a guide strand (antisense strand) comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 29 and a passenger strand (sense strand) complementary to the guide strand

[0142] The sequence identity of the sequence contained in the passenger strand (sense strand) contained in the double-stranded nucleic acid molecule to the sequence of SEQ ID NOs. 1 to 2 may be 90% or more, 92% or more, 95% or more, 98% or more, or 100%.

[0143] In addition, the siRNA that inhibits the expression of the NDR2 gene can be a single-stranded nucleic acid molecule with a hairpin RNA structure formed by connecting the 3' end of the passenger chain (sense chain) and the 5' end of the guide chain (antisense chain) via a linker sequence composed of nucleotide residues and / or a linker with a non-nucleotide structure, or connecting the 3' end of the guide chain (antisense chain) and the 5' end of the passenger chain (sense chain) via a linker sequence composed of nucleotide residues and / or a linker with a non-nucleotide structure.

[0144] The length of the linker sequence composed of nucleotide residues is not particularly limited, and is preferably a length that allows the passenger strand and the guide strand to form a double strand. The number of bases in the linker sequence is, for example, 1 base or more, preferably 2 bases or more, more preferably 3 bases or more, and for example, 100 bases or less, preferably 80 bases or less, more preferably 50 bases or less. Specific examples of the number of bases in the linker sequence are 1 to 100 bases, 2 to 80 bases, or 3 to 50 bases.

[0145] Examples of the non-nucleotide structure linker include chemical linkers such as hexaethylene glycol linkers, poly-(oxyphosphoenyl-oxy-1,3-propylene glycol) linkers, allyl linkers, and polyethylene glycol linkers, and amino linkers having a carbamate structure, etc. The length of the non-nucleotide structure linker is not particularly limited, but is preferably a length that allows the passenger strand and the guide strand to form a double strand.

[0146] (4) Nucleotide residues used in nucleic acids

[0147] In the present embodiment, the nucleotide residues used as the nucleic acid of the active ingredient include sugar, base and phosphoric acid as constituent elements. The above-mentioned nucleotide residues can include, for example, ribonucleotide residues and deoxyribonucleotide residues. The above-mentioned ribonucleotide residues, for example, have ribose residues as sugar, and as bases, have adenine (A), guanine (G), cytosine (C) and uracil (U), and the above-mentioned deoxyribonucleotide residues, for example, have deoxyribose residues as sugar, and as bases, have adenine (A), guanine (G), cytosine (C) and thymine (T).

[0148] The nucleotide residues include unmodified nucleotide residues and modified nucleotide residues. In the unmodified nucleotide residues, the constituent elements may be, for example, the same or substantially the same as naturally occurring constituent elements, preferably the same or substantially the same as naturally occurring constituent elements in the human body.

[0149] The modified nucleotide residues are, for example, nucleotide residues modified from the non-modified nucleotide residues. The modified nucleotides may be, for example, any one of the constituent elements of the non-modified nucleotide residues is modified. In the present invention, "modification" is, for example, substitution, addition and / or deletion of the constituent elements, substitution, addition and / or deletion of atoms and / or functional groups in the constituent elements, which may be referred to as "change". The modified nucleotide residues may include, for example, naturally occurring nucleotide residues, artificially modified nucleotide residues, etc. The modified nucleotide residues of natural origin may be, for example, referred to Limbach et al. (Limbach et al. 1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196). In addition, the modified nucleotide residues may be, for example, residues of substitutes for the nucleotides.

[0150] The modification of the above-mentioned nucleotide residues can be, for example, the modification of the ribose-phosphate backbone (hereinafter, the ribose phosphate backbone). In the above-mentioned ribose phosphate backbone, for example, the ribose residue can be modified. The above-mentioned ribose residue can, for example, modify the 2' carbon, specifically, for example, the hydroxyl group bound to the 2' carbon can be replaced by hydrogen or fluorine. By replacing the hydroxyl group of the above-mentioned 2' carbon with hydrogen, the ribose residue can be replaced by deoxyribose. In addition, it is also possible to modify by introducing a methyl or methoxyethyl group into the 2' hydroxyl group, for example, 2'-F, 2'-O-methyl (2'-OMe) and 2'-O methoxyethyl (2'-MOE) can be listed. The above-mentioned ribose residue can, for example, be replaced by a stereoisomer, for example, it can be replaced by an arabinose residue.

[0151] The above-mentioned ribose phosphate backbone can be replaced by a non-ribose phosphate backbone having a non-ribose residue and / or a non-phosphate. For example, the above-mentioned non-ribose phosphate backbone can include the non-charged body of the above-mentioned ribose phosphate backbone. For example, the substitutes of the above-mentioned nucleotides substituted by the above-mentioned non-ribose phosphate backbone can include morpholino, cyclobutyl, pyrrolidine, etc. In addition to the above-mentioned substitutes, for example, artificial nucleic acid monomer residues can also be listed. As specific examples, for example, PNA (peptide nucleic acid), 2', 4'-BNA [Bridged Nucleic Acid, alias LNA (locked nucleic acid, Locked Nucleic Acid)] as a cross-linked artificial nucleic acid, AmNA, ENA (2'-O, 4'-C-ethylene bridged nucleic acid (2'-O, 4'-C-EthylenebridgedNucleic Acids)), GuNA and scpBNA can be listed. When the nucleic acid involved in the present embodiment is an antisense nucleic acid that utilizes the mechanism of RNA degradation by RNaseH, it is preferably an antisense nucleic acid into which an artificial nucleic acid is introduced, more preferably an antisense nucleic acid into which an artificial nucleic acid is introduced at both ends of the sequence, and particularly preferably an antisense nucleic acid into which an artificial nucleic acid is introduced only at both ends of the sequence. This is because by setting it as an antisense nucleic acid into which an artificial nucleic acid is introduced only at both ends of the sequence, it is possible to maintain the characteristics of the artificial nucleic acid while RNA degradation by RNaseH occurs, and a high antisense effect can be expected.

[0152] On the other hand, when the nucleic acid involved in this embodiment is an antisense nucleic acid utilizing the mechanism of induction of exon skipping, it is preferably designed not to undergo RNA degradation by RNase H like Mixmer or fully modified oligonucleotide in order not to cleave the target RNA.

[0153] In the above-mentioned ribose phosphate backbone, for example, the phosphate group can be modified. In the above-mentioned ribose phosphate backbone, the phosphate group closest to the sugar residue is called the α-phosphate group. The above-mentioned α-phosphate group is negatively charged, and its charge is evenly distributed on the two oxygen atoms that are not bound to the sugar residue. Among the four oxygen atoms of the above-mentioned α-phosphate group, in the phosphodiester bond between the nucleotide residues, the following two oxygen atoms that are not bound to the sugar residue are also referred to as "non-linking oxygen". On the other hand, in the phosphodiester bond between the above-mentioned nucleotide residues, the following two oxygen atoms that are bound to the sugar residue are also referred to as "linking oxygen". The above-mentioned α-phosphate group is preferably modified to be non-charged, or the charge distribution of the above-mentioned non-bound atoms is modified to be asymmetric.

[0154] The above-mentioned phosphate group can replace the above-mentioned non-bonded oxygen, for example. The above-mentioned oxygen can be substituted by any atom of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is, for example, an alkyl group or an aryl group), preferably substituted by S. The above-mentioned non-bonded oxygen is preferably, for example, both are substituted, and more preferably both are substituted by S. The above-mentioned modified phosphate group can be listed, for example: thiophosphate, dithiophosphate, selenophosphate, boranephosphate, boranephosphate, hydrogenphosphonate, phosphoramidite, alkyl or arylphosphonate, and phosphate triester, among which dithiophosphate in which both of the above-mentioned two non-bonded oxygens are substituted by S is preferred.

[0155] The above-mentioned phosphate group can, for example, replace the above-mentioned combined oxygen. The above-mentioned oxygen can, for example, be replaced by any atom of S (sulfur), C (carbon) and N (nitrogen), or by borane (BH3). The above-mentioned modified phosphate group can, for example, be listed as cross-linked phosphoramidites replaced by N, cross-linked phosphorothioates replaced by S, and cross-linked methylene phosphonates replaced by C. The phosphorothioate modification (Sization) after replacing the above-mentioned oxygen atom with an S (sulfur) atom is a modification of the phosphodiester bond portion connecting nucleic acids and nucleic acids, and the phosphodiester bond in the sequence is changed to a thiophosphate bond. Thus, it is expected that the resistance to nucleases will be improved, so it is preferred. The above-mentioned combined oxygen substitution can, for example, be carried out in at least one of the 5' terminal nucleotide residue and the 3' terminal nucleotide residue of the nucleic acid involved in the present embodiment, or it can be carried out in both. In addition, for example, all phosphodiester bonds in the nucleic acid involved in the present embodiment can be changed to thiophosphate bonds.

