Antisense oligonucleotides and uses thereof

Abstract

Disclosed herein is a novel single-stranded antisense oligonucleotide (ASO) that can reduce the transcription of thioredoxin domain-containing protein 5 (TXNDC5) mRNA. Disclosed herein is also the use of the single-stranded ASO as disclosed herein for the manufacture of a medicament suitable for treating diseases associated with upregulation of TXNDC5. Accordingly, provided are pharmaceutical compositions comprising the disclosed ASO molecules and methods for treating subjects suffering from TXNDC5-mediated diseases by administering the disclosed single-stranded ASO molecules to the subjects.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 294,835, filed December 29, 2021, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the treatment of disease. More specifically, the present disclosure relates to the treatment of disease through the use of single-stranded antisense oligonucleotides (ASOs) that inhibit expression of thioredoxin domain containing protein 5 (TXNDC5) mRNA. [Background technology]

[0003] Thioredoxin domain-containing protein 5 (TXNDC5) is a protein disulfide isomerase that catalyzes its thioredoxin activity and enables it to act as a chaperone in the endoplasmic reticulum. It has been confirmed that the upregulated expression of TXNDC5 is associated with various types of diseases, including cancer, diabetes, arthritis, neurodegenerative diseases, organ fibrosis-related diseases (e.g., pulmonary fibrosis, renal fibrosis, liver fibrosis and myocardial fibrosis) and vitiligo.

[0004] The emerging use of post-transcriptional gene silencing techniques, particularly antisense oligonucleotides (i.e., ASOs), as a tool to knock out the expression of specific genes in various organisms, now makes it possible to map protein interactions in cell signaling pathways by systematically silencing functional genes, thereby providing a novel way to develop treatments for a myriad of diseases. After extensive research and experimentation, the inventors of this study have identified short oligonucleotide molecules that can target the pre-mRNA or mRNA of TXNDC5 and reduce the RNA levels of TXNDC5, as well as reduce TXNDC5 protein levels. Antisense molecules can act on target sequences through different mechanisms of action: degradation of mRNA through RNaseH, steric hindrance of ribosomal subunit binding, alteration of mRNA maturation, inhibition of 5'-capping, and termination of translation. These identified short nucleic acid molecules are therefore useful for the development of pharmaceuticals to treat diseases associated with overexpression of TXNDC5, thereby reducing or minimizing the symptoms associated therewith in subjects in need of such treatment. Summary of the Invention

[0005] The present disclosure is directed to single stranded nucleic acids for the treatment or prevention of diseases, particularly diseases associated with upregulation of TXNDC5.

[0006] Accordingly, one aspect of the present invention is directed to an isolated single-stranded antisense oligonucleotide (ASO) that reduces TXNDC5 mRNA expression, the ASO molecule being approximately 16-21 nucleotides in length and comprising a deoxyribonucleotide sequence having at least 80% sequence identity to any of SEQ ID NO: (sequence number) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0007] According to a preferred embodiment of the present disclosure, the ASO molecule has a deoxyribonucleotide sequence having at least 90% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0008] According to embodiments of the present disclosure, the ASO molecule comprises at least one locked nucleic acid (LNA) molecule, a 2'-sugar modification, a modified internucleotide linkage, or a combination thereof. In some embodiments of the present disclosure, the ASO molecule comprises six LNA molecules. In other embodiments of the present disclosure, the ASO molecule comprises ten 2'-sugar modifications.

[0009] In another aspect, the disclosure is directed to a method of treating a subject suffering from a disease mediated through upregulation of TXNDC5, comprising administering to the subject a therapeutically effective amount of an ASO molecule of the invention to suppress transcription of the TXNDC5 gene.

[0010] According to an embodiment of the present disclosure, the ASO molecule of the present invention is a single-stranded oligonucleotide that reduces TXNDC5 mRNA expression. The ASO molecule is about 16-21 nucleotides in length and has a deoxyribonucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0011] According to embodiments of the present disclosure, the ASO molecule comprises at least one LNA molecule, a 2'-sugar modification, a modified internucleotide linkage, or a combination thereof. In some embodiments, the ASO molecule comprises six LNA molecules. In other embodiments, the ASO molecule comprises ten 2'-sugar modifications.

