Antisense oligonucleotide targeting SLC7a5 as target molecule

Abstract

The purpose of the present invention is to provide an antisense oligonucleotide that targets SLC7A5 as a target molecule and exhibits a pharmacological effect based on suppression of SLC7A5 expression in vivo.　Provided is an antisense oligonucleotide that targets SLC7A5 as a target molecule, the antisense oligonucleotide comprising a continuous nucleotide sequence of not less than 12 nucleotides included in the nucleotide sequence represented by SEQ ID NO: 1.

Description

Antisense oligonucleotides targeting SLC7A5

[0001] This invention relates to an antisense oligonucleotide that targets SLC7A5 (the gene encoding LAT1). Furthermore, this invention relates to a nucleic acid drug containing the antisense oligonucleotide.

[0002] Cells need to take in amino acids as nutrients, and this function is carried out by amino acid transporters, which are membrane proteins located in the cell membrane. Amino acid transporters are classified as "systems" based on their substrate selectivity. Among amino acid transporters, L-type amino acid transporter 1 (LAT1), which belongs to the System L family, is Na + As an independent neutral amino acid transporter, it is one of the most important transport mechanisms in cellular nutrition, responsible for supplying many essential amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine to cells.

[0003] LAT1 is located upstream of mTOR (mammalian target of rapamycin), which is responsible for regulating cell growth and proliferation, and is known to be specifically expressed in cancer cells. Its expression is upregulated in many cancers, including colorectal cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, pancreatic cancer, kidney cancer, laryngeal cancer, esophageal cancer, and brain tumors, and it has been reported that patients with high LAT1 expression have a poor prognosis in many cancers, including pancreatic cancer. Conventionally, LAT1 has been studied as a molecular target for cancer treatment. For example, it has been reported that the LAT1 high-affinity inhibitor KYT-0353 (JPH203) suppressed tumor growth in tumor-bearing nude mice. Furthermore, it has been reported that antisense oligoDNA against SLC7A5 (solute carrier type 7A5), the gene encoding LAT1, reduces LAT1 expression, thereby suppressing cancer cell proliferation and potentially extending the lifespan of peritoneal dissemination tumor-bearing mice (Non-Patent Literature 1). However, the base sequence of the antisense oligoDNA in question and specific experimental data have not been provided.

[0004] In recent years, SLC7A5 expression has been correlated with decreased lung function and increased severity in patients with idiopathic pulmonary fibrosis (IPF). It has been reported that inhibiting LAT1 activity using the high-affinity LAT1 inhibitor JPH203, or suppressing SLC7A5 expression using shRNA, significantly suppresses the activation of TGF-β-stimulated pro-fibrosis molecules such as mTOR, HIF, and c-Myc (Non-Patent Literature 2). Furthermore, pharmacological inhibition of LAT1 by JPH203 is sufficient to alleviate bleomycin-induced pulmonary fibrosis, suggesting that LAT1-targeted cell-selective therapy for IPF is a promising approach.

[0005] On the other hand, conventional attempts have been made to use oligonucleotides such as antisense oligonucleotides, siRNAs, aptamers, and miRNAs to treat intractable diseases. Currently, therapeutic agents using antisense oligonucleotides have been approved in Japan for amyotrophic lateral sclerosis, Duchenne muscular dystrophy, and spinal muscular atrophy (Non-Patent Literature 3).

[0006] Furthermore, therapeutic agents using oligonucleotides are sensitive to nuclease degradation, which can lead to inefficient delivery to target cells after systemic administration, posing a barrier to practical application. Therefore, efforts have been made to overcome these barriers by chemically modifying oligonucleotides. For example, it is known that introducing phosphorothioate bonds as modifications to the phosphate group of antisense oligonucleotides, and introducing 2'-F, 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), or 2',4'-crosslinked nucleic acids as modifications to the sugar group, can result in high binding ability to target RNA and excellent resistance to nucleases and pharmacokinetics (Non-Patent Literature 4).

[0007] Nagamori, Osamu; Cancer and Amino Acid Transporters; Biochemistry, Vol. 86, No. 3, pp. 338-344 (2014); M. Choudhury; Targeting the Amino Acid Transporter SLC7A5 for Pulmonary Fibrosis; American Journal of Respiratory and Critical Care Medicine 2024; 209:A6648; National Institute of Health Sciences, Department of Genetics and Medicine; Nucleic Acid Drugs Approved in Japan, the United States, or Europe (https: / / www.nihs.go.jp / mtgt / pdf / section2-1.pdf); Inoue, Takao; "Development and Market of Biopharmaceuticals, 2019, Chapter 4 Development Trends of Nucleic Acid Drugs," Reprint, CMC Publishing Co., Ltd.

[0008] In conventional technology, designing and chemically modifying antisense oligonucleotides often fails to produce sufficient efficacy in vitro. Furthermore, even when sufficient efficacy is obtained in vitro, it often fails to produce sufficient efficacy in vivo. Therefore, as mentioned above, SLC7A5 has been suggested as a potential novel target molecule for fibrosis treatment, and while methods for chemically modifying oligonucleotides have been developed, no antisense oligonucleotides targeting SLC7A5 that are clinically practical for the treatment of fibrosis and similar conditions have been developed.

[0009] Therefore, the object of the present invention is to provide an antisense oligonucleotide that targets SLC7A5 and exerts pharmacological effects in vivo based on the suppression of SLC7A5 expression.

[0010] The present inventors conducted diligent studies to solve the aforementioned problems and found that antisense oligonucleotides containing a sequence of 12 or more consecutive bases, as shown in Sequence ID No. 1, exert SLC7A5 expression inhibitory effects not only in vitro but also in vivo, and that in vivo, they can effectively produce antifibrotic and antitumor effects through the mechanism of suppressing SLC7A5 expression. The present invention was completed by further studies based on these findings.

[0011] That is, the present invention provides the invention in the following embodiments: Item 1. An antisense oligonucleotide targeting SLC7A5, having a continuous sequence of 12 or more bases included in the base sequence shown in Sequence ID No. 1. Item 2. The antisense oligonucleotide according to Item 1, having 12 to 40 bases. Item 3. The antisense oligonucleotide according to Item 1 or 2, consisting of the base sequence shown in Sequence ID No. 1. Item 4. The antisense oligonucleotide according to any one of Items 1 to 3, wherein at least one nucleotide is a 2',4'-bridged nucleotide. Item 5. The antisense oligonucleotide according to any one of Items 1 to 4, wherein at least one of the nucleoside binding sites is a phosphorothioate bond. Item 6. The antisense oligonucleotide according to any one of Items 1 to 5, wherein all nucleoside binding sites are phosphorothioate bonds, and the 1st to 3rd nucleotides from the 5' end and the 1st to 3rd nucleotides from the 3' end are 2',4'-bridged nucleotides. Item 7. An antisense oligonucleotide according to any one of items 1 to 6, which is a chemically modified antisense oligonucleotide consisting of the following sequence 1': Sequence 1': T^T^G^a^g^c^a^g^g^t^a^G^G^T [In sequence 1', "G" is guanine of the 2',4'-BNA structure, "T" is thymine of the 2',4'-BNA structure, "a" is adenine of unmodified DNA, "g" is guanine of unmodified DNA, "c" is cytosine of unmodified DNA, "t" is thymine of unmodified DNA, and "^" indicates a phosphorothioate bond.] Item 8. A nucleic acid drug comprising an antisense oligonucleotide according to any one of items 1 to 7. Item 9. A nucleic acid drug according to item 8, used for the treatment of fibrosis or a disease accompanied by fibrosis. Item 10. A nucleic acid drug according to item 9, wherein the fibrosis or disease accompanied by fibrosis is pulmonary fibrosis, hepatic fibrosis, interstitial lung disease accompanied by fibrosis, or liver disease accompanied by fibrosis. Item 11. The nucleic acid drug according to item 10, wherein the fibrosis or disease associated with fibrosis is idiopathic pulmonary fibrosis, interstitial pneumonia, interstitial lung disease associated with systemic scleroderma, hepatitis, cirrhosis, hepatolenticular degeneration, or biliary atresia. Item 12. The nucleic acid drug according to item 8, used for the treatment of tumors.Item 13. The nucleic acid drug according to Item 12, wherein the tumor is cervical cancer, colorectal cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, pancreatic cancer, kidney cancer, liver cancer, laryngeal cancer, esophageal cancer, or brain tumor. Item 14. Use of an antisense oligonucleotide according to any one of Items 1 to 7 for the manufacture of a treatment for fibrosis or a disease involving fibrosis. Item 15. A method for treating fibrosis or a disease involving fibrosis, comprising administering a therapeutically effective amount of an antisense oligonucleotide according to any one of Items 1 to 7 to a patient with fibrosis or a disease involving fibrosis.