[0156] The phosphate group may be replaced by the phosphorus-free linker. The linker includes, for example, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiol acetal, methylal, oxime, methylene imino, methylene methyl imino, methylene hydrazone, methylene dimethyl hydrazone, and methylene oxy methyl imino, preferably methylene carbonyl amino and methylene methyl imino.

[0157] The modification of the terminal nucleotide residues may include, for example, the addition of other molecules. Examples of the other molecules include functional molecules such as the labeling substances and protective groups described above. Examples of the protective groups include S (sulfur), Si (silicon), B (boron), ester groups, and the like.

[0158] In the above-mentioned nucleotide residues, the above-mentioned base is not particularly limited. The above-mentioned base may be, for example, a natural base or a non-natural base. The above-mentioned base may be, for example, a natural origin or a synthetic product. The above-mentioned base may be, for example, a common base, a modified analog thereof, etc.

[0159] (5) Therapeutic agents for fibrotic diseases

[0160] The therapeutic agent for fibrotic diseases involved in this embodiment is for liver fibrosis, liver cirrhosis, viral hepatitis, autoimmune hepatitis, primary biliary hepatitis, nonalcoholic fatty liver disease, alcoholic liver disease, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, α1-antitrypsin deficiency, non-viral congestive cirrhosis, drug-induced liver injury, pancreatitis, pancreatic fibrosis, retinal fibrosis, vocal cord scar, vocal cord mucosal fibrosis, laryngeal fibrosis, pulmonary fibrosis, interstitial pneumonia, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, idiopathic organizing pneumonia, exfoliative interstitial pneumonia, interstitial pneumonia associated with respiratory bronchiolitis, acute interstitial pneumonia, lymphocytic interstitial pneumonia, sarcoidosis, chronic eosinophilic pneumonia, Acute eosinophilic pneumonia, lymphangioleiomyomatosis, pulmonary alveolar proteinosis, Hermansky-Pudlak syndrome, Langerhans cell histiocytosis, iron lung disease, amyloidosis, alveolar microlithiasis, allergic pneumonia, pneumoconiosis, infectious lung disease, drug-induced pneumonia, radiation pneumonia, cystic fibrosis, myelofibrosis, cardiac fibrosis, heart failure, renal fibrosis, chronic renal failure, diabetic nephropathy, chronic glomerulonephritis, malignant nephrosclerosis, polycystic kidney, drug-induced nephropathy, retroperitoneal fibrosis, collagen disease, scleroderma, congenital keratosis, renal systemic fibrosis, and a wide range of fibrosis-related diseases including airway fibrosis, intestinal fibrosis, bladder fibrosis, prostate fibrosis and skin fibrosis. Preferably, it is a liver fibrosis disease, liver cirrhosis, pulmonary fibrosis, interstitial pneumonia, renal fibrosis or chronic renal failure, and more preferably pulmonary fibrosis, liver fibrosis or renal fibrosis.

[0161] The administration method of the therapeutic agent for fibrosis diseases involved in this embodiment is not particularly limited, and is preferably non-oral administration such as inhalation, intravenous administration (intravenous injection), intramuscular administration, subcutaneous administration, intradermal administration, intramedullary administration and transdermal administration. The therapeutic agent for fibrosis diseases involved in this embodiment is preferably administered to a subject who needs the therapeutic agent for fibrosis diseases, such as a subject with the above-mentioned fibrosis-related disease onset and a subject judged to have a high risk of onset. The subject can be a human or a non-human animal. The dosage of the nucleic acid molecule of this embodiment in the treatment method involved in this embodiment is not particularly limited as long as it is an effective amount for the treatment of the above-mentioned disease. It varies according to the type of disease, severity, age, body weight, route of administration, etc., and is usually about 0.0001 to about 100 mg / kg body weight per time for adults, for example, about 0.001 to about 10 mg / kg body weight, preferably about 0.005 to about 5 mg / kg body weight. The amount can be administered, for example, 3 times a day to once a month, preferably once a day to once a week.

[0162] The therapeutic agent for fibrotic diseases according to the present embodiment is usually formulated into a suitable pharmaceutical composition together with a pharmaceutically acceptable carrier and administered orally or parenterally.

[0163] The antisense nucleic acid or siRNA according to the present embodiment is preferably administered by being encapsulated in a drug delivery carrier such as liposomes, poly(glycolic acid copolymer) (PLGA) microspheres, lipid microspheres, polymer micelles, gels or exosomes.

[0164] (6) Low molecular weight compounds with NDR2 inhibitory activity

[0165] In addition to nucleic acids that inhibit the expression of the NDR2 gene, low molecular weight compounds having NDR2 inhibitory activity can also be active ingredients of therapeutic agents for fibrotic diseases. Low molecular weight compounds refer to compounds with a molecular weight of less than 1,000.

[0166] Hereinafter, the present invention will be described in detail using examples, etc., but the present invention is not limited thereto. In addition, in the present embodiment, "antisense oligonucleotide" and "ASO" have the same meaning as "antisense nucleic acid". In addition, in the embodiment, for example, #3 refers to the antisense nucleic acid shown in sequence number 3 or the siRNA containing the guide chain shown in sequence number 3. In addition, unless otherwise specified, the nucleic acid control uses ON-TARGET plus Control Pool Non-Targeting siRNA (D-001810-10-05, Dharmacon), Antisense LNAGapmeR Negative control A (339515LG00000002-DDA, Qiagen), Ambion in vivo Negative Control siRNA (4459405, Thermo Fisher Scientific) or Silenser select Nontarget siRNA (4390846, Thermofisher Scientific). In addition, the cell culture conditions are carried out at 37°C and 5% CO2 unless otherwise specified.

[0167] Example

[0168] [Example 1: Knockdown of NDR2 in human IPF lung fibroblasts using ASO]

[0169] The effect of ASO on the expression of NDR2 in LL29 cells (ATCC), a human lung fibroblast cell line established from the lungs of IPF patients, was analyzed.

[0170] Lipofectamine RNAiMAX (Thermo Fisher Scientific) was used to transfect LL29 cells with ASO (Antisense LNAGapmeR, Qiagen) against human NDR2 according to the instructions of the Lipofectamine RNAiMAX reagent. To prepare the ASO transfection solution, 50 μL of Opti-MEM solution was used to mix the two at a ratio of 2 μL of RNAiMAX transfection reagent to make an RNAiMAX prepared solution. The prepared solution (ASO dilution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, LL29 cells were plated at a rate of 2.5×10 4500 μL of cell suspension was inoculated in cells / well and allowed to stand in a CO2 incubator (final concentration of ASO 30 nM). 48 hours after transfection, the culture medium was replaced with F-12K culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 24 hours after TGF-β1 stimulation, RNA was extracted from the cells using RNeasy96Kit (Qiagen). The obtained RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to obtain cDNA. The obtained cDNA was used as a template, and real-time PCR was performed using Taqman Gene Expression Assays (Thermo Fisher Scientific), TaqMan Fast Universal PCR master mix (Thermo Fisher Scientific) and Applied Bio systems 7500Fast Real-Time PCR Systems (Applied Biosystems) to investigate the effect of NDR2 knockdown on the transcription level of the NDR2 gene. The transcription level of the NDR2 gene was calculated by dividing the measured value of NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific) by the measured value of GAPDH Taqman Gene Expression Assays (Hs02786624_g1).

[0171] Antisense LNA GapmeR (SEQ ID NOs: 3 to 5) was used as the ASO sequence.

[0172] <Antisense LNA GapmeR>

[0173] ASONDR2: 5'-CAGTTGACTAGATAGG-3' (SEQ ID NO: 3)

[0174] ASONDR2: 5'-GATCGACGTAACTTTT-3' (SEQ ID NO: 4)

[0175] ASONDR2: 5'-TTAACCAGCCATGATA-3' (SEQ ID NO: 5)

[0176] Figure 5 The results of real-time PCR of NDR2 when Antisense LNA GapmeR against NDR2 was introduced into LL29 cells. The introduction of NDR2 ASO suppressed the transcription of NDR2 gene. Therefore, it was shown that the used NDR2 ASO effectively suppressed the expression of the target gene.