[0012] According to an embodiment of the present disclosure, the disease associated with overexpression of TXNDC5 is selected from the group consisting of aging, arthritis (e.g., rheumatoid arthritis), cancer, diabetes (e.g., type II diabetes), neurodegenerative disease, fibrosis, vitiligo, and viral infection. In a preferred embodiment, the subject suffers from organ fibrosis, such as pulmonary fibrosis, renal fibrosis, liver fibrosis, or myocardial fibrosis.

[0013] In another aspect, the disclosure is directed to a pharmaceutical composition for treating, preventing or ameliorating a disease associated with overexpression of TXNDC5, comprising an ASO molecule of the invention and a pharma- ceutical acceptable carrier.

[0014] The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features and advantages of the invention will be apparent from the description, and from the claims.

[0015] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1A] Figure 1A shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB11111128235. [Figure 1B] Figure 1B shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB11111128255. [Figure 1C]Figure 1C shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128252. [Figure 1D] Figure 1D shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128266. [Figure 1E] Figure 1E shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128265. [Figure 1F] Figure 1F shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128238. [Figure 1G] Figure 1G shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128279. [Figure 1H] Figure 1H shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128280. [Figure 1I]Figure 1I shows the effect of the ASO-MOE or ASO-LNA on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction via Western blot analysis according to some embodiments of the present invention, where the ASO is DCB1111128281. [Figure 2A] FIG. 2A shows the effect of the ASO-MOE on lung function, a compliance factor of lung function, in mice with bleomycin (BLM)-induced pulmonary fibrosis according to one embodiment of the present invention. [Figure 2B] Figure 2B shows the effect of the ASO-MOE on lung function, a resistance factor for lung function, in mice with bleomycin (BLM)-induced pulmonary fibrosis according to one embodiment of the present invention. [Figure 2C] Figure 2C shows the effect of the ASO-MOE on lung function, an elastance factor of lung function, in mice with bleomycin (BLM)-induced pulmonary fibrosis according to one embodiment of the present invention. [Diagram 3] FIG. 3 shows pressure-volume curves in bleomycin-induced fibrotic mice, according to one embodiment of the present invention. [Figure 4] FIG. 4 shows the effect of the present ASO-MOE in reducing the fibrotic area in BLM-induced fibrotic mice according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The detailed description given below in connection with the accompanying drawings is intended as a description of the present embodiment and is not intended to represent the only manner in which the embodiment may be constructed or utilized. The description sets forth functions of the invention and sequences of steps for constructing and operating the embodiment, although the same or equivalent functions and sequences may be accomplished by different embodiments.

[0018] 1.Definition For convenience, certain terms employed in the context of this disclosure are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0019] The term "nucleic acid" is defined as a molecule formed by the covalent linkage of two or more nucleotides, including DNA, RNA, and variants or analogs of such DNA or RNA. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein. The term "oligonucleotide" is defined as a nucleic acid consisting of less than 25 nucleotides, such as 20 nucleotides.

[0020] Antisense oligonucleotides (ASOs) as used herein refer to single-stranded DNA or RNA that are complementary to pre-mRNA or mRNA sequences and can reduce RNA levels, thereby reducing protein levels. According to a preferred embodiment of the present disclosure, ASOs are generated that are complementary to the nucleobases at positions 337-356, 670-689, 675-694, 862-881, 879-898, 1003-1022, 1007-1026, 1278-1297, 2864-2883, 2865-2884, 2868-2887, and 2873-2892 of TXNDC5 mRNA (NCBI Reference Sequence: NM_030810.4), resulting in downregulation of TXNDC5 mRNA.

[0021] As used herein, a "sequence" of a nucleic acid refers to the order of nucleotides that make up the nucleic acid. Throughout this application, nucleic acids are designated as having a 5' end and a 3' end. Unless otherwise specified, the left-hand end of a single-stranded nucleic acid is the 5' end and the right-hand end of a single-stranded nucleic acid is the 3' end.

[0022] The term "locked nucleic acid (LNA)" as used herein refers to a nucleic acid in which some nucleotides of the nucleic acid are locked nucleic acid monomers (i.e., bicyclic nucleotides or analogs thereof). The ribose moiety of the LNA nucleotide is modified with an additional bridge connecting the 2'-oxygen and 4'-carbon. This bridge "locks" the ribose in the 3'-endo(north) conformation, which is often found in A-form duplexes. These LNA monomers are described, inter alia, in WO 2001 / 25248, WO 2003 / 006475 and WO 2003 / 095467, the disclosures of each of which are incorporated herein by reference.