[0012] The antisense oligonucleotides of the present invention can effectively suppress the expression of SLC7A5 in vivo, and can effectively exert antifibrotic and antitumor effects through the mechanism of suppressing SLC7A5 expression. Therefore, they can be used in the treatment of fibrosis; diseases involving airway remodeling, including fibrosis (thickening of the airway wall, etc.); diseases involving hepatic fibrosis; tumors, etc. Furthermore, even after chemical modification, the antisense oligonucleotides of the present invention effectively exert the suppressive effect on SLC7A5 expression, antifibrotic effect, and antitumor effect in vivo. Chemical modification can improve resistance to nucleases, affinity to target genes, and pharmacokinetics, thus meeting the required characteristics for clinical application.

[0013] This report presents the results of measuring the amount of SLC7A5 mRNA in human cervical cancer-derived cells (HeLa cells) treated with modified oligonucleotides that are candidate ASOs for SLC7A5. It also presents the results of measuring the amount of SLC7A5 mRNA in HeLa cells treated with 39 modified oligonucleotides that are candidate ASOs for SLC7A5. Furthermore, it describes the results of administering 11 modified oligonucleotides that are candidate ASOs for SLC7A5 to human SLC7A5-high-expression mice and measuring the suppression rate of SLC7A5 mRNA expression in the lungs. Finally, it presents the results of measuring the amount of LAT1 protein in HeLa cells treated with oligonucleotide (TR32110) by Western blotting. Lastly, it describes the results of observing HeLa cells treated with TR32110 after immunostaining and DAPI staining for LAT1. This document presents the results of measuring TGFβ1 / SMAD signal intensity in HeLa cells treated with TR32110 or a small molecule inhibitor of LAT1 (JPH203). It also presents the results of measuring the mRNA levels of SLC7A5, COL1A1, and ACTA2 in normal human lung fibroblasts (NHLF cells) treated with TR32110 and human TGF-β1. Furthermore, it shows the results of measuring collagen protein levels in NHLF cells treated with TR32110 and human TGF-β1. Finally, it presents the results of observing cells after staining collagen fibers in NHLF cells treated with TR32110 and human TGF-β1. The document also presents the results of measuring α-SMA protein levels in NHLF cells treated with TR32110 and human TGF-β1. Lastly, it shows the results of observing cells after immunostaining for α-SMA in NHLF cells treated with TR32110 and human TGF-β1. This report describes the results of administering 11 modified oligonucleotides, including TR32110, to human SLC7A5-high-expression mice and measuring the suppression rate of Col1a1 mRNA expression in the lungs. It also describes the results of measuring the mRNA levels of SLC7A5, COL1A1, and ACTA2 in normal human dermal fibroblasts (NHDF cells) treated with TR32110 and human TGF-β1.This document presents the results of measuring collagen protein levels in NHDF cells treated with TR32110 and human TGF-β1. It also presents the results of observing cells after staining collagen fibers in NHDF cells treated with TR32110 and human TGF-β1. Furthermore, it presents the results of observing cells after immunostaining for α-SMA in NHDF cells treated with TR32110 and human TGF-β1. Finally, it presents the results of measuring the mRNA levels of SLC7A5, COL1A1, and ACTA2 in human hepatic stellate cells (LX-2 cells) treated with TR32110 and human TGF-β1. The document also presents the results of measuring collagen protein levels in LX-2 cells treated with TR32110 and human TGF-β1. The document also presents the results of observing cells after staining collagen fibers in LX-2 cells treated with TR32110 and human TGF-β1. Finally, it presents the results of measuring α-SMA protein levels in LX-2 cells treated with TR32110 and human TGF-β1. This report presents the results of observing LX-2 cells treated with TR32110 and human TGF-β1 after immunostaining for α-SMA. It also shows the cell viability of HeLa cells treated with TR32110, and the time-dependent measurements of apoptosis and necrosis in HeLa cells treated with TR32110.

[0014] 1. Notation In this specification, antisense oligonucleotides may be abbreviated as "ASO". Also, the base sequences of nucleic acids (ASO, etc.) shown in this specification have the 5' end on the left and the 3' end on the right.

[0015] 2. Antisense Oligonucleotides The ASO of the present invention is an ASO that targets human SLC7A5 and is characterized by containing a sequence of 12 or more bases included in ttgagcaggtaggt (SEQ ID NO: 1). The ASO of the present invention will be described in detail below.

[0016] [Target Molecule] The ASO of the present invention is an ASO that targets human SLC7A5, and is a nucleic acid molecule that can exert anti-fibrotic and antitumor effects by suppressing the expression of SLC7A5.

[0017] SLC7A5 is the gene that codes for LAT1, and the mRNA of human SLC7A5 (Accession ID: NM_003486.7) has the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 1 is complementary to the nucleotide sequence at positions 545-558 in SEQ ID NO: 2.

[0018] The number of bases in the ASO of the present invention is not particularly limited, as long as it includes a continuous base sequence of 12 or more bases as shown in Sequence ID No. 1 and is capable of knocking down SLC7A5, but preferably 13 or more, more preferably 14 or more. Specifically, the number of bases in the ASO of the present invention can be 12 to 40, preferably 12 to 30, more preferably 12 to 20, even more preferably 13 to 18, even more preferably 14 to 18, particularly preferably 14 to 16 or 14 to 15, and most preferably 14 (i.e., consisting of the base sequence shown in Sequence ID No. 1).

[0019] If the ASO of the present invention has 12 to 13 bases, the base sequence of the ASO of the present invention may be set to a continuous base sequence of 12 to 13 bases included in the base sequence shown in Sequence ID No. 1.

[0020] Furthermore, if the ASO of the present invention has 14 bases, the base sequence of the ASO of the present invention may be set to consist of the base sequence shown in Sequence ID No. 1.

[0021] Furthermore, if the ASO of the present invention has 15 or more bases, the base sequence of the ASO of the present invention includes the base sequence shown in Sequence ID No. 1, and one or more bases are ligated to its 5' end and / or 3' end. The sequence of one or more bases ligated to the 5' end and / or 3' end is not particularly limited as long as it can knock down SLC7A5, but it is preferable that it is complementary to the base sequence of the mRNA of the target molecule SLC7A5, and that the entire ASO of the present invention can hybridize to the mRNA of SLC7A5.

[0022] The ASO of the present invention may be either oligodeoxyribonucleic acid (oligo DNA) or oligoribonucleic acid (oligo RNA), and is preferably oligo DNA. When the ASO of the present invention is provided as an oligo RNA, thymine (t) may be substituted with uracil (u) in the nucleotide sequence shown in SEQ ID NO: 1.

[0023] [Chemical Modification] The ASO of the present invention may be an unchemically modified ASO (i.e., an ASO consisting of natural nucleotides), but it is preferable that the ASO is chemically modified to improve or enhance resistance to nucleases, affinity for target genes, pharmacokinetics and the like.

[0024] When chemical modification is performed on the ASO of the present invention, it is sufficient that at least one selected from the base moiety of a nucleotide, the sugar moiety, and the internucleoside linkage moiety is chemically modified.

[0025] Examples of nucleotides having a chemically modified base moiety include nucleotides in which a substituent is introduced into the base moiety. Specific examples of the substituent include a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, and a halogen atom. When the base is cytosine (C), preferred examples of chemically modified cytosine include 5-methylcytidine, 2'-O-methylcytidine, and the like.

[0026] Examples of nucleotides having a chemically modified sugar moiety include 2',4'-bridged nucleotides, and nucleotides in which the hydroxyl group at the 2' position of the sugar moiety is substituted with an alkoxy group (e.g., an alkoxy group having 1 to 5 carbon atoms such as a methoxy group or an ethoxy group) or a halogen atom (e.g., a fluorine atom). Among these, 2',4'-bridged nucleotides are preferred.

[0027] Preferable examples of 2',4'-bridged nucleotides include the structures shown below.

[0028] In the above structural formula, Base represents a base corresponding to the base sequence, specifically a purine-9-yl group or a 2-oxo-1,2-dihydropyrimidine-1-yl group, which may be substituted with a substituent. Specific examples of substituents include hydroxyl groups, linear alkyl groups having 1 to 6 carbon atoms, linear alkoxy groups having 1 to 6 carbon atoms, mercapto groups, linear alkylthio groups having 1 to 6 carbon atoms, amino groups, linear alkylamino groups having 1 to 6 carbon atoms, and halogen atoms. Specifically, the Base in the above structural formula may be a substituted 6-aminopurine-9-yl group if the base is A (adenine); a substituted 2-amino-6-hydroxypurine-9-yl group if the base is G (guanine); a substituted 2-oxo-4-amino-1,2-dihydropyrimidine-1-yl group if the base is C (cytosine) (for example, a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidine-1-yl group (including a 5-methylcytosine-1-yl group, a 2'-O-methylcytosine-1-yl group, etc.); or a substituted 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidine-1-yl group if the base is T (thymine).