[0177] [Example 2: Effects on collagen secretion caused by knockdown of NDR2 in human IPF lung fibroblasts]

[0178] To investigate the possibility that NDR2 controls collagen secretion in LL29 cells (ATCC), a human lung fibroblast cell line established from the lungs of IPF patients, the amount of collagen secreted by cells transfected with NDR2 ASO was analyzed.

[0179] ASO for human NDR2 (Antisense LNA GapmeR, Qiagen) was transfected into LL29 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions for the Lipofectamine RNAiMAX reagent. To prepare the ASO transfection solution, 50 μL of Opti-MEM solution was used to make 2 μL of RNAiMAX transfection reagent to make an RNAiMAX prepared solution. The prepared solution (ASO dilution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts and 100 μL was added to each well of a 24-well plate. Among them, LL29 cells were plated at a rate of 2.5×10 4500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (final concentration of ASO 30 nM). 48 hours after transfection, the culture medium was replaced with F-12K culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 400 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind test tube (Eppendorf) and mixed. The test tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. Add 750 μL of cooled acid-salt wash reagent (Biocolor) and centrifuge (4°C, 12,000×g, 10 minutes) to remove the supernatant. Add 250 μL of Sircol Collagen Assay ALKALIReagent (Biocolor) to mix. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 555 nm using an absorbance microplate reader.

[0180] Antisense LNA GapmeR (SEQ ID NOs: 3 to 5) was used as the ASO sequence.

[0181] Figure 6 The results of measuring collagen secretion when NDR2 was knocked down are shown. By using ASO of NDR2, the gene expression of NDR2 was reduced, and the collagen secretion increased by TGF-β1 was reduced. Therefore, it was shown that the collagen secretion induced by TGF-β1 in human lung fibroblast cell line was inhibited by knocking down NDR2.

[0182] [Example 3: ASO concentration-dependent knockdown of NDR2 in human IPF lung fibroblasts]

[0183] ASO for human NDR2 (Antisense LNA GapmeR, Qiagen) was adjusted to a final concentration of 10, 30 or 50 nM, and transfected into LL29 cells (ATCC), a human lung fibroblast cell line constructed from the lungs of IPF patients, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. In order to prepare the ASO transfection solution, the two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent (Transfection Reagent) to prepare an RNAiMAX prepared solution. The prepared solution (ASO dilution solution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, LL29 cells were cultured at a rate of 2.5×10 4 500 μL of the cell suspension was inoculated in a cell / well manner and allowed to stand in a CO2 incubator (the final concentration of ASO was adjusted to the target concentration). 48 hours after transfection, the culture medium was replaced with F-12K culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. After 18 hours of TGF-β1 stimulation, RNA was extracted from the cells using RNeasy 96 Kit (Qiagen). The obtained RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific), and the transcription level of GAPDH as an internal standard was compared with the value measured by GAPDH Taqman Gene Expression Assays (Hs02786624_g1) as the transcription level of the gene.

[0184] Antisense LNA GapmeR (SEQ ID NO: 5) was used as the ASO sequence.

[0185] Figure 7The results of real-time PCR of NDR2 when Antisense LNA GapmeR against NDR2 was introduced into LL29 cells. The introduction of NDR2 ASO inhibited the transcription amount of NDR2 gene in a concentration-dependent manner. Therefore, it was shown that the NDR2 ASO used inhibited the expression of the target gene in a concentration-dependent manner.

[0186] [Example 4: Effects of ASO concentration-dependent knockdown of NDR2 on collagen secretion in human IPF lung fibroblasts]

[0187] ASO for human NDR2 (Antisense LNA GapmeR, Qiagen) was prepared to a final concentration of 10, 30 or 100 nM, and transfected into LL29 cells (ATCC), a human lung fibroblast cell line constructed from the lungs of IPF patients, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. In order to prepare the ASO transfection solution, 50 μL of Opti-MEM solution was used to form 2 μL of RNAiMAX transfection reagent (Transfection Reagent), and the two were mixed to make an RNAiMAX prepared solution. The prepared solution (ASO dilution solution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, LL29 cells were cultured at a rate of 2.5×10 4500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (the final concentration of ASO was adjusted to the target concentration). 48 hours after transfection, the culture medium was replaced with F-12K culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 400 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a ProteinLoBind test tube (Eppendorf) and mixed. The test tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. Add 750 μL of cooled acid-salt wash reagent (Biocolor) and centrifuge (4°C, 12,000×g, 10 minutes) to remove the supernatant. Add 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) to mix. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 555 nm using an absorbance microplate reader.

[0188] Antisense LNAGapmeR (SEQ ID NO: 5) was used as the ASO sequence.

[0189] Figure 8 The results of collagen secretion when the Antisense LNA GapmeR against NDR2 was introduced into LL29 cells are shown. Collagen secretion was inhibited in a concentration-dependent manner of NDR2 ASO. Therefore, it was shown that collagen secretion was inhibited by concentration-dependent knockdown of NDR2 ASO.

[0190] [Example 5: Knockdown of NDR2 using siRNA in human IPF lung fibroblasts]

[0191] The effect of siRNA on the expression of NDR2 was analyzed in LL29 cells (ATCC), a human lung fibroblast cell line established from the lungs of IPF patients.

[0192] siRNA against human NDR2 (ON-TARGET plus siRNA or ON-TARGET plus pool siRNA, Dharmacon or Silencer Select siRNA, Thermo Fisher Scientific) was transfected into LL29 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. To prepare the siRNA transfection solution, 50 μL of Opti-MEM solution was used to form 2 μL of RNAiMAX transfection reagent (Transfection Reagent), and the two were mixed to make an RNAiMAX prepared solution. The prepared solution (siRNA diluent) prepared by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, LL29 cells were plated at a rate of 2.5×10 4 500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (the final concentration of ON-TARGET plus siRNA and ON-TARGET plus pool siRNA was 30 nM, and the final concentration of Silencer Select siRNA was 10 nM). 48 hours after transfection, the culture medium was replaced with F-12K culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 48 hours or 24 hours after TGF-β1 stimulation, RNA was extracted from the cells using RNeasy 96 Kit (Qiagen). The obtained RNA was subjected to a reverse transcription reaction using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific), and the transcription level of GAPDH as an internal standard was compared with the value measured by GAPDH Taqman Gene Expression Assays (Hs02786624_g1) as the transcription level of the gene.

[0193] The siRNA sequences used were ON-TARGET plus siRNA (SEQ ID NOs. 23 and 30), ON-TARGET plus siRNA (SEQ ID NOs. 24 and 31), ON-TARGET plus pool siRNA (SEQ ID NOs. 23 to 26 and SEQ ID NOs. 30 to 33 mixed in equal amounts), and Silencer Select siRNA (SEQ ID NOs. 27 and 34). However, the presence or absence of additional sequences was unknown for ON-TARGET plus siRNA.

[0194] <ON-TARGET plus siRNA>

[0195] siNDR2: Sense strand: 5'-ACAACACGCUCGCAAAGAA-3' (SEQ ID NO: 30)

[0196] Antisense strand: 5'-UUCUUUGCGAGCGUGUUGU-3' (SEQ ID NO: 23)

[0197] siNDR2: positive strand: 5'-GAAAGAAACUCUGGUAUUU-3' (SEQ ID NO: 31)

[0198] Antisense strand: 5'-AAAUACCAGAGUUUCUUUC-3' (SEQ ID NO: 24)

[0199] siNDR2: positive strand: 5'-GAAACACAGUUCUACAUUU-3' (SEQ ID NO: 32)

[0200] Antisense strand: 5'-AAAUGUAGAACUGUGUUUC-3' (SEQ ID NO: 25)

[0201] siNDR2: positive strand: 5'-GCAGACUGGUUACAACAAA-3' (SEQ ID NO: 33)

[0202] Antisense strand: 5'-UUUGUUGUAACCAGUCUGC-3' (SEQ ID NO: 26)

[0203] <Silencer Select siRNA>

[0204] siNDR2: positive strand: 5'-GGUUUGAAGGGUUGACUCAtt-3' (SEQ ID NO: 34)

[0205] Antisense strand: 5'-UGAGUCAACCCUUCAAACCtt-3' (SEQ ID NO: 27)

[0206] Fig. 9 The graph shows the results of real-time PCR for NDR2 measured from RNA extracted 48 hours after the addition of TGF-β1 protein when ON-TARGET plus siRNA against NDR2 was introduced into LL29 cells. Fig.10 The graph shows the results of real-time PCR for NDR2 measured from RNA extracted 24 hours after the addition of TGF-β1 protein when ON-TARGETplus pool siRNA was introduced. Fig.11 The results of real-time PCR of NDR2 obtained by measuring RNA extracted 24 hours after the addition of TGF-β1 protein when Silencer Select siRNA was introduced. The introduction of NDR2 siRNA inhibited the transcription of NDR2 gene induced by TGF-β1. Therefore, it was shown that the NDR2 siRNA used effectively inhibited the expression of the target gene.