[0023] The term "complementary" refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Polynucleotides are described as "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides.

[0024] "Percent (%) sequence identity" for any nucleotide sequence identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues in a particular nucleotide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be accomplished in various ways within the skill of the art using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR, Inc.) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, sequence comparison between two nucleotide sequences was performed using the computer program Blastn (nucleotide-nucleotide BLAST), provided online by the National Center for Biotechnology Information (NCBI). The percent sequence identity of a given nucleotide sequence A to a given nucleotide sequence B (which may alternatively be expressed as a given nucleotide sequence A having a particular % nucleotide sequence identity to a given nucleotide sequence B) is calculated according to the following formula: X / Y×100% where X is the number of nucleotide residues scored as identical matches by the sequence alignment program BLAST in its alignment of A and B, and Y is the total number of nucleotide residues in the shorter of A or B.

[0025] As used herein, the term "treat" or "treating" or "treatment" refers to preventative (e.g., prophylactic), curative, or palliative treatment. As used herein, the term "treating" refers to the application or administration of an ASO of the disclosure to a subject having a medical condition, a symptom of a condition, a predisposition to a disease or disorder secondary to a condition, with the intent of partially or completely alleviating, ameliorating, relieving, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or subjects who show only early signs of the disease, disorder, and / or condition with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced, as that term is defined herein. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the amelioration of symptoms or reduction in markers of a disease, but also the halting or slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a reduction in the extent of disease, a stabilized (i.e., non-worsening) state of disease, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or total).

[0026] As used herein, the term "effective amount" refers to an amount of a component sufficient to produce a desired response. As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., the present ASO) that produces a desired therapeutic "effective treatment" as defined above. The specific therapeutically effective amount will vary depending on factors such as the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of concurrent treatment (if any), and the specific formulation employed. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the compound or composition are outweighed by the therapeutically beneficial effects.

[0027] As used herein, the term "subject" or "patient" refers to a human or non-human animal that has dysregulated expression of TXNDC5, particularly upregulation of TXNDC5, compared to a healthy subject, and undergoes the method of the present invention. The term "subject" or "patient" is intended to refer to both male and female genders, unless one gender is specifically indicated. Examples of non-human animals include all vertebrates, such as mammals, for example, primates, dogs, rodents (e.g., mice or rats), cats, sheep, horses, or pigs, as well as non-mammals, such as birds and amphibians.

[0028] Unless otherwise specified herein, scientific and technical terms employed in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art. Unless otherwise required by context, it is understood that singular terms include their plurals and plural terms include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include plural references unless the context clearly indicates otherwise.

[0029] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean as would be considered by one of ordinary skill in the art. Other than in the effective / effective examples, or unless expressly stated otherwise, all numerical ranges, amounts, values ​​and percentages, such as those for amounts of materials, durations, temperatures, operating conditions, ratios of amounts, and the like, disclosed herein, should be understood in all instances as being modified by the term "about." Thus, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0030] 2. Antisense Oligonucleotide (ASO) The present disclosure is directed to a novel solution for treating diseases related to upregulated TXNDC5 by using antisense oligonucleotide (ASO) molecules.Therefore, in the broadest aspect, the present invention relates to a single-stranded deoxyribonucleic acid that is complementary to at least a portion of a target gene, such as TXNDC5 mRNA, and when transfected into a host cell, the single-stranded deoxyribonucleic acid can suppress the expression of target gene mRNA.

[0031] The single stranded deoxyribonucleic acid or ASO molecule of the present disclosure is about 16-21 nucleotides in length and comprises a deoxyribonucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. According to a preferred embodiment of the present disclosure, the ASO molecule comprises a deoxyribonucleotide sequence having at least 90% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In a particular preferred embodiment, the ASO molecule comprises a deoxyribonucleotide sequence that is 100% identical to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. The ASO of the present disclosure can be 16-21 nucleotides in length, such as 16, 17, 18, 19, 20, or 21 nucleotides in length. In some preferred embodiments, the ASOs of the disclosure are 20 nucleotides in length, hi other preferred embodiments, the ASOs of the disclosure are 16 nucleotides in length.