[0029] In the 5'amino-2',4'-BNA shown in the structural formula above, R represents a hydrogen atom; a C1-C7 alkyl group which may be branched or ring-forming; a C2-C7 alkenyl group which may be branched or ring-forming; a C3-C12 aryl group which may have substituents and may contain heteroatoms; or an aralkyl group having a C3-C12 aryl moiety which may have substituents and may contain heteroatoms. Specifically, substituents that may be included in the aryl group or aralkyl group include a hydroxyl group, a C1-C6 linear alkyl group, a C1-C6 linear alkoxy group, a mercapto group, a C1-C6 linear alkylthio group, an amino group, a C1-C6 linear alkylamino group, and a halogen atom. Preferably, R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, more preferably a hydrogen atom or a methyl group, and particularly preferably a methyl group.

[0030] Another example of a 2',4'-crosslinked nucleotide is a nucleotide having the structure shown in the following general formula (1).

[0031] In the general formula (1) above, Base is as described above. Also, in the general formula (1) above, R is the same as R in 5'amino-2',4'-BNA above. A 2',4'-bridged nucleotide in the general formula (1) above, in which R is a methyl group, is a modified nucleotide sometimes called "AmNA".

[0032] Furthermore, another example of a 2',4'-bridged nucleotide is a nucleotide having the structure shown in the following general formula (2). This nucleotide is a known 2',4'-bridged nucleotide, also known as a guanidine-bridged nucleic acid (International Publication No. 2014 / 046212).

[0033] In the general formula (2) above, Base is as described above. In the general formula (2) above, R 1 , R 12 , and R 13, which are the same or different, each represent a hydrogen atom, or an alkyl group having 1 to 7 carbon atoms which may be branched or form a ring, R 14 represents a hydrogen atom.

[0034] Further, as another example of 2',4'-bridged nucleotides, nucleotides having a structure represented by the following general formula (3) can be mentioned. Said nucleotide is a known 2',4'-bridged nucleotide, which is also called spirocyclopropane-bridged nucleic acid (International Publication No. WO 2015 / 125783).

[0035] In the general formula (3), Base is as defined above. In the general formula (3), R 21 and R 22 , which are the same or different, are each a hydrogen atom; an alkyl group having 1 to 7 carbon atoms that may be substituted with an aryl group having 3 to 12 carbon atoms which may contain a hetero atom, and may be branched or form a ring; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom; or alternatively, R 21 and R 22 together form a group -(CH2) n - [wherein n is an integer of 2 to 5].

[0036] Further, as another example of 2',4'-bridged nucleotides, nucleotides having a structure represented by the following general formula (4) or (4') can be mentioned. Said nucleotide is a known 2',4'-bridged nucleotide which is also called ethyleneoxy-bridged nucleic acid (International Publication No. WO 2016 / 017422).

[0037] In the general formulas (4) and (4'), Base is as defined above. In the general formulas (4) and (4'), X 3 represents an oxygen atom or a sulfur atom. In the general formulas (4) and (4'), R 31 and R 32 , which are the same or different, each represent a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may be branched or form a ring; an alkoxy group having 1 to 7 carbon atoms which may be branched or form a ring; or an amino group. Further, in the case of general formula (4), R31 and R 32 Together, the base = C(R 35 )R 36 [In the formula, R 35 and R 36 R may be the same or different, representing a hydrogen atom, a hydroxyl group, a mercapto group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a linear or branched alkylamino group having 1 to 6 carbon atoms. In the above general formulas (4) and (4'), R 33 R represents a hydrogen atom, a C1-C7 alkyl group which may be branched or ring-forming, a C1-C7 alkoxy group which may be branched or ring-forming, or a C1-C6 linear or branched alkylthio group. In the above general formula (4), R 34 This represents a hydrogen atom, a C1-C7 alkyl group which may be branched or ring-forming, a C1-C7 alkoxy group which may be branched or ring-forming, or a C1-C6 linear or branched alkylthio group.

[0038] Furthermore, examples of chemical modifications to the nucleoside bond include those in which the nucleoside bond is formed by phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, 3'-aminophospholamidates, aminoalkyl phosphorlamidates, thionophospholamidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, boranophosphates, etc. Phosphothioates are preferred as the chemical modification of the nucleoside bond.

[0039] In the ASO of the present invention, chemical modification may be applied to some of the nucleotide and / or nucleoside binding sites, or to all of the nucleotide and / or nucleoside binding sites.

[0040] A preferred example of the chemical modification applied to the ASO of the present invention is the inclusion of at least one, preferably 1 to 10, more preferably 2 to 8, even more preferably 4 to 8, and particularly preferably 6 2',4'-crosslinked nucleotides. A preferred example when 2',4'-crosslinked nucleotides are included is that at least one of the 1st to 3rd nucleotides from the 5' end and at least one of the 1st to 3rd nucleotides from the 3' end are 2',4'-crosslinked nucleotides (preferably 2',4'-BNA(LNA)). A more preferred example when 2',4'-crosslinked nucleotides are included is that the 1st to 3rd nucleotides from the 5' end and the 1st to 3rd nucleotides from the 3' end are 2',4'-crosslinked nucleotides (preferably 2',4'-BNA(LNA)).

[0041] Furthermore, a preferred example of the chemical modification applied to the ASO of the present invention is that at least one of the nucleoside bonding sites is a phosphorothioate bond. In addition, a preferred example of the case in which phosphorothioate bonds are included is that, of 100% of the total number of nucleoside bonding sites, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and especially preferably 100% (all nucleoside bonding sites) are phosphorothioate bonds.

[0042] A preferred example of the ASO of the present invention is a chemically modified ASO in which all nucleoside bonds are phosphorothioate bonds and the 1st to 3rd nucleotides from the 5' end and the 1st to 3rd nucleotides from the 3' end are 2',4'-bridged nucleotides (preferably 2',4'-BNA(LNA)).

[0043] A suitable example of the ASO of the present invention is a chemically modified ASO consisting of the following sequence 1'. The chemically modified ASO consisting of the following sequence 1' can significantly suppress the expression of SLC7A5, exhibiting significantly superior anti-fibrotic and anti-tumor effects in vivo, and is particularly suitable for use. Sequence 1': T^T^G^a^g^c^a^g^g^t^a^G^G^T

[0044] In sequence 1', "G" represents guanine in the 2',4'-BNA(LNA) structure, "T" represents thymine in the 2',4'-BNA(LNA) structure, "a" represents adenine in unmodified DNA, "g" represents guanine in unmodified DNA, "c" represents cytosine in unmodified DNA, "t" represents thymine in unmodified DNA, and "^" indicates a phosphorothioate bond.

[0045] [Uses and Application Methods] The ASO of the present invention is suitably used as a nucleic acid drug for human use.

[0046] The ASO of the present invention exerts an anti-fibrotic effect by suppressing the expression of SLC7A5 in vivo, and can therefore be used to treat fibrosis. Fibrosis is a disease in which fibrosis (replacement with fibrotic tissue) progresses in tissues. The types of fibrosis that can be treated with the ASO of the present invention are not particularly limited, but include, for example, pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF); hepatic fibrosis; renal fibrosis; cutaneous fibrosis; cardiac fibrosis such as atrial fibrosis and endocardial fibrosis; vascular fibrosis; cerebral fibrosis; cystic fibrosis; myelofibrosis; articular fibrosis; intestinal fibrosis; peritoneal fibrosis; retroperitoneal fibrosis, etc. Among these types of fibrosis, pulmonary fibrosis (especially idiopathic pulmonary fibrosis) and hepatic fibrosis are preferred as targets for treatment with the ASO of the present invention.

[0047] Furthermore, the ASO of the present invention can be used not only to treat diseases characterized by fibrosis, but also to treat diseases accompanied by fibrosis. Examples of diseases accompanied by fibrosis include interstitial lung diseases accompanied by fibrosis, such as interstitial pneumonia and interstitial lung disease associated with systemic sclerosis; liver diseases accompanied by fibrosis, such as hepatitis (viral, alcoholic, or non-alcoholic fatty liver disease), cirrhosis, and hepatolenticular degeneration; and biliary tract diseases accompanied by fibrosis, such as biliary atresia. Among these diseases accompanied by fibrosis, suitable examples of targets for treatment with the ASO of the present invention include interstitial lung diseases or liver diseases accompanied by fibrosis. Another suitable example of a target for treatment with the ASO of the present invention is interstitial lung disease associated with systemic sclerosis. Specifically, interstitial lung diseases associated with systemic sclerosis include those accompanied by pulmonary fibrosis.