[0207] [Example 6: Effect of NDR2 siRNA on collagen secretion in human IPF lung fibroblasts]

[0208] To investigate the possibility that NDR2 controls collagen secretion in LL29 cells (ATCC), a human lung fibroblast cell line established from the lungs of IPF patients, the amount of collagen secreted by cells transfected with NDR2 siRNA was analyzed.

[0209] siRNA against human NDR2 (ON-TARGET plus siRNA or ON-TARGET plus pool siRNA (Dharmacon) or Silencer Select siRNA (Thermo Fisher Scientific)) was transfected into LL29 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. To prepare the siRNA transfection solution, Opti-MEM solution was mixed at a ratio of 50 μL to 2 μL of RNAiMAX transfection reagent to prepare an RNAiMAX prepared solution. The prepared solution (siRNA dilution solution) obtained by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. LL29 cells were plated at a rate of 2.5×10 4500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (the final concentration of ON-TARGET plus siRNA or ON-TARGET plus pool siRNA was 30 nM, and the final concentration of Silencer Select siRNA was 10 nM). 48 hours after transfection, the medium was replaced with F-12K medium containing 0.2% FBS. 24 hours after the medium replacement, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 400 μL of the culture supernatant after 48 hours or 72 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind test tube (Eppendorf) and mixed. The test tube was placed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. Add 750 μL of cooled acid-salt washing reagent (Acid-Salt Wash Reagent) (Biocolor) and centrifuge (4°C, 12,000×g, 10 minutes) to remove the supernatant. Add 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) to mix. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 555 nm using an absorbance microplate reader.

[0210] As siRNA sequences, ON-TARGET plus siRNA (SEQ ID NOs. 23 and 30), ON-TARGET plus siRNA (SEQ ID NOs. 24 and 31), ON-TARGET plus pool siRNA (SEQ ID NOs. 23 to 26 and SEQ ID NOs. 30 to 33 mixed in equal amounts), or Silencer Select siRNA (SEQ ID NOs. 27 and 34) were used.

[0211] Fig.12 The graph shows the results of collagen secretion in the supernatant collected 48 hours after the addition of TGF-β1 protein when ON-TARGET plus siRNA against NDR2 was introduced into LL29 cells. Fig.13 The results show the secretion of collagen in the supernatant collected 72 hours after the addition of TGF-β1 protein when ON-TARGET plus pool siRNA was introduced. Fig.14The results show the secretion of collagen in the supernatant collected 48 hours after the addition of TGF-β1 protein when Silencer Select siRNA was introduced. The introduction of NDR2 siRNA inhibited the secretion of collagen. Therefore, it is shown that the knockdown of NDR2 by NDR2 siRNA inhibits the secretion of collagen.

[0212] [Example 7: Knockdown of NDR2 by ASO in human hepatic stellate cells]

[0213] The effects of ASOs on NDR2 expression were analyzed in primary human hepatic stellate cells isolated from human liver (Samsara Sciences).

[0214] ASO for human NDR2 (Antisense LNA GapmeR, Qiagen) was transfected into human primary hepatic stellate cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions for the Lipofectamine RNAiMAX reagent. In order to prepare the ASO transfection solution, the two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent (Transfection Reagent) to make an RNAiMAX prepared solution. The prepared solution (ASO dilution solution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, human primary hepatic stellate cells were cultured at a rate of 2.5×10 4500 μL of cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (final concentration of ASO 100 nM). 48 hours after transfection, the medium was replaced with D-MEM medium containing 0.2% FBS. 24 hours after the medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. RNA was extracted from the cells using RNeasy 96 Kit (Qiagen) 48 hours after TGF-β1 stimulation. The obtained RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific), and the transcription level of GAPDH as an internal standard was compared with the value measured by GAPDH Taqman Gene Expression Assays (Hs02786624_g1) as the transcription level of the gene.

[0215] Antisense LNAGapmeR (SEQ ID NOs. 3, 4, 5, and 6) was used as the ASO sequence.

[0216] <Antisense LNA GapmeR>

[0217] ASONDR2: 5'-CAGTTGACTAGATAGG-3' (SEQ ID NO: 3)

[0218] ASONDR2: 5'-GATCGACGTAACTTTT-3' (SEQ ID NO: 4)

[0219] ASONDR2: 5'-TTAACCAGCCATGATA-3' (SEQ ID NO: 5)

[0220] ASONDR2: 5'-CCATCTTCAGGAGTAT-3' (SEQ ID NO: 6)

[0221] Fig.15 The results of real-time PCR of NDR2 when Antisense LNA GapmeR against NDR2 was introduced into human hepatic stellate cells. The introduction of NDR2 ASO suppressed the transcription of NDR2 gene. Therefore, it was shown that the used NDR2 ASO effectively suppressed the expression of target gene in human hepatic stellate cells.

[0222] [Example 8: Effect of NDR2 ASO on collagen secretion in human hepatic stellate cells]

[0223] To investigate the possibility that NDR2 controls collagen secretion in primary human hepatic stellate cells isolated from human liver (Samsara Sciences), the amount of collagen secreted by cells transfected with NDR2 ASO was analyzed.

[0224] ASO for human NDR2 (Antisense LNA GapmeR, Qiagen) was transfected into human primary hepatic stellate cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions for the Lipofectamine RNAiMAX reagent. In order to prepare the ASO transfection solution, the two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent (Transfection Reagent) to make an RNAiMAX prepared solution. The prepared solution (ASO dilution solution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, human primary hepatic stellate cells were cultured at a rate of 2.5×10 4 500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (final concentration of ASO 100 nM). 48 hours after transfection, the medium was replaced with D-MEM medium containing 0.2% FBS. 24 hours after the medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 300 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind test tube (Eppendorf) and mixed. The test tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. Add 750 μL of cooled acid-salt wash reagent (Biocolor) and centrifuge (4°C, 12,000×g, 10 minutes) to remove the supernatant. Add 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) to mix. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 555 nm using an absorbance microplate reader.

[0225] Antisense LNA GapmeR (SEQ ID NOs. 3, 4, 5, and 6) was used as the ASO sequence.

[0226] Fig.16 The results of measuring collagen secretion when Antisense LNA GapmeR against NDR2 was introduced into human hepatic stellate cells. By using NDR2 ASO, the gene expression of NDR2 was reduced, and the collagen secretion increased by TGF-β1 was reduced. Therefore, it was shown that the knockdown of NDR2 by NDR2 ASO in human hepatic stellate cells inhibited collagen secretion.

[0227] [Example 9: Knockdown of NDR2 by siRNA in human hepatic stellate cells]

[0228] The effects of siRNA on NDR2 expression in primary human hepatic stellate cells (Samsara Sciences) isolated from human liver were analyzed.

[0229] siRNA against human NDR2 (ON-TARGET plus siRNA (Dharmacon) or SilencerSelect siRNA (Thermo Fisher Scientific)) was transfected into human primary hepatic stellate cells using Lipofectamine RNAiMAX (ThermoFisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. In order to prepare the siRNA transfection solution, the two were mixed at a ratio of 50 μL of Opti-MEM solution to 1 μL of RNAiMAX transfection reagent (Transfection Reagent) to prepare an RNAiMAX prepared solution. The prepared solution (siRNA dilution solution) obtained by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, human primary hepatic stellate cells were cultured at a rate of 2.5×10 4 500 μL of the cell suspension was inoculated per cell / well and allowed to stand in a CO2 incubator (the final concentration of ON-TARGET plus siRNA was 10 nM, and the final concentration of SilencerSelect siRNA was 3 nM). 48 hours after transfection, the culture medium was replaced with D-MEM containing 0.2% FBS.