[0032] 3. Qualified ASO Alternatively or additionally, the ASO may include modifications (e.g., substitutions) at its backbone nucleobase or at its sugar ring, such as by introducing substitutions or changes to the internucleotide linkage, sugar moiety, or nucleobase. In some embodiments, the nucleic acid of the ASO is comprised of a DNA molecule in combination with one or more LNA molecules. In other embodiments, the nucleic acid of the ASO is comprised of a DNA molecule in combination with one or more 2'-sugar modifications (e.g., 2'-O-methoxyethyl substitutions). Modified ASOs are often preferred over native forms due to desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0033] (i) Modified internucleotide linkages The naturally occurring internucleotide bond of RNA and DNA is a 3' to 5' phosphodiester bond. Oligonucleotides with modified internucleotide bonds include those that retain a phosphorus atom and those that do not have a phosphorus atom. Representative phosphorus-containing internucleotide bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for the preparation of phosphorus-containing and non-phosphorus-containing conjugates are known.

[0034] In certain embodiments, the ASO comprises one or more modified internucleotide linkages, such as one or more phosphorothioate linkages.

[0035] (ii) Modified sugar moiety The ASO may optionally include one or more nucleosides with modified sugars. In certain embodiments, the ASO comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (e.g., 5'- or 2'-substituents), bridging of non-geminal ring atoms to form locked nucleic acids (LNAs), R, R a and R b C 1-12 The S, N(R) or C(R) of the ribosyl ring oxygen atom is an alkyl, a protecting group or a combination thereof. a )(R b )2.

[0036] Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides or replacement of the ribosyl ring oxygen atom with S with additional substitution at the 2'-position.

[0037] According to an alternative embodiment, the chemically modified sugar comprises a 5'-substitution of an LNA molecule. Examples of LNA include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the ASO herein comprises one or more LNA nucleosides, wherein the bridge has one of the formulas: 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2, 4'-(CH2)-O-2'(LNA), 4'-(CH2)2-O-2'(ENA), 4'-C(CH3)2-O-2', 4'-CH(CH3)-O-2', and 4'-CH(CHOCH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-NR-O-2', 4'-CH2-C(CH3)-2', and 4'-CH2-C(=CH2)-2'; R is independently H, C, 1-12 Each of the aforementioned LNAs contains a variety of stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose.

[0038] In certain embodiments, nucleosides are modified by replacing the ribosyl ring with a sugar surrogate. Such modifications include, but are not limited to, those having the following formula for the ribosyl ring: [ka] This includes replacement with surrogate ring systems (sometimes referred to as DNA analogs) such as morpholino, cyclohexenyl, cyclohexyl, or tetrahydropyranyl rings, such as those having one of the following ring systems:

[0039] Many other bicyclo- and tricyclo-sugar surrogate ring systems are known in the art that can also be used to modify nucleosides for incorporation into the present ASOs.

[0040] Thus, the ASO may be composed entirely of DNA molecules or may be composed of DNA molecules in combination with at least one modified nucleotide. In some embodiments, the ASO is composed of DNA molecules in combination with LNA molecules, such as 2'-O-, 4'-C methylene bicyclonucleoside monomers. One beneficial effect of having LNA monomers in a nucleic acid is that the stability of the nucleic acid is improved, and therefore the ASO of the present invention may include the incorporation of LNA monomers into standard DNA oligonucleotides to increase the stability of the resulting molecule, such as by increasing the resistance of the ASO to proteases (endonucleases and exonucleases), thereby increasing its circulating half-life in biological samples. In general, the single-stranded ASO of the present invention may include at least about 5%, 10%, 15%, 20%, 25% or 30% LNA monomers, based on the total number of nucleotides in the strand. In certain embodiments, the ASO of the invention will comprise at least about 25%, 30%, 40%, 50% or 60% LNA monomers, preferably about 40% LNA monomers, more preferably about 60% LNA monomers, based on the total number of nucleotides in the strand. In one embodiment of the invention, the ASO of the invention is entirely composed of DNA molecules, with one strand comprising a nucleotide sequence having at least 90% sequence identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In another embodiment of the invention, the ASO of the invention is composed of DNA molecules combined with LNA molecules, with the single stranded ASO being composed of DNA molecules combined with at least about 30% LNA molecules. In one embodiment of the invention, the single stranded ASO comprises at least one LNA monomer. In other embodiments, the single stranded ASO comprises six LNA monomers.