[0048] Furthermore, since the ASO of the present invention exhibits an anti-fibrotic effect, it can also be used to treat diseases involving airway remodeling, including fibrosis (such as thickening of the airway wall). Specific examples of diseases involving airway remodeling, including fibrosis, include bronchial asthma, chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis, and chronic sinusitis, among other chronic airway inflammations.

[0049] Furthermore, the ASO of the present invention exerts an antitumor effect by suppressing the expression of SLC7A5 in vivo, and can therefore be used to treat tumors. The types of tumors that can be treated with the ASO of the present invention are not particularly limited, but examples include cervical cancer, colorectal cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, pancreatic cancer, kidney cancer, liver cancer, laryngeal cancer, esophageal cancer, brain tumor, cervical cancer, tongue cancer, laryngeal cancer, thyroid cancer, breast cancer, small intestine cancer, bladder cancer, kidney cancer, liver cancer, gallbladder cancer, ovarian cancer, lymphoma, myeloma, myelosarcoma, retinoplasm, reticuloma, melanoma, fibroid, neuroma, glioma, schwannoma, sarcoma, osteosarcoma, fibroid, fibrosarcoma, papilloma, adenoma, cyst, etc. These tumors may be primary or metastatic. Among these tumors, cervical cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, pancreatic cancer, kidney cancer, laryngeal cancer, esophageal cancer, and brain tumors are particularly suitable as targets for ASO treatment according to the present invention, as many cases show high expression of LAT1 in tumor cells, and excellent antitumor effects can be observed by suppressing the expression of SLC7A5.

[0050] The method of administering the ASO of the present invention is not particularly limited, as long as the ASO of the present invention is delivered to the affected area, but examples include systemic administration such as intravenous injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection; and local administration such as local injection to the affected area, transpulmonary administration, and suppository administration.

[0051] The dosage of the ASO of the present invention can be appropriately determined according to the type of active ingredient used, the form of administration, the type of disease to be treated, the severity of the patient's symptoms, etc. For example, the single dose of the ASO of the present invention is usually set to about 0.1 to 10 mg / kg, preferably about 2.3 to 4.7 mg / kg body weight, and administered at a frequency of about once every 4 to 7 days.

[0052] Furthermore, the ASO of the present invention may be used alone, or it may be used in combination with one or more other agents.

[0053] [Formulation Form] The ASO of the present invention is prepared in a formulation form according to the administration method and used as a nucleic acid drug. Examples of formulation forms of nucleic acid drugs containing the ASO of the present invention include liquid formulations such as liquid preparations, suspensions, and liposome preparations, or powder formulations such as dry powder.

[0054] Furthermore, the nucleic acid drug containing ASO of the present invention is formulated by adding pharmaceutically acceptable carriers and additives depending on the formulation form. For example, if it is to be a liquid formulation, it can be formulated using physiological saline, buffer solution, etc.

[0055] Furthermore, it is desirable that the nucleic acid drug containing the ASO of the present invention be formulated together with a nucleic acid delivery aid so that the ASO can be easily transferred into target cells. Specific examples of nucleic acid delivery aids include lipofectamine, oligofectamine, RNAifect, liposomes, polyamines, DEAE dextran, calcium phosphate, and dendrimers.

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0057] 1. Test Materials and Test Methods 1-1. Cells Human cervical cancer-derived cells (HeLa cells, JCRB Cell Bank) and normal human dermal fibroblasts (NHDF cells, PromoCell) were grown in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) at 37°C under a 5% CO2 atmosphere. Normal human lung fibroblasts (NHLF cells, American Type Culture Collection) were grown in fibroblast growth medium 2 (PromoCell). Established human hepatic stellate cells (LX-2 cells, MilliporeSigma) were grown in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque Co., Ltd.) containing 2% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco).

[0058] 1-2. SLC7A5 knock-in mice were created by introducing the human SLC7A5 gene into C57BL / 6J mice, which are overexpressing SLC7A5, according to known methods. These SLC7A5 knock-in mice were confirmed to express human SLC7A5 throughout the body. When bleomycin was administered to these SLC7A5 knock-in mice, SLC7A5 was highly expressed.

[0059] 1-3. Quantification of mRNA Amount in Cells (1) Measurement of mRNA Amount in HeLa Cells mRNA was extracted from cells and cDNA was prepared from the mRNA using the SuperPrep II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.) and Veriti Thermal Cycler (Applied Biosystems). The prepared cDNA was mixed with TaqMan Fast Advanced Master Mix (Applied Biosystems) and a TaqMan probe / primer set [TaqMan Gene Expression Assays (Applied Biosystems)] appropriate for the target gene. Real-time PCR was then performed using StepOnePlus Real-Time PCR Systems (Applied Biosystems) to measure the mRNA amount of the target gene.

[0060] (2) Measurement of mRNA levels in NHDF cells, NHLF cells, or LX-2 cells Total RNA was extracted from cells using the PureLink RNA Mini Kit (Invitrogen). Using the obtained RNA as a template, cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems) and the Veriti Thermal Cycler (Applied Biosystems). The synthesized cDNA was mixed with TaqMan Fast Advanced Master Mix (Applied Biosystems) and a TaqMan probe / primer set [TaqMan Gene Expression Assays (Applied Biosystems)] appropriate for the target gene. Real-time PCR was then performed using StepOnePlus Real-Time PCR Systems (Applied Biosystems) to measure the mRNA level of the target gene.

[0061] 1-4. Quantitative Analysis of mRNA Amount in Lung Tissue Homogenization was performed by adding TRIzol Reagent (Invitrogen) at a ratio of 1 ml / 100 mg per lung to the collected lung tissue. Then, p-bromoanisole (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at a ratio of 50 μl / 1 ml of TRIzol. Next, total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). Using the obtained total RNA as a template, cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems) and the Veriti Thermal Cycler (Applied Biosystems). The synthesized cDNA was mixed with TaqMan Fast Advanced MasterMix (Applied Biosystems) and a TaqMan probe / primer set [TaqMan Gene Expression Assays (Applied Biosystems)] appropriate for the target gene. Real-time PCR was then performed using the StepOnePlus Real-Time PCR Systems (Applied Biosystems) to measure the amount of mRNA of the target gene.

[0062] 1-5. Determination of LAT1 and α-SMA protein levels in cells Cells were lysed using M-PER Reagent (Thermo Scientific), and the supernatant was collected by centrifugation. The proteins in the obtained supernatant were converted to SDS and subjected to SDS-PAGE, followed by Western blotting. To detect LAT1 protein, anti-LAT1 antibody (Transgenic Corporation) and anti-GAPDH antibody (Cell Signaling Technology) (loading control) were used as primary antibodies, and IRDye800CW-labeled anti-rabbit IgG antibody (LI-COR) and IRDye680RD-labeled anti-mouse IgG antibody (LI-COR) were used as secondary antibodies. For the detection of α-SMA protein, anti-α-SMA antibody (abcam) and anti-GAPDH antibody (abcam) (loading control) were used as primary antibodies, and IRDye800CW-labeled anti-rabbit IgG antibody (LI-COR) and IRDye680RD-labeled anti-mouse IgG antibody (LI-COR) were used as secondary antibodies. Protein quantification was performed by quantifying the signal obtained from the band using an Odyssey Fc Imager (LI-COR).

[0063] 1-6. Immunofluorescence Staining For immunofluorescence staining against LAT1 protein, anti-LAT1 antibody (Transgenic Corporation) was used as the primary antibody, and Alexa Fluor594-labeled anti-rabbit IgG antibody (Invitrogen) was used as the secondary antibody. For immunofluorescence staining against α-SMA protein, anti-α-SMA antibody (abcam) was used as the primary antibody, and Alexa Fluor594-labeled anti-rabbit IgG antibody (Invitrogen) was used as the secondary antibody. Immunofluorescence-stained skin and lung-derived cells were observed using an inverted fluorescence microscope IX71 (Olympus Corporation). Immunofluorescence-stained liver-derived cells were observed using an inverted fluorescence microscope Mica (Leica Microsystems K.K.).

[0064] 1-7. Determination of Collagen Protein Amount in Cells (1) Determination of Collagen Protein Amount in NHDF or NHLF Cells 100 μl of 0.5 M acetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) aqueous solution containing 0.1 mg / ml pepsin (Sigma-Aldrich) was added to the cells and treated overnight at 4°C. The supernatant was then separated into supernatant and cell residue. The amount of collagen protein in the recovered supernatant was measured using the Sigma-Aldrich Soluble Collagen Assay Kit (Biocolor) according to the protocol provided by the manufacturer. A microplate reader (Synergy HTX Multimode Reader, BioTek) was used for the measurement. To correct for the cell number during the assay, the total protein amount contained in the recovered supernatant and cell residue was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the protocol provided by the manufacturer. For the measurement of proteins in cell residue, 50 μl of M-PER Reagent (Thermo Scientific) was added to the cell residue to dissolve it, and the supernatant obtained by centrifugation (14,000 × g, 5 minutes) was used. The amount of collagen protein was corrected based on the sum of the amount of protein measured from the recovered supernatant and the amount of protein measured from the cell residue.