[0230] After 24 hours of medium replacement, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. RNA was extracted from cells using RNeasy96Kit (Qiagen) 48 hours after TGF-β1 stimulation. The obtained RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (High Capacity cDNA Reverse Transcription Kit) (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific), and the transcription level of GAPDH as an internal standard was compared with the value measured by GAPDH Taqman Gene Expression Assays (Hs02786624_g1) as the transcription level of the gene.

[0231] As siRNA sequences, ON-TARGET plus siRNA (SEQ ID NOs. 23 and 30), ON-TARGET plus siRNA (SEQ ID NOs. 24 and 31), and Silencer Select siRNA (SEQ ID NOs. 27 and 34) were used.

[0232] Fig.17 The results of real-time PCR of NDR2 when ON-TARGET plus siRNA against NDR2 was introduced into human hepatic stellate cells. Fig.18 The results of real-time PCR of NDR2 when Silencer Select siRNA was introduced into human hepatic stellate cells. The introduction of NDR2 siRNA suppressed the transcription of NDR2 gene. Therefore, it was shown that the NDR2 siRNA used suppressed the expression of target gene in human hepatic stellate cells.

[0233] [Example 10: Effect of knockdown of NDR2 by siRNA on collagen secretion in human hepatic stellate cells]

[0234] To investigate the possibility that NDR2 controls collagen secretion in primary human hepatic stellate cells isolated from human liver (Samsara Sciences), the amount of collagen secreted by cells transfected with NDR2 siRNA was analyzed.

[0235] siRNA against human NDR2 (ON-TARGET plus siRNA (Dharmacon) or SilencerSelect siRNA (Thermo Fisher Scientific)) was transfected into human primary hepatic stellate cells using Lipofectamine RNAiMAX (ThermoFisher Scientific) according to the instructions for the Lipofectamine RNAiMAX reagent. To prepare the siRNA transfection solution, the two were mixed at a ratio of 50 μL of Opti-MEM solution to 1 μL of RNAiMAX transfection reagent to prepare an RNAiMAX prepared solution. The prepared solution (siRNA dilution solution) obtained by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, human primary hepatic stellate cells were cultured at a rate of 2.5×10 4 500 μL of the cell suspension was inoculated per cell / well and allowed to stand in a CO2 incubator (the final concentration of ON-TARGET plus siRNA was 10 nM, and the final concentration of SilencerSelect siRNA was 3 nM). 48 hours after transfection, the culture medium was replaced with D-MEM containing 0.2% FBS.

[0236] 24 hours after the medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 300 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind test tube (Eppendorf) and mixed. The test tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. 750 μL of cooled acid-salt wash reagent (Acid-Salt Wash Reagent) (Biocolor) was added thereto, and after centrifugation (4°C, 12,000×g, 10 minutes), the supernatant was removed. 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) was added thereto and mixed. 200 μL of the mixed solution was transferred into a 96-well plate, and the absorbance at 555 nm was measured using an absorbance microplate reader.

[0237] As siRNA sequences, ON-TARGET plus siRNA (SEQ ID NOs. 23 and 30), ON-TARGET plus siRNA (SEQ ID NOs. 24 and 31), and Silencer Select siRNA (SEQ ID NOs. 27 and 34) were used.

[0238] Fig.19 The results show the results of collagen secretion when ON-TARGET plus siRNA against NDR2 was introduced into human hepatic stellate cells. Fig. 20 The results show the results of collagen secretion when Silencer Select siRNA was introduced into human hepatic stellate cells. The introduction of NDR2 siRNA inhibited collagen secretion. Therefore, it is shown that knockdown of NDR2 by NDR2 siRNA can inhibit collagen secretion in human hepatic stellate cells.

[0239] [Example 11: Effect of knockdown of NDR2 by siRNA on collagen secretion in human renal fibroblasts]

[0240] The effects of siRNA on NDR2 expression in cultured human renal fibroblast HRF cells (Cell Biologics) and on collagen secretion were analyzed.

[0241] siRNA against human NDR2 (ON-TARGET plus pool siRNA (Dharmacon)) was transfected into HRF cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of Lipofectamine RNAiMAX reagent. HRF cells were plated in 24-well plates at 6.0×10 4500 μL of cell suspension was inoculated in cells / well and left to stand in a CO2 incubator for 24 hours. RNAiMAX preparation solution was prepared by mixing Opti-MEM solution at a ratio of 25 μL to RNAiMAX transfection reagent transfection (Transfection Reagent Transfection) 1 μL. The preparation solution (siRNA dilution solution) obtained by diluting siRNA with Opti-MEM was mixed with RNAiMAX preparation solution in equal amounts. Maintenance medium (basic medium containing 9% fetal bovine serum, 0.9% L-glutamine (L-Glutamone), 0.09% FGF and 0.09% hydrocortisone) was added to 50 μL of the mixed solution at a ratio of 450 μL, and 500 μL (final concentration of siRNA 30 nM) was added to each well of a 24-well plate. After 24 hours of transfection, the medium was replaced with Complete Fibroblast Medium (Complete Fibroblast Medium) / w (CellBiologics). 24 hours after the medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL.

[0242] RNA was extracted from cells using RNeasy Mini Kit (Qiagen) after 48 hours of TGF-β1 stimulation. The obtained RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Hs00960027_m1, Thermo Fisher Scientific), and the transcription level of GAPDH as an internal standard was measured by GAPDH Taqman Gene Expression Assays (Hs02786624_g1) as the relative ratio of the value obtained.

[0243] In the collagen secretion assay, Anero Pack Kenki 5% and O2 meter were added to the Pouch bag to set hypoxia (1% O2) conditions, and a 24-well plate was placed therein and allowed to stand in a CO2 incubator. 350 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind test tube (Eppendorf) and mixed. The test tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. 750 μL of cooled acid-salt wash reagent (Acid-Salt Wash Reagent) (Biocolor) was added thereto, and after centrifugation (4°C, 12,000×g, 10 minutes), the supernatant was removed. 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) was added thereto and mixed, and 200 μL of the mixed solution was transferred to a 96-well plate, and the absorbance at 555 nm was measured using an absorbance microplate reader.

[0244] As the siRNA sequence, ON-TARGET plus pool siRNA (sequence numbers 23 to 26 and sequence numbers 30 to 33 mixed in equal amounts) was used.

[0245] Fig.21 The results of real-time PCR of NDR2 when ON-TARGET plus pool siRNA against NDR2 was introduced into human renal fibroblasts are shown. Fig. 22 The results show collagen secretion. The introduction of NDR2 siRNA inhibited the transcription of NDR2 gene in human renal fibroblasts. In addition, the introduction of NDR2 siRNA inhibited collagen secretion. Therefore, it is shown that the secretion of collagen in human renal fibroblasts can be inhibited by knocking down NDR2.

[0246] [Example 12: ASO concentration-dependent knockdown of NDR2 in mouse lung fibroblasts]

[0247] The effect of ASO on the expression of NDR2 in cultured lung fibroblasts MLg-2908 (ATCC) from normal mice was analyzed.

[0248] The ASO for mouse NDR2 (Antisense LNA GapmeR, Qiagen) was transfected into MLg-2908 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the Lipofectamine RNAiMAX Reagent Protocol. The two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent (Transfection Reagent) to prepare an RNAiMAX prepared solution. The prepared solution (ASO dilution) obtained by diluting ASO with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, MLg-2908 cells were placed in a 24-well plate at a rate of 4.0×10 4 500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator (adjusted to the target final concentration of ASO). 24 hours after transfection, the culture medium was replaced with MEM culture medium containing 0.2% FBS. 24 hours after the culture medium was replaced, human TGF-β1 protein (Peprotech) was added to a final concentration of 3 ng / mL. 18 hours after TGF-β1 stimulation, RNA was extracted from the cells using RNeasy96Kit (Qiagen). The obtained RNA was subjected to reverse transcription reaction using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Mm00730941_s1, Thermo Fisher Scientific), and the relative ratio of the transcription level of 18S as an internal standard to the value measured by 18S Taqman Gene Expression Assays (Hs99999901_s1) was used as the transcription level of the gene.