[0041] Alternatively, the single-stranded ASO of the invention may be comprised of a DNA molecule combined with at least one 2'-sugar modification, such as a 2'-O-methoxyethyl (2'-O-MOE) modified sugar. In general, each individual strand of a single-stranded ASO of the invention may contain at least about 5%, 10%, 15%, 20%, 25% or 30% 2'-sugar modification, based on the total number of nucleotides in the strand. In certain embodiments, the single-stranded ASO of the invention will contain at least about 25%, 30%, 40%, 50% or 60% 2'-sugar modification, preferably about 40% 2'-sugar modification, more preferably about 50% 2'-sugar modification, based on the total number of nucleotides in the strand. In one embodiment of the invention, the single stranded ASO of the invention is composed entirely of DNA molecules and comprises a nucleotide sequence having at least 90% sequence identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In another embodiment of the invention, the single stranded ASO of the invention is composed of DNA molecules in combination with one or more 2'-sugar modifications, and the strand of deoxyribonucleic acid is composed of DNA molecules in combination with at least 20% 2'-sugar modifications. In one embodiment of the invention, the single stranded ASO comprises only one 2'-O-MOE modified sugar. In another embodiment, the single stranded ASO comprises 10 2'-O-MOE modified sugars.

[0042] (iii) modified nucleobase Chemically modified nucleosides may also be included in the ASO to increase their binding affinity to target nucleic acids. Nucleobase (or base) modifications or substitutions are functionally interchangeable but structurally distinct from naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may affect nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of antisense oligomers to target nucleic acids.

[0043] Thus, the present single-stranded ASOs include any of the following: 5-methylcytosine, 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-halouracil; 5-halocytosine; 5-propynyluracil and 5-propynylcytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, 6-azocytosine and 6-azothymine; 5-uracil; 4 The single-stranded ASO of the present invention may be comprised of DNA molecules in combination with at least one modified nucleobase, such as -thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine; 7-methyladenine; 2-F-adenine; 2-aminoadenine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine; 3-deazaguanine and 3-deazaadenine. In general, the single-stranded ASO of the present invention may contain at least about 5%, 10%, 15%, 20%, 25% or 30% modified nucleobases, based on the total number of nucleotides in the strand. In certain embodiments, the single-stranded ASO of the invention comprises at least about 25%, 30%, 40%, 50% or 60% modified nucleobases, preferably about 40% modified nucleobases, more preferably about 50% modified nucleobases, based on the total number of nucleotides in the strand. In one embodiment of the invention, the single-stranded ASO of the invention is entirely composed of DNA molecules and comprises a nucleotide sequence having at least 90% sequence identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In other embodiments of the invention, the single-stranded ASO of the invention is composed of DNA molecules in combination with 5-methylcytosine (e.g., at least 20% 5-methylcytosine). In some embodiments of the invention, the single-stranded ASO comprises only one 5-methylcytosine. In other embodiments, the single stranded ASO contains 10 5-methylcytosines.

[0044] 4. How to generate this ASO Currently, there are various methods to generate ASOs for gene silencing studies, including chemical synthesis or digestion with DNaseIII family enzymes of long dsDNA, with or without the chemical modifications mentioned above. These methods involve in vitro preparation of nucleic acids that are then directly introduced into cells by lipofection, electroporation or other techniques.

[0045] The ASO molecules of the present invention are obtained by in vitro preparation and / or chemical synthesis using protocols well known in the art. For example, the single-stranded nucleic acid of the present invention can be produced using polymerization techniques of nucleic acid chemistry known to those skilled in the art of organic chemistry. In general, the standard oligomerization cycle of phosphoramidite approach can be used, but other chemistries such as H-phosphonate chemistry or phosphotriester chemistry can also be used. The ASO molecules can be delivered directly to the subject or via a delivery vehicle such as liposomes. Furthermore, the ASO can be formulated with suitable carriers and / or diluents to give a pharma-ceutically acceptable preparation. Methods for delivering nucleic acid molecules are known in the art and include, but are not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other vesicles such as biodegradable polymers, hydrogels, or cyclodextrins.