[0065] (2) Quantification of collagen protein content in LX-2 cells: 600 μl of 0.5 M acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) aqueous solution containing 0.1 mg / ml pepsin (Sigma-Aldrich) was added to the cells and treated overnight at 4°C. The collagen protein content in the recovered supernatant was then measured using the Sircol Soluble Collagen Assay Kit (Biocolor) according to the protocol provided by the manufacturer. A microplate reader (Varioskan LUX multimode microreader, Thermo Scientific) was used for the measurement.

[0066] 1-8. Staining of Cellular Collagen Fibers Cellular collagen fibers were stained using the Picro-Sirius Red Stain Kit For Collagen (ScyTek Laboratories) according to the manufacturer's protocol. Cells stained for collagen fibers were observed using an inverted fluorescence microscope IX71 (Olympus Corporation).

[0067] 1-9. Cell Viability Cells were cultured under specified conditions in a 96-well plate (Thermo Scientific). After 24 or 72 hours of culture, 10 μl of Cell Counting Kit-8 (Dojin Chemical Research Institute Co., Ltd.) was added to each well and allowed to stand at 37°C for 1 hour. Subsequently, absorbance was measured using a microplate reader (Infinite M200, Tecan) according to the protocol provided by the manufacturer. The absorbance at the start of culture was set as 100% cell viability, and the cell viability after the specified time was determined.

[0068] 1-10. Number of Apoptotic and Necrotic Cells Cells were cultured under specified conditions in 96-well plates (white plates, Thermo Scientific). The number of apoptotic and necrotic cells was measured using the RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega) according to the manufacturer's protocol.

[0069] 2. Test Results 2-1. Screening of ASOs for SLC7A5 1 (in vitro) Based on the structure of SLC7A5 mRNA, 90 14mer oligonucleotides were designed as candidate ASOs for SLC7A5. These oligonucleotides were used in which all phosphodiester bonds were replaced with phosphorothioate bonds. These oligonucleotides were synthesized by Gene Design Co., Ltd.

[0070] These 90 oligonucleotides were transfected into human cervical cancer-derived cells (HeLa cells, from the JCRB cell bank). Specifically, 4 × 10⁶ HeLa cells were transfected. 4Cells were seeded in 195 μl of DMEM medium (antibiotic-free) to a concentration of cells / ml and cultured overnight. Then, each oligonucleotide was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 5 μM, and cultured for 24 hours (n=3). Next, the culture medium was removed, and the amount of SLC7A5 mRNA in the cells was measured. The SLC7A5 gene expression level in cells transfected with each oligonucleotide was determined, with the SLC7A5 mRNA level in cells not transfected with an oligonucleotide set as 100%.

[0071] The results obtained are shown in Figures 1-1 and 1-2. As a result, 39 oligonucleotides were selected from among 90 types that showed an SLC7A5 gene expression level of 85% or less. The sequences of the 39 selected oligonucleotides are shown in Table 1.

[0072]

[0073] 2-2. Screening of ASOs against SLC7A5 2 (in vitro) The 39 oligonucleotides shown in Table 1 were chemically modified for optimization. Specifically, three nucleotides from each end of each oligonucleotide, a total of six oligonucleotides, were replaced with 2',4'-BNA(LNA) modified nucleotides, and all phosphodiester bonds were replaced with phosphorothioate bonds. These oligonucleotides were synthesized by Gene Design Co., Ltd. The sequences of the 39 chemically modified oligonucleotides are shown in Table 2.

[0074]

[0075] The 39 oligonucleotides shown in Table 2 were transfected into HeLa cells. Specifically, 4 × 10⁶ HeLa cells were transfected. 4Cells were seeded in 195 μl of DMEM medium (antibiotic-free) to a concentration of cells / ml and cultured overnight. Then, each oligonucleotide was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 100 nM, and cultured for 24 hours (n=3). Next, the culture medium was removed, and the amount of SLC7A5 mRNA in the cells was measured. The SLC7A5 gene expression level in cells transfected with each oligonucleotide was determined, with the SLC7A5 mRNA level in cells not transfected with an oligonucleotide set as 100%.

[0076] The results obtained are shown in Figure 2. From among 39 oligonucleotides, 11 were selected that showed low SLC7A5 gene expression levels. The sequences of the 11 selected oligonucleotides are shown in Table 3.

[0077]

[0078] 2-3. Screening of ASOs against SLC7A5 3 (in vivo) The in vivo activity of 11 oligonucleotides shown in Table 3 was investigated using SLC7A5-high-expression mice (n=4-10). Under anesthesia, 50 μl of bleomycin (Bleo for injection; manufactured by Nippon Kayaku Co., Ltd.) was administered to the lungs of 10-week-old male SLC7A5 knock-in mice at a dose of 3 mg / kg. Four, five, and seven days later, the 11 oligonucleotides shown in Table 3 were administered intraperitoneally at a dose of 20 mg / kg each. Six days after bleomycin administration, 10 ml / kg of physiological saline was administered. Eight days after bleomycin administration, the mice were euthanized, their lungs were collected, and the amount of SLC7A5 mRNA in the lungs was measured. In the positive control group, the test was performed under the same conditions as above, except that physiological saline was administered instead of oligonucleotides. In the negative control group, the test was performed under the same conditions as above, except that physiological saline was administered instead of bleomycin and oligonucleotides. The inhibition rate for each oligonucleotide administration group was calculated by setting the SLC7A5 mRNA level in the positive control group as 0% inhibition rate and the SLC7A5 mRNA level in the negative control group as 100% inhibition rate.

[0079] The results are shown in Figure 3. TR32091, TR32094, TR32104, TR32111, TR32112, TR32115, TR32126, and TR32127 showed excellent suppression of SLC7A5 mRNA expression in vitro, but no suppression of SLC7A5 mRNA expression was observed in vivo. Similarly, TR32109 and TR32118 showed excellent suppression of SLC7A5 mRNA expression in vitro, but only limited suppression was observed in vivo. In contrast, TR32110 showed significantly superior suppression of SLC7A5 mRNA expression even in vivo. Based on these test results, TR32110 was selected as the ASO for SLC7A5.

[0080] 2-4. Effects of TR32110 on LAT1 protein expression in HeLa cells. TR32110 was transfected into HeLa cells, and the effect of TR32110 on LAT1 protein expression was investigated (n=2). Specifically, 8 × 10⁶ HeLa cells were transfected. 4 Cells were seeded in 10 ml of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and cultured overnight. Then, TR32110 was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 10 nM, and cultured for 24 hours. The amount of LAT1 protein in the cultured cells was measured by Western blotting. A control was also tested under the same conditions as above, except that TR32110 was not added. The amount of LAT1 protein was calculated with the signal intensity of the control group set to 100%.

[0081] The results obtained are shown in Figure 4. These results confirmed that the amount of LAT1 protein was reduced in cells into which TR32110 was introduced.

[0082] 2-5. Effects of TR32110 on the expression distribution of LAT1 protein in HeLa cells. TR32110 was transfected into HeLa cells, and its effect on the expression distribution of LAT1 protein in the cells was investigated (n=2). Specifically, HeLa cells were transfected with 1.9 × 10⁶ HeLa cells. 4 Cells were seeded in 500 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a cell / ml concentration and cultured overnight. Then, TR32110 was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 30 nM or 100 nM, and cultured for 24 hours. Immunofluorescence staining for LAT1 and DAPI staining were performed on the cultured cells. As a control, the test was conducted under the same conditions as above, except that TR32110 was not added.

[0083] The results obtained are shown in Figure 5. These results confirm that the expression distribution of the LAT1 protein is reduced in cells into which TR32110 was introduced.