[0249] The sequence of ASO uses Antisense LNA GapmeR. (Sequence No. 15 and 17)

[0250] <Antisense LNA GapmeR>

[0251] ASONDR2: 5'-AGTTGATTAAGATAGG-3' (SEQ ID NO: 15)

[0252] ASONDR2: 5'-TTACCTGGATTGACAA-3' (SEQ ID NO: 17)

[0253] Fig.23 The results of real-time PCR of NDR2 when Antisense LNA GapmeR against NDR2 was introduced into MLg-2908 cells. Introduction of NDR2 ASO suppressed the transcription level of NDR2 gene in mouse lung fibroblasts in an ASO concentration-dependent manner.

[0254] [Example 13: Effect of NDR2 ASO on procollagen secretion in mouse lung fibroblasts]

[0255] To investigate the possibility that NDR2 controls collagen secretion in cultured lung fibroblasts MLg-2908 (ATCC) from normal mice, the amount of procollagen secreted from cells transfected with NDR2 ASO was analyzed.

[0256] ASO for mouse NDR2 (Antisense LNA GapmeR, Qiagen) was transfected into MLg-2908 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent transfection (Transfection Reagent Transfection) to prepare RNAiMAX prepared solution. The prepared solution (ASO dilution) obtained by diluting ASO with Opti-MEM was mixed with RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, MLg-2908 cells were placed in a 24-well plate at a rate of 4.0×10 4 500 μL of cell suspension was inoculated in a cell / well manner and allowed to stand in a CO2 incubator (adjusted to the target final concentration of ASO). 24 hours after transfection, the culture medium was replaced with MEM culture medium containing 0.2% FBS. Human TGF-β1 protein (Peprotech) was added 24 hours after the culture medium was replaced to a final concentration of 3 ng / mL. The culture supernatant after 48 hours of TGF-β1 stimulation was treated according to the method of Mouse Pro-Collagen I alpha 1SimleStep ELISA Kit (Abcam), and the absorbance of the obtained sample was measured at 450 nm using an absorbance microplate reader.

[0257] The sequence of ASO used was Antisense LNA GapmeR (SEQ ID NOs. 15 and 17).

[0258] Fig.24 The results of procollagen secretion when Antisense LNA GapmeR against NDR2 was introduced into MLg-2908 cells. By using ASO against NDR2, the gene expression of NDR2 was reduced, and the amount of procollagen secretion increased by TGF-β was reduced in an ASO concentration-dependent manner. Therefore, it was shown that procollagen secretion can be inhibited by knocking down NDR2 in mouse lung fibroblasts.

[0259] [Example 14: Knockdown of NDR2 in mouse hepatic stellate cells using siRNA]

[0260] The effect of siRNA on the expression of NDR2 in mouse primary hepatic stellate cells (ScienCell) isolated from mouse liver was analyzed.

[0261] siRNA for mouse NDR2 (Ambion In Vivo siRNA, Thermo Fisher Scientific) was transfected into mouse primary hepatic stellate cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions for the Lipofectamine RNAiMAX reagent. The two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent transfection (Transfection Reagent Transfection) to prepare an RNAiMAX prepared solution. The prepared solution (siRNA dilution solution) obtained by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts and 100 μL was added to each well of a 24-well plate. Mouse primary hepatic stellate cells were plated in a 24-well plate at a rate of 2.8×10 4500 μL of cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator for 24 hours (final concentration of siRNA 10 nM). 24 hours after transfection, the culture medium was replaced with a stellate cell culture medium (StellateCell Medium) (ScienCell) containing 0.2% FBS. Human TGF-β1 protein (Peprotech) was added 24 hours after the culture medium was replaced to a final concentration of 10 ng / mL. RNA was extracted from the cells using RNeasy 96 Kit (Qiagen) 24 hours after TGF-β1 stimulation. The obtained RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Mm00730941_s1, Thermo Fisher Scientific), and the ratio of the transcription level of 18S as an internal standard to the value measured by 18S Taqman Gene Expression Assays (Hs99999901_s1) was used as the transcription level of the gene.

[0262] As siRNA sequences, Ambion In Vivo siRNA (Thermo Fisher Scientific) (SEQ ID NOs. 28 and 35, and SEQ ID NOs. 29 and 36) were used.

[0263] <Ambion In Vivo siRNA>

[0264] siNDR2: positive strand: 5'-CAGACAACCUUUUACUGGAtt-3' (SEQ ID NO: 35)

[0265] Antisense strand: 5'-UCCAGUAAAAGGUUGUCUGgt-3' (SEQ ID NO: 28)

[0266] siNDR2: positive strand: 5'-CCAUACCCGGGAAAGAGUAtt-3' (SEQ ID NO: 36)

[0267] Antisense strand: 5'-UACUCUUUCCCGGGUAUGGtt-3' (SEQ ID NO: 29)

[0268] Fig.25The figure shows the results of real-time PCR of NDR2 when Ambion In Vivo siRNA against NDR2 was introduced into mouse hepatic stellate cells. The introduction of NDR2 siRNA suppressed the transcription level of NDR2 gene in mouse hepatic stellate cells.

[0269] [Example 15: Effects of NDR2 knockdown on collagen secretion in mouse hepatic stellate cells]

[0270] To investigate the possibility that NDR2 controls collagen secretion in mouse hepatic stellate cells, the amount of collagen secreted by cells transfected with NDR2 siRNA (Ambion In Vivo siRNA (Thermo Fisher)) was analyzed.

[0271] siRNA for mouse NDR2 (Ambion In Vivo siRNA, Thermo Fisher Scientific) was transfected into mouse primary hepatic stellate cells isolated from mouse liver (ScienCell) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the instructions of the Lipofectamine RNAiMAX reagent. The two were mixed at a ratio of 50 μL of Opti-MEM solution to 2 μL of RNAiMAX transfection reagent transfection (Transfection Reagent Transfection) to prepare RNAiMAX prepared solution. The nucleic acid dilution obtained by diluting siRNA with Opti-MEM was mixed with the RNAiMAX prepared solution in equal amounts, and 100 μL was added to each well of a 24-well plate. Among them, mouse primary hepatic stellate cells were plated in a 24-well plate at a ratio of 2.8×10 4500 μL of the cell suspension was inoculated in a cell / well format and allowed to stand in a CO2 incubator for 24 hours (final concentration of siRNA: 10 nM). 24 hours after transfection, the culture medium was replaced with a stellate cell culture medium (ScienCell) containing 0.2% FBS. Human TGF-β1 protein (Peprotech) was added 24 hours after the culture medium was replaced to a final concentration of 10 ng / mL. 400 μL of the culture supernatant after 48 hours of TGF-β1 stimulation was added to 1 mL of ice-cold Sircol Dye Reagent (Biocolor) in a Protein LoBind tube (Eppendorf) and mixed. The tube was installed in a small rotary incubator, mixed at 25°C for 30 minutes, and then centrifuged (4°C, 12,000×g, 10 minutes), and the supernatant was removed. Add 750 μL of cooled acid-salt wash reagent (Acid-Salt Wash Reagent) (Biocolor) and centrifuge (4°C, 12,000×g, 10 minutes) to remove the supernatant. Add 250 μL of Sircol Collagen Assay ALKALI Reagent (Biocolor) to mix. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 555 nm using an absorbance microplate reader.

[0272] As siRNA sequences, Ambion In Vivo siRNA (Thermo Fisher Scientific) (SEQ ID NOs. 28 and 35, and SEQ ID NOs. 29 and 36) were used.

[0273] Fig.26 The results of collagen secretion when Ambion In Vivo siRNA against NDR2 was introduced into mouse hepatic stellate cells. By using siRNA against NDR2, the gene expression of NDR2 was reduced, and the collagen secretion increased by TGF-β1 was reduced. Therefore, it was shown that collagen secretion can be inhibited in mouse hepatic stellate cells by knocking down NDR2.

[0274] [Example 16: Knockdown of NDR2 in mouse lung using ASO]

[0275] The effects of NDR2 ASO on the transcriptional levels of target genes in mouse lungs were analyzed.

[0276] 9-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan) were divided into groups according to the body weight of the day before the first administration of NDR2 ASO, and the groups were set as D-PBS administration group (n=6), nucleic acid single (QW) administration group (n=6) and nucleic acid 3 times a week (TIW) administration group (n=6). NDR2 ASO was prepared into a 10 mg / mL nucleic acid stock solution using ultrapure water. In the case of the QW administration group, the nucleic acid administration group was orally administered with a nucleic acid administration solution containing NDR2 ASO prepared using D-PBS (-) (Nacalai Tesque) as an administration solvent in a manner of 1, 3 or 10 mg / kg body weight (mpk), and the solvent administration group was orally administered with an equal amount of administration solvent as the nucleic acid administration group on day 0. In the TIW administration group, a nucleic acid administration solution containing NDR2ASO prepared using D-PBS(-) (Nacalai Tesque) as an administration solvent was orally administered at 1 mg / kg body weight on days 0, 2, and 4. Gene expression analysis was performed 72 hours after the final administration.