[0084] 2-6. Dose-response study of TR32110 on the TGF-β1 / SMAD signaling pathway The effect of TR32110 on the TGFβ-1 / SMAD signaling pathway was investigated (n=4). Specifically, first, 40 μl of DMEM medium (Nacalai Tesque Co., Ltd.) containing penicillin (Gibco) at a final concentration of 100 Unit / ml and streptomycin (Gibco) at a final concentration of 100 μg / ml was added to each well of a 96-well plate, and 2.5 × 10⁶ HeLa cells were placed in each well. 5Cells were seeded to a concentration of cells / ml and cultured overnight. Subsequently, 2.5 μl each of pGL4.48[luc2P / SBE / Hygro] and pGL4.74[hRluc / TK] plasmids (Promega) were added using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the attached protocol, and cultured overnight. Next, TR32110 or JPH203 (MedKoo Biosciences) was added and cultured. In the test group with TR32110, 2.5 μl was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to achieve final concentrations of 6.25 nM, 12.5 nM, 25 nM, 50 nM, or 100 nM, and cultured for 24 hours. In the test group treated with JPH203, 2.5 μl of JPH203 was added along with Opti-MEM (gibco) to achieve a final concentration of 0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM of JPH203 and a final concentration of 1 w / v% of DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.), and the cells were cultured for 1 hour. Cells cultured with TR32110 or JPH203 were then treated with 5 μl of human TGF-β1 (PeproTech) to a final concentration of 50 ng / ml and cultured for 3 hours. Next, 35 μl of Dual-Glo Luciferase Reagent (Promega) was added, and the mixture was allowed to react at room temperature for 10 minutes. Chemiluminescence was then measured using a microplate reader (Infinite M200, Tecan). Subsequently, 35 μl of Dual-Glo Stop&Glo Reagent (Promega) was added, and the mixture was allowed to react at room temperature for 20 minutes. Chemiluminescence was then measured using a microplate reader (Infinite M200, Tecan). The untreated control was tested under the same conditions as above, except that TR32110 or JPH203 and TGF-β1 were not added. After normalizing each well based on the amount of chemiluminescence derived from sea urticaria luciferase, the ratio (%) of chemiluminescence in cells treated with each concentration of TR32110 or JPH203 was calculated as the TGFβ-1 / SMAD signal intensity, with the chemiluminescence in the untreated control set as 100%.

[0085] The results obtained are shown in Figure 6. These results confirmed that TR32110 has an inhibitory effect on the TGF-β1 / SMAD signaling pathway, which is a fibrosis signaling pathway. Furthermore, under these test conditions, TR32110 showed a higher inhibitory effect on TGF-β1 / SMAD signaling than JPH203, a small molecule inhibitor of LAT1.

[0086] 2-7. Effects of TR32110 on the expression of fibrosis marker genes in lung-derived cells The effects of TR32110 on the expression of fibrosis marker genes in normal human lung fibroblasts (NHLF cells) were investigated (n=6). Specifically, first, NHLF cells were divided into 4 × 10⁶ cells. 4 Cells were seeded in 1 ml of fibroblast growth medium 2 (antibiotic-free) (PromoCell) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 1 ml of fibroblast basal medium 2 (PromoCell) containing 30 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added and cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 20 ng / ml and cultured for another 24 hours. After culture, the culture medium was removed, and the mRNA levels of SLC7A5, COL1A1, and ACTA2 in the cells were measured. For the positive control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. Each mRNA level was calculated with the mRNA level in positive control NHLF cells set to 100%.

[0087] The results obtained are shown in Figure 7. The results showed that NHLF cells introduced with TR32110 had lower levels of the fibrosis markers COL1A1 and ACTA2 mRNA compared to positive control NHLF cells. In other words, these results confirm that TR32110 has the effect of suppressing the expression of fibrosis markers in lung fibroblasts.

[0088] 2-8. Effects of TR32110 on collagen protein expression in lung-derived cells The effects of TR32110 on collagen protein expression in normal human lung fibroblasts were investigated (n=3). Specifically, first, NHLF cells were divided into 4 × 10⁶ cells. 4 Cells were seeded in 100 μl of fibroblast growth medium 2 (antibiotic-free) (PromoCell) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 100 μl of fibroblast basal medium 2 (PromoCell) containing 30 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added and cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 20 ng / ml and cultured for another 24 hours. After culture, the culture medium was removed, and the amount of collagen protein in the cells was measured. For the positive control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1.

[0089] The results obtained are shown in Figure 8. The results showed that NHLF cells introduced with TR32110 had lower levels of collagen protein compared to positive control NHLF cells. In other words, these results confirm that TR32110 suppresses collagen protein expression in lung fibroblasts, and that TR32110 is effective in treating diseases involving airway remodeling, including fibrosis.

[0090] 2-9. Effects of TR32110 on Extracellular Matrix Generation in Lung-Derived Cells The effects of TR32110 on extracellular matrix generation in normal human lung fibroblasts were investigated (n=2). Specifically, first, NHLF cells were subjected to 3 × 10⁶ 4Cells were seeded in 500 μl of fibroblast growth medium 2 (antibiotic-free) (PromoCell) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 500 μl of fibroblast basal medium 2 (PromoCell) containing 30 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added, followed by 24-hour culture. Next, human TGF-β1 (PeproTech) was added to a final concentration of 20 ng / ml, and cultured for 48 hours. After culture, the culture medium was removed, and the collagen fibers of the cells were stained. For the positive control (TGFβ1), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control 1 (Vehicle), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. In negative control 2 (TR32110), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of human TGF-β1.

[0091] The results obtained are shown in Figure 9. These results show that NHLF cells introduced with TR32110 exhibited suppressed collagen fiber formation compared to positive control NHLF cells, indicating that TR32110 has activity to suppress extracellular matrix generation. In other words, these results support the idea that TR32110 is also effective in treating diseases involving airway remodeling, including fibrosis.

[0092] 2-10. Effects of TR32110 on α-SMA protein expression in lung-derived cells The effects of TR32110 on α-SMA protein expression in normal human lung fibroblasts were investigated (n=2). Specifically, first, NHLF cells were subjected to 4 × 10⁶ 4Cells were seeded in 2 ml of fibroblast growth medium 2 (antibiotic-free) (PromoCell) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 500 μl of fibroblast basal medium 2 (PromoCell) containing 30 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added and cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 20 ng / ml and cultured for another 24 hours. The amount of α-SMA protein in the cultured cells was measured by Western blotting. For the positive control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control 1, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. In negative control 2, the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of human TGF-β1. The amount of α-SMA protein was calculated with the signal intensity of the positive control set to 100%.

[0093] The results obtained are shown in Figure 10. The results showed that NHLF cells introduced with TR32110 had reduced levels of α-SMA protein, a myofibroblast marker, compared to positive control NHLF cells. In other words, these results confirm that TR32110 has an effect of suppressing the differentiation of fibroblasts into myofibroblasts.

[0094] 2-11. Effects of TR32110 on the differentiation of lung-derived fibroblasts into myofibroblasts The effects of TR32110 on the differentiation of normal human lung fibroblasts into myofibroblasts were investigated (n=2). Specifically, first, NHLF cells were divided into 3 × 10⁶ cells. 4Cells were seeded in 500 μl of fibroblast growth medium 2 (antibiotic-free) (PromoCell) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 500 μl of fibroblast basal medium 2 (PromoCell) containing 30 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added and cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 20 ng / ml and cultured for another 24 hours. Immunostaining for α-SMA protein was performed on the cultured cells. For the positive control (TGFβ1), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control 1 (vehicle), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. Furthermore, in negative control 2 (TR32110), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of human TGF-β1.

[0095] The results obtained are shown in Figure 11. The results showed that in NHLF cells introduced with TR32110, the expression distribution of α-SMA protein was reduced compared to positive control NHLF cells, and the number of cells exhibiting myofibroblast-like morphology was also decreased. In other words, these results confirm that TR32110 has the effect of suppressing the differentiation of fibroblasts into myofibroblasts, which is considered a major morphological change in fibrosis.

[0096] 2-12. Investigation of the fibrosis-inhibiting effect of TR32110 in a fibrotic mouse model The fibrosis-inhibiting effect of TR32110 was investigated in a fibrotic mouse model (n=4-10). Under anesthesia, 50 μl of bleomycin (Bleo for injection; manufactured by Nippon Kayaku Co., Ltd.) was administered to the lungs of 10-week-old male SLC7A5 knock-in mice at a dose of 3 mg / kg. Four, five, and seven days later, 11 oligonucleotides shown in Table 3 were administered intraperitoneally at a dose of 20 mg / kg each. Six days after bleomycin administration, 10 ml / kg of physiological saline was administered. Eight days after bleomycin administration, the mice were euthanized, their lungs were collected, and the amount of Col1a1 mRNA in the lungs was measured. In the positive control group, the test was conducted under the same conditions as above, except that physiological saline was administered instead of oligonucleotides. In the negative control group, the test was conducted under the same conditions as above, except that physiological saline was administered instead of bleomycin and oligonucleotides. The inhibition rate for each oligonucleotide was calculated by setting the Col1a1 mRNA expression level in the positive control group as 0% inhibition and the Col1a1 mRNA expression level in the negative control group as 100% inhibition.