[0277] The lungs collected 72 hours after the final administration were crushed with a frozen SK mill under liquid nitrogen, and RNA was extracted using QIAZOLLysis reagent (Qiagen). RNA was then purified using RNeasy mini Kit (Qiagen), and reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Mm00730941_s1, Thermo Fisher Scientific), and the relative ratio of the transcription level of 18S as an internal standard to the value measured by 18S Taqman Gene Expression Assays (Hs99999901_s1) was used as the transcription level of the gene.

[0278] Antisense LNAGapmeR (SEQ ID NO: 14) was used as the ASO sequence.

[0279] <Antisense LNA GapmeR>

[0280] ASONDR2: 5'-TATGCCCAGTTGATTA-3' (SEQ ID NO: 14)

[0281] Fig. 27The figure shows the amount of NDR2 gene transcription in the lungs when the Antisense LNA GapmeR against NDR2 was administered to normal mice. The amount of NDR2 gene transcription decreased by the administration of NDR2 ASO. This indicates that the NDR2 Antisense LNA GapmeR used effectively inhibited the expression of the target gene in the lungs.

[0282] [Example 17: Knockdown of NDR2 in mouse liver using ASO]

[0283] The effects of NDR2 ASO on the transcriptional levels of target genes in mouse liver were analyzed.

[0284] For 9-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan), the body weight of the mice measured on the day of the first administration of NDR2 ASO was used to group them according to the stratified random grouping of EXSUS (n=3 for each group). NDR2 ASO was made into a 10 mg / mL nucleic acid stock solution using ultrapure water. For the nucleic acid administration group, a nucleic acid administration solution containing NDR2 ASO prepared using D-PBS (-) (Nacalai Tesque) as an administration solvent was subcutaneously administered (sc administration) or caudal vein administration (iv administration) in a manner of 10 or 30 mg / kg body weight. For the solvent administration group, an equal amount of administration solvent as the nucleic acid administration group was administered subcutaneously or by caudal vein administration. Analysis of gene expression was performed 48 hours after administration.

[0285] The liver collected 48 hours after administration was crushed under liquid nitrogen using a frozen SK mill, and RNA was extracted using QIAZOLLysis reagent (Qiagen). Then, RNA was purified using RNeasy mini Kit (Qiagen), and reverse transcription was performed using a high-capacity cDNA reverse transcription kit (High Capacity cDNA Reverse Transcription Kit) (Applied Biosystems). The transcription amount of NDR2 was measured using NDR2 Taqman Gene Expression Assays (Mm00730941_s1, Thermo Fisher Scientific), and the relative ratio of the transcription amount of 18S as an internal standard to the value measured by 18S Taqman Gene Expression Assays (Hs9999901_s1) was used as the transcription amount of the gene.

[0286] Antisense LNA GapmeR (SEQ ID NO: 14) was used as the ASO sequence.

[0287] Fig.28 It shows the amount of NDR2 gene transcription in the liver when NDR2 ASO is administered subcutaneously or intravenously. The amount of NDR2 gene transcription is reduced by the administration of NDR2 ASO. This shows that the NDR2 Antisense LNAGapmeR used effectively inhibits the expression of the target gene in the liver during subcutaneous administration or intravenous administration.

[0288] [Example 18: Effects of NDR2 knockdown in mouse liver on similar genes]

[0289] The effect of NDR2 siRNA on the transcription level of the similar gene NDR1 in normal mouse liver was analyzed.

[0290] 8-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan) were divided into PBS-administered groups (n=3) or nucleic acid-administered groups (n=3) according to their body weight the day before NDR2 siRNA administration. For Ambion In Vivo siRNA (Thermo Fisher Scientific) as NDR2 siRNA and Ambion In Vivo Negative control siRNA (Thermo Fisher Scientific) as control siRNA, invivofectamine 3.0 Reagent (Thermo Fisher Scientific) was used to prepare 0.2 or 0.3 mg / mL nucleic acid administration solutions according to the product instructions of invivofectamine 3.0. For the nucleic acid administration group, the nucleic acid administration solution containing Ambion InVivo siRNA was administered via the tail vein in a manner of 2 or 3 mg / kg body weight, and for the solvent administration group, PBS (Thermo Fisher Scientific) equal to that of the nucleic acid administration group was administered via the tail vein.

[0291] The liver collected 72 hours after administration was crushed under liquid nitrogen using a cryo-crushing SK mill, and RNA was extracted using QIAZOL Lysisreagent (Qiagen). RNA was then purified using RNeasy mini Kit (Qiagen), and reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The transcription level of the NDR2 gene was determined by the relative ratio of the value measured by NDR2 Taqman Gene Expression Assays (Mm00730941_s1, Thermo Fisher Scientific) and the value measured by 18S Taqman Gene Expression Assays (Hs99999901_s1). The transcription level of the NDR1 gene was determined by the relative ratio of the value measured by NDR1 Taqman Gene Expression Assays (Mm00506497_m1, Thermo Fisher Scientific) and the value measured by 18S Taqman Gene Expression Assays (Hs99999901_s1).

[0292] As the siRNA sequence, Ambion In Vivo siRNA (SEQ ID NOs. 28 and 35) was used.

[0293] Fig.29 represents the amount of NDR2 gene transcription when NDR2 siRNA is administered to normal mice, Fig.30 It shows the transcription amount of NDR1 gene when NDR2 siRNA is administered. The transcription amount of NDR2 gene in liver is reduced by the administration of NDR2 siRNA, but the transcription amount of NDR1 gene does not change. This shows that the NDR2 Ambion In Vivo siRNA used has no effect on the expression of NDR1 gene as a similar gene.

[0294] [Example 19: Effects on drug metabolizing enzymes in normal mice by knockdown of NDR2]

[0295] In order to confirm the effect of NDR2 knockdown on drug-metabolizing enzymes, the effect of NDR2 siRNA administration via the tail vein of normal mice on the drug-metabolizing enzyme CYP2E1 (Cytochrome P450 Family 2 Subfamily E Member 1) protein was analyzed.

[0296] 8-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan) were grouped according to the body weight on the day before the administration of NDR2 siRNA, as a PBS administration group (n=3) or a nucleic acid administration group (n=3). Ambion In Vivo siRNA (Thermo Fisher Scientific) as NDR2 siRNA and Ambion In Vivo Negative control siRNA (Thermo Fisher Scientific) as control siRNA were used. Invivofectamine 3.0 Reagent (Thermo Fisher Scientific) was used to prepare a 0.2 or 0.3 mg / mL nucleic acid administration solution according to the product instructions of invivofectamine 3.0. For the nucleic acid administration group, the nucleic acid administration solution containing Ambion In Vivo siRNA or Ambion In Vivo Negative control siRNA was administered by tail vein administration at 2 or 3 mg / kg body weight, and for the solvent administration group, PBS (Thermo Fisher Scientific) was administered by tail vein administration in an amount equal to that of the nucleic acid administration group.

[0297] The liver collected 72 hours after administration was frozen and then crushed using a freeze-crushing SK mill. A lysis buffer (150 mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM benzylsulfonyl fluoride, 2% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail) was added to the liver crushed material, and an organ extract was prepared using a handheld ultrasonic generator. β-mercaptoethanol and bromophenol blue were added to the supernatant obtained by centrifugation of the organ extract to a final concentration of 5% and 0.025%, respectively. Then, it was heated at 95°C for 4 minutes to prepare a sample. The obtained sample was subjected to SDS-PAGE to separate the proteins contained in the sample according to size. Then, the separated proteins were transferred to a PVDF membrane and western blotting was performed using an anti-CYP2E1 antibody (Abcam) and an anti-vinculin antibody (Sigma).

[0298] As the siRNA sequence, Ambion In Vivo siRNA (SEQ ID NOs. 28 and 35) was used.

[0299] Fig.31 The results of Western blotting of NDR2 protein and CYP2E1 protein when NDR2 was knocked down are shown. Fig.32 represents the value of NDR2 protein corrected with vinculin, Fig.33 The values ​​of CYP2E1 protein after correction with vinculin are shown in a graph. NDR2 protein was reduced by using siRNA against NDR2, but the amount of CYP2E1 protein did not change. Therefore, it was shown that knockdown of NDR2 had no effect on CYP2E1 protein in normal mice.