[0097] The results are shown in Figure 12. In fibrosis model mice administered TR32110, the expression of Col1a1, a fibrosis marker, was effectively suppressed. On the other hand, while the other 10 oligonucleotides showed superior inhibitory effects on SLC7A5 mRNA expression in vitro, they were unable to suppress Col1a1 expression in fibrosis model mice. In other words, these results confirm that TR32110 can effectively exert its fibrosis-inhibiting effect in vivo.

[0098] 2-13. Effects of TR32110 on the expression of fibrosis marker genes in skin-derived cells. The effects of TR32110 on the expression of fibrosis marker genes in normal human dermal fibroblasts (NHDF cells) were investigated (n=2). Specifically, NHDF cells were 1 × 10⁶ 5Cells were seeded in 100 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and cultured overnight. The culture medium was then removed, and 10 μl of DMEM medium containing 86 nM TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added and cultured for 12 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 200 ng / ml and cultured for 24 hours. The mRNA levels of SLC7A5, COL1A1, and ACTA2 in the cultured cells were measured. For the positive control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. mRNA levels were calculated with the positive control mRNA level set to 100%.

[0099] The results obtained are shown in Figure 13. The results showed that NHDF cells introduced with TR32110 had reduced levels of COL1A1 mRNA and ACTA2 mRNA compared to positive control NHDF cells. In other words, these results confirm that TR32110 has the effect of suppressing the expression of fibrosis marker genes in dermal fibroblasts.

[0100] 2-14. Effects of TR32110 on collagen protein expression in skin-derived cells The effects of TR32110 on collagen protein expression in normal human dermal fibroblasts were investigated (n=3). Specifically, first, NHDF cells were subjected to 8 × 10⁶ 4Cells were seeded in 100 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a cell / ml concentration and cultured overnight. Next, 5 μl of a mixture of TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added (final concentration of TR32110: 100 nM) and cultured for 24 hours. Subsequently, human TGF-β1 (PeproTech) was added to a final concentration of 200 ng / ml and cultured for 48 hours. Then, the culture medium was removed and the amount of collagen protein in the cells was measured. A positive control was tested under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. A negative control was tested under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1.

[0101] The results obtained are shown in Figure 14. The results showed that NHDF cells introduced with TR32110 had lower levels of collagen protein compared to positive control NHDF cells. In other words, these results confirm that TR32110 has an effect of suppressing collagen protein expression in dermal fibroblasts.

[0102] 2-15. Effects of TR32110 on Extracellular Matrix Generation in Skin-Derived Cells The effects of TR32110 on extracellular matrix generation in normal human dermal fibroblasts were investigated (n=2). Specifically, NHDF cells were subjected to 8 × 10⁶ 4Cells were seeded in 100 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and cultured overnight. Next, 5 μl of a mixture of TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added (final concentration of TR32110 200 nM) and cultured for 24 hours. Then, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml and cultured for 48 hours. After culture, the culture medium was removed and the collagen fibers of the cells were stained. For the positive control (TGFβ1), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control 1 (Vehicle), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. In negative control 2 (TR32110), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of human TGF-β1.

[0103] The results obtained are shown in Figure 15. These results show that in NHDF cells introduced with TR32110, collagen fiber formation was suppressed compared to positive control NHDF cells, revealing that TR32110 has the activity to suppress extracellular matrix generation by dermal fibroblasts.

[0104] 2-16. Effects of TR32110 on differentiation of skin-derived cells into myofibroblasts The effects of TR32110 on differentiation of normal human skin fibroblasts into myofibroblasts were investigated (n=2). Specifically, first, NHDF cells were divided into 2 × 10⁶ cells. 4Cells were seeded in 500 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and cultured overnight. Next, 24 μl of a mixture of TR32110 and X-tremeGENE HP DNA Transfection Reagent (Roche) was added (final concentration of TR32110: 100 nM) and cultured for 24 hours. Then, human TGF-β1 (PeproTech) was added to a final concentration of 200 ng / ml and cultured for 48 hours. Immunostaining for α-SMA protein was performed on the cultured cells. For the positive control (TGFβ1), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. For the negative control 1 (vehicle), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. Furthermore, in negative control 2 (TR32110), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of human TGF-β1.

[0105] The results obtained are shown in Figure 16. The results showed that in NHDF cells introduced with TR32110, the expression distribution of α-SMA protein was reduced compared to positive control NHDF cells, and the number of cells exhibiting myofibroblast-like morphology was also decreased. In other words, these results confirm that TR32110 also has an effect of suppressing the differentiation of cutaneous fibroblasts into myofibroblasts.

[0106] 2-17. Effects of TR32110 on the expression of fibrosis marker genes in liver-derived cells. The effects of TR32110 on the expression of fibrosis marker genes in a cell line of human hepatic stellate cells (LX-2 cells) were investigated (n=2). Specifically, first, LX-2 cells were expressed in 5 × 10⁶ cells. 4Cells were seeded in 1 ml of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco) to a concentration of cells / ml, and cultured overnight. The culture medium was then removed, and 1 ml of DMEM medium (Nacalai Tesque Co., Ltd.) containing 2 mM L-glutamine (Gibco) with 3 nM TR32110 and Lipofectamine 3000 Transfection Reagent (Invitrogen) was added, and the cells were cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml, and the cells were cultured for another 24 hours. After culturing, the culture medium was removed, and the mRNA levels of SLC7A5, COL1A1, and ACTA2 in the cells were measured. For the positive control, the same conditions as above were used, except that DNase / RNase-free water was used instead of TR32110. In the negative control group, the tests were conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. Each mRNA level was calculated with the mRNA level in the positive control LX-2 cells set to 100%.

[0107] The results obtained are shown in Figure 17. The results showed that LX-2 cells introduced with TR32110 had lower levels of the fibrosis markers COL1A1 and ACTA2 mRNA compared to positive control LX-2 cells. In other words, these results confirm that TR32110 has an effect of suppressing the expression of fibrosis markers in hepatic stellate cells.

[0108] 2-18. Effects of TR32110 on collagen protein expression in hepatic stellate cells The effects of TR32110 on collagen protein expression in cultured human hepatic stellate cells (LX-2 cells) were investigated. Specifically, first, LX-2 cells were divided into 1.7 × 10⁶ cells. 5Cells were seeded in 2 ml of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco) to a concentration of cells / ml, and cultured overnight. The culture medium was then removed, and 2.5 ml of DMEM medium (Nacalai Tesque Co., Ltd.) containing 2 mM L-glutamine (Gibco) with 12 nM TR32110 and Lipofectamine 3000 Transfection Reagent (Invitrogen) was added, and the cells were cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml, and the cells were cultured for another 24 hours. After culture, the culture medium was removed, and the amount of collagen protein in the cells was measured. For the positive control, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. In the negative control group, the tests were conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1.

[0109] The results obtained are shown in Figure 18. The results showed that LX-2 cells introduced with TR32110 had lower levels of collagen protein compared to positive control LX-2 cells. In other words, these results confirm that TR32110 suppresses collagen protein expression in hepatic stellate cells, and that TR32110 is effective in treating diseases involving liver remodeling, including fibrosis.

[0110] 2-19. Effects of TR32110 on Extracellular Matrix Generation in Hepatic Stellary Cells The effects of TR32110 on extracellular matrix generation in cultured human hepatic stellate cells (LX-2 cells) were investigated (n=2). Specifically, first, LX-2 cells were divided into 1.8 × 10⁶ cells. 5Cells were seeded in 0.5 ml of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco) to a concentration of cells / ml, and cultured overnight. The culture medium was then removed, and 0.5 ml of DMEM medium (Nacalai Tesque Co., Ltd.) containing 6 or 12 nM TR32110 and 2 mM L-glutamine (Gibco) with Lipofectamine 3000 Transfection Reagent (Invitrogen) was added, and the cells were cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml, and the cells were cultured for another 24 hours. After culture, the culture medium was removed, and the collagen fibers of the cells were stained. For the positive control (TGFβ1), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. In the negative control (Vehicle), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1.

[0111] The results obtained are shown in Figure 19. These results show that in LX-2 cells introduced with TR32110, collagen fiber formation was suppressed compared to positive control LX-2 cells, revealing that TR32110 has activity to suppress extracellular matrix generation. In other words, these results support the idea that TR32110 is also effective in treating liver diseases accompanied by fibrosis.

[0112] 2-20. Effects of TR32110 on α-SMA protein expression in liver-derived cells. The effects of TR32110 on α-SMA protein expression in a cell line of human hepatic stellate cells (LX-2 cells) were investigated. Specifically, first, LX-2 cells were divided into 5 × 10⁶ cells. 4Cells were seeded in 2.5 ml of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco) to a concentration of cells / ml, and cultured overnight. The culture medium was then removed, and 2.5 ml of DMEM medium (Nacalai Tesque Co., Ltd.) containing 3 or 6 nM TR32110 and 2 mM L-glutamine (Gibco) with Lipofectamine 3000 Transfection Reagent (Invitrogen) was added, and the cells were cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml, and the cells were cultured for another 24 hours. The amount of α-SMA protein in the cultured cells was measured by Western blotting. For the positive control, the same conditions as above were used, except that DNase / RNase-free water was used instead of TR32110. In the negative control group, the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1. The amount of α-SMA protein was calculated with the signal intensity of the positive control group set to 100%.

[0113] The results obtained are shown in Figure 20. The results showed that LX-2 cells introduced with TR32110 had reduced levels of α-SMA protein, a marker for active hepatic stellate cells, compared to positive control LX-2 cells. In other words, these results confirm that TR32110 has an effect of suppressing the differentiation of quiescent hepatic stellate cells into active hepatic stellate cells.

[0114] 2-21. Effects of TR32110 on the differentiation of quiescent hepatic stellate cells into activated hepatic stellate cells The effects of TR32110 on the differentiation of quiescent human hepatic stellate cell lines (LX-2 cells) into activated LX-2 cells were investigated (n=2). Specifically, first, LX-2 cells were divided into 4 × 10⁶ cells. 4Cells were seeded in 500 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) containing 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco) to a cell / ml concentration, and cultured overnight. The culture medium was then removed, and 500 μl of DMEM medium (Nacalai Tesque Co., Ltd.) containing 3 or 6 nM TR32110 and 2 mM L-glutamine (Gibco) with Lipofectamine 3000 Transfection Reagent (Invitrogen) was added, and the cells were cultured for 24 hours. Next, human TGF-β1 (PeproTech) was added to a final concentration of 5 ng / ml, and the cells were cultured for another 24 hours. Immunostaining for α-SMA protein was performed on the cultured cells. For the positive control (TGFβ1), the test was conducted under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110. In the negative control (vehicle), the test was performed under the same conditions as above, except that DNase / RNase-free water was used instead of TR32110 and human TGF-β1.

[0115] The results obtained are shown in Figure 21. In LX-2 cells introduced with TR32110, the expression distribution of α-SMA protein was reduced compared to positive control LX-2 cells, and the number of cells exhibiting the morphology of active hepatic stellate cells was also decreased. In other words, these results also confirm that TR32110 has the effect of suppressing the differentiation of quiescent hepatic stellate cells into active hepatic stellate cells, which is considered to be a major morphological change in hepatic fibrosis.

[0116] 2-22. Examination of the antitumor effect of TR32110 The antitumor effect of TR32110 on human cervical cancer-derived cells was examined (n=3). Specifically, HeLa cells were subjected to 5 × 10⁶ 4Cells were seeded in 100 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and incubated overnight. Subsequently, TR32110 was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 100 nM, and incubated for 24 or 72 hours. Cell viability was then measured after 24 and 72 hours. As a control, the same conditions as above were used, except that an oligonucleotide (non-target ASO) prepared from nematode miRNA (cel-miR-239b) was used instead of TR32110. The base sequence of the oligonucleotide (non-target ASO) used as a control is 5'-T^A^mC^t^t^t^t^g^t^g^t^A^G^T-3' [where "A" is adenine in the 2',4'-BNA(LNA) structure, "G" is guanine in the 2',4'-BNA(LNA) structure, "mC" is 5-methylcytosine in the 2',4'-BNA(LNA) structure, "T" is thymine in the 2',4'-BNA(LNA) structure, "g" is guanine in unmodified DNA, "t" is thymine in unmodified DNA, and "^" indicates a phosphorothioate bond.]

[0117] The results obtained are shown in Figure 22. HeLa cells introduced with TR32110 had a reduced number of viable cells compared to control HeLa cells. In other words, these results clearly demonstrate that TR32110 has antitumor activity.

[0118] 2-23. Effects of TR32110 on tumor cell apoptosis The effects of TR32110 on human cervical cancer-derived cells on apoptosis were investigated (n=3). Specifically, first, 6 × 10⁶ HeLa cells were placed in each well of a 96-well plate (white plate, Thermo Scientific). 4Cells were seeded in 100 μl of DMEM medium (antibiotic-free) (Nacalai Tesque Co., Ltd.) to a concentration of cells / ml and incubated overnight. Next, the culture medium was removed, and 90 μl of CO2 Independent Medium (Gibco) containing L-glutamine (Gibco) added to a concentration of 4 mM at the time of apoptosis measurement was added. Furthermore, 100 μl of 2× Detection Reagent prepared according to the protocol attached to the RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega) was added, and the mixture was allowed to stand at 37°C for 2 hours. Subsequently, TR32110 was added together with Lipofectamine 3000 Transfection Reagent (Invitrogen) to a final concentration of 100 nM, and then the chemiluminescence intensity reflecting apoptosis and the fluorescence intensity reflecting necrosis were measured over 48 hours using a microplate reader (Synergy HTX Multimode Reader, BioTek). Furthermore, as Control 1, the test was conducted under the same conditions as above, except that an oligonucleotide (non-target ASO) prepared from nematode miRNA (cel-miR-239b) was used instead of TR32110. The oligonucleotide used as Control 1 is the same as that used in Test Example 2-22 above. Furthermore, as Control 2, the test was conducted under the same conditions as above, except that TR32110 was not added. The fluorescence intensity and emission intensity of Control 2 were set to 1, and the relative values ​​of fluorescence intensity and emission intensity under each condition were calculated.

[0119] The results obtained are shown in Figure 23. The results showed that HeLa cells introduced with TR32110 exhibited increased chemiluminescence intensity due to apoptosis compared to HeLa cells introduced with non-target ASO, while no significant difference was observed in fluorescence intensity due to necrosis. In other words, these results demonstrate that TR32110 has an apoptosis-inducing effect on tumor cells expressing LAT1, and that this apoptosis-inducing effect contributes to its antitumor effect.

Claims

1. An antisense oligonucleotide targeting SLC7A5, having a continuous sequence of 12 or more bases contained in the base sequence shown in Sequence ID No.

1.

2. The antisense oligonucleotide according to claim 1, wherein the number of bases is 12 to 40.

3. An antisense oligonucleotide according to claim 1 or 2, comprising the base sequence shown in Sequence ID No.

1.

4. The antisense oligonucleotide according to claim 1 or 2, wherein at least one nucleotide is a 2',4'-crosslinked nucleotide.

5. The antisense oligonucleotide according to claim 1 or 2, wherein at least one of the nucleoside binding sites is a phosphorothioate bond.

6. The antisense oligonucleotide according to claim 1 or 2, wherein all nucleoside bonds are phosphorothioate bonds, and the 1st to 3rd nucleotides from the 5' end and the 1st to 3rd nucleotides from the 3' end are 2',4'-bridged nucleotides.

7. The antisense oligonucleotide according to claim 1 or 2, comprising the following sequence 1': Sequence 1': T^T^G^a^g^c^a^g^g^t^a^G^G^T [In sequence 1', "G" represents guanine in the 2',4'-BNA structure, "T" represents thymine in the 2',4'-BNA structure, "a" represents adenine in unmodified DNA, "g" represents guanine in unmodified DNA, "c" represents cytosine in unmodified DNA, "t" represents thymine in unmodified DNA, and "^" represents a phosphorothioate bond.] 8. A nucleic acid drug comprising the antisense oligonucleotide according to claim 1 or 2.

9. The nucleic acid drug according to claim 8, used for the treatment of fibrosis or a disease involving fibrosis.

10. The nucleic acid drug according to claim 9, wherein the fibrosis or disease accompanied by fibrosis is pulmonary fibrosis, hepatic fibrosis, interstitial lung disease accompanied by fibrosis, or liver disease accompanied by fibrosis.

11. The nucleic acid drug according to claim 10, wherein the fibrosis or disease accompanied by fibrosis is idiopathic pulmonary fibrosis, interstitial pneumonia, interstitial lung disease associated with systemic scleroderma, hepatitis, cirrhosis, hepatolenticular degeneration, or biliary atresia.

12. The nucleic acid drug according to claim 8, used for the treatment of tumors.

13. The nucleic acid drug according to claim 12, wherein the tumor is cervical cancer, colorectal cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, pancreatic cancer, kidney cancer, liver cancer, laryngeal cancer, esophageal cancer, or brain tumor.

14. Use of the antisense oligonucleotide according to claim 1 or 2 for the manufacture of a treatment for fibrosis or a disease involving fibrosis.

15. A method for treating fibrosis or a disease involving fibrosis, comprising administering a therapeutically effective amount of the antisense oligonucleotide described in claim 1 or 2 to a patient with fibrosis or a disease involving fibrosis.

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