[0300] [Example 20: Effect of NDR2 siRNA on fibrosis disease-related genes in carbon tetrachloride (CCl4)-induced liver fibrosis model]

[0301] In order to confirm that knockdown of NDR2 is effective against fibrotic diseases, NDR2 siRNA was intravenously administered to carbon tetrachloride (CCl4)-induced liver fibrosis model mice (hereinafter referred to as CCl4 model mice), and the effect on the transcription levels of fibrotic disease-related genes was analyzed.

[0302] For 8-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan), an olive oil solution containing 12.5 v / v% CCl4 (Fujifilm Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at 10 mL / kg body weight on the 3rd and 6th days, with the initial nucleic acid administration day as day 0, to prepare liver fibrosis model mice. The mice were divided into groups according to the body weight on the day before the initial administration of NDR2 siRNA, and the groups were set as a PBS administration group (n=3) without CCl4 administration, a PBS administration group (n=3) with CCl4 administration, and a nucleic acid administration group (n=3) with CCl4 administration. For Ambion In Vivo siRNA (Thermo Fisher Scientific) as NDR2 siRNA and Ambion In Vivo Negative control siRNA (Thermo Fisher Scientific) as control siRNA, invivofectamine 3.0 Reagent (Thermo Fisher Scientific) was used to prepare a 0.2 mg / mL nucleic acid administration solution according to the product instructions of invivofectamine 3.0. For the nucleic acid administration group, the nucleic acid administration solution containing Ambion In Vivo siRNA or Ambion In Vivo Negative control siRNA was administered by tail vein at 2 mg / kg body weight, and the solvent administration group was administered with PBS (Thermo Fisher Scientific) in the same amount as the nucleic acid administration group on days 0 and 4. Evaluation of fibrosis was implemented on the 8th day.

[0303] RNA was extracted from the liver collected on day 8 using QIAZOL Lysis reagent (Qiagen). RNA was then purified using RNeasy mini Kit (Qiagen) and reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to obtain cDNA. The obtained cDNA was subjected to real-time PCR using TaqMan Fast Universal PCR master mix and Applied Biosystems 7500 Fast Real-Time PCR Systems (Applied Biosystems) to investigate the effect of knockdown of NDR2 on the transcription level of each gene. TaqMan Gene Expression Assays were used to measure the transcription levels of NDR2 Taqman Probe (Mm00491127_m1, Thermo Fisher Scientific), CYR61 Taqman Probe (Mm00487498_m1, Thermo Fisher Scientific), CTGF Taqman Probe (Mm01192933_g1, Thermo Fisher Scientific), TNF Taqman Probe (Mm00443258_m1, Thermo Fisher Scientific), CCL2 Taqman Probe (Mm00441242_m1, Thermo Fisher Scientific), COL1A1 Taqman Probe (Mm801666_g1, Thermo Fisher Scientific), and TGF-β1 Taqman Probe (Mm01178820_m1, Thermo Fisher Scientific), which are genes associated with fibrosis, and the expression of 18S The relative ratio of the transcription levels of Taqman Probe (Hs99999901_s1) was taken as the transcription level of each gene.

[0304] As the siRNA sequence, Ambion In Vivo siRNA (SEQ ID NOs. 28 and 35) was used.

[0305] Fig.34The graph shows the results of real-time PCR of NDR2 when Ambion In Vivo siRNA against NDR2 was administered to CCl4 model mice via the tail vein. Figures 35-40 The results of real-time PCR of CYR61, CTGF, TNF, CCL2, COL1A1 and TGF-β1, which are genes related to fibrosis diseases, under the same conditions are shown respectively. By using siRNA of NDR2, the gene expression of fibrosis promoting factors (CYR61 and CTGF), inflammatory markers (TNF and CCL2), and fibrosis markers (COL1A1 and TGF-β1) increased by CCl4 was reduced. Therefore, it is shown that the knockdown of NDR2 tends to improve fibrosis diseases in the liver of CCl4 model mice.

[0306] [Example 21: Analysis of fibrosis marker (αSMA) in CCl4 model]

[0307] To confirm that knockdown of NDR2 is effective in treating fibrotic diseases, NDR2 siRNA was intravenously administered to CCl4 model mice and the effects on fibrotic disease marker proteins were analyzed.

[0308] For 8-week-old male C57BL / 6J mice (Charles River Co., Ltd., Japan), an olive oil solution containing 12.5 v / v% CCl4 (Fujifilm Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at 10 mL / kg body weight on the 3rd and 6th days, with the first nucleic acid administration day as day 0, to prepare liver fibrosis model mice. The mice were divided into groups according to the body weight on the day before the first administration of NDR2 siRNA, and the groups were set as a PBS administration group (n=3) without CCl4 administration, a PBS administration group (n=3) with CCl4 administration, and a nucleic acid administration group (n=3) with CCl4 administration. For Ambion In Vivo siRNA (Thermo Fisher Scientific) as NDR2 siRNA and Ambion In Vivo Negative control siRNA (Thermo Fisher Scientific) as control siRNA, invivofectamine 3.0 Reagent (Thermo Fisher Scientific) was used to prepare a 0.2 mg / mL nucleic acid administration solution according to the product instructions of invivofectamine 3.0. For the nucleic acid administration group, the nucleic acid administration solution containing Ambion In Vivo siRNA or Ambion In Vivo Negative control siRNA was administered by tail vein at 2 mg / kg body weight, and for the solvent administration group, PBS (Thermo Fisher Scientific) of the same amount as the nucleic acid administration group was administered by tail vein on days 0 and 4.

[0309] The liver collected on the 8th day was frozen and then crushed using a freeze-crushing SK mill. Lysis buffer (150mM NaCl, 1% NP40, 0.1% SDS, 50mM Tris-HCl pH7.5, 1mM EDTA, 1mM benzylsulfonyl fluoride, 2% protease inhibitor mixture, 1% phosphatase inhibitor mixture) was added to the liver crushed material, and the organ extract was prepared using a handheld ultrasonic generator. β-Mercaptoethanol and bromophenol blue were added to the supernatant obtained by centrifugation of the organ extract to a final concentration of 5% and 0.025%, respectively. Then, it was heated at 95°C for 4 minutes as a sample. SDS-PAGE was performed using the obtained sample to separate the proteins contained in the sample according to size. Then, the separated proteins were transferred to a PVDF membrane and protein blotting was performed using anti-αSMA antibodies (Sigma) and anti-vinculin antibodies (Sigma).

[0310] As the siRNA sequence, Ambion In Vivo siRNA (SEQ ID NOs. 28 and 35) was used.

[0311] Fig.41 The results of Western blotting of αSMA protein when NDR2 was knocked down are shown. Fig.42 The values ​​of αSMA protein corrected by vinculin are shown in a graph. The amount of αSMA protein increased by CCl4 was reduced by using siRNA of NDR2. Therefore, it was shown that knockdown of NDR2 suppressed αSMA protein in the liver of the CCl4 model.

Claims

1. A therapeutic agent for fibrotic diseases, wherein: The therapeutic agent for fibrotic diseases contains a nucleic acid that inhibits the expression of the NDR2 gene as an effective ingredient.

2. The therapeutic agent according to claim 1, wherein The fibrotic disease is a pulmonary fibrotic disease, a liver fibrotic disease or a kidney fibrotic disease.

3. The therapeutic agent according to claim 1 or 2, wherein The nucleic acid that inhibits the expression of the NDR2 gene is an antisense nucleic acid.

4. The therapeutic agent according to claim 1 or 2, wherein The nucleic acid that inhibits the expression of NDR2 gene is siRNA.

5. A method for treating a fibrotic disease, wherein: The method for treating fibrotic diseases comprises the step of administering a nucleic acid that inhibits the expression of the NDR2 gene to a subject.

6. A nucleic acid, wherein The nucleic acid is used in the treatment of fibrotic diseases and inhibits the expression of NDR2 gene.

7. Use of a nucleic acid that inhibits the expression of NDR2 gene in the manufacture of a therapeutic agent for fibrotic diseases.

8. The method, nucleic acid or use according to any one of claims 5 to 7, wherein The fibrotic disease is a pulmonary fibrotic disease, a liver fibrotic disease or a kidney fibrotic disease.

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