[0046] All ASO molecules used in the present invention are listed in Table 1. The modified ASOs of the present invention (i.e., ASO-LNA or ASO-MOE) were obtained by modifying the corresponding ASOs with at least one LNA molecule or 2'-MOE modified sugar as listed in Table 1. Table 2 lists the modified ASO molecules of the present invention. Table 1. Deoxyribonucleotide sequences of the ASOs [Table 1] JPEG2025501269000004.jpg175170 Table 2. Deoxyribonucleotide sequences of ASO-LNA or ASO-MOE [Table 2]

[0047] Therefore, a further aspect of the present invention relates to the use of the single-stranded deoxyribonucleic acid of the present invention for the manufacture of medicines for the treatment of diseases associated with upregulation of TXNDC5, such as aging, arthritis (e.g., rheumatoid arthritis), cancer, diabetes (e.g., type II diabetes), neurodegenerative diseases, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, liver fibrosis, atherosclerosis, vitiligo and viral infection. Examples of cancers that can be treated by the single-stranded ASO of the present invention include, but are not limited to, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, multiple myeloma, non-small cell lung cancer, pancreatic cancer, prostate cancer, kidney cancer and uterine cancer. Examples of neurodegenerative diseases that can be treated by the single-stranded ASO of the present invention include, but are not limited to, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease and prion disease. In a preferred embodiment, the single-chain ASO of the invention is for the manufacture of a medicament for the treatment of pulmonary fibrosis, renal fibrosis, hepatic fibrosis or myocardial fibrosis.

[0048] Therefore, a pharmaceutical composition is provided for treating or preventing diseases mediated through upregulation of TXNDC5.The pharmaceutical composition comprises at least one single-stranded deoxyribonucleic acid of the present invention as an active ingredient and a pharmaceutically acceptable carrier.Optionally, the pharmaceutical composition may further comprise other drugs suitable for promoting the treatment of the above-mentioned diseases, such as antidiabetic drugs for the treatment of diabetes, chemotherapeutic drugs for the treatment of cancer, nonsteroidal anti-inflammatory drugs (NSAIDs) for the treatment of arthritis, antifibrotic therapeutic drugs such as nintedanib or pirfenidone for fibrotic lung disease, to name a few.

[0049] The nucleic acid of the present invention may be suspended in a suitable dispersion medium, such as water, PBS, saline, oil or fatty acid. The resulting pharmaceutical composition may be administered parenterally, by inhalation spray, topically, rectally, nasally, orally or vaginally. As used herein, the term "parenterally" includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered intramuscularly, intraperitoneally or intravenously, and most preferably, the composition is administered intramuscularly. In one embodiment, the composition of the present invention is injected intramuscularly at a site in one limb (i.e., arm or leg) of the subject. The body part suitable for injection is selected based on the following: choice of nucleic acid to be released, personal condition of the subject including sex, age, weight and / or current and previous medical conditions, etc. A skilled physician can determine the body part suitable for injection without undue experimentation. The sterile injectable form of the composition of the present invention may be an aqueous or oily suspension. These suspensions may be formulated by techniques well known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, phosphate buffer and isotonic sodium chloride solution (i.e., physiological saline). In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma-ceutically acceptable oils such as olive oil or castor oil, particularly in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharma-ceutically acceptable dosage forms, including emulsions and suspensions.Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The required dosage will depend on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's weight, body surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages are 0.15 mg to 1.5 mg of nucleic acid / Kg body weight, such as 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 and 1.5 mg of nucleic acid / Kg body weight, preferably 0.3 mg to 1.2 mg of nucleic acid / Kg body weight, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 mg of nucleic acid / Kg body weight, more preferably 0.5 mg to 1.0 mg of nucleic acid / Kg body weight, such as 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg of nucleic acid / Kg body weight. In view of the different efficiencies of various administration routes, variations in the required dosage are expected. A person skilled in the art can easily evaluate the relevant factors and determine the dosage to be used for the intended purpose based on this information.

[0050] The present invention also features a method of treating a subject exhibiting upregulation of TXNDC5, comprising administering the single-stranded deoxyribonucleic acid of the present invention or the composition of the present invention to a subject in need thereof, and further comprising administering an additional pharmaceutical agent (e.g., a chemotherapeutic agent for the treatment of cancer) to the subject. The subject here refers to humans and non-human animals. In a preferred embodiment, the subject is a human. In one embodiment, the subject is diagnosed with pulmonary fibrosis. In another embodiment, the subject is diagnosed with cancer. In yet another embodiment, the subject is diagnosed with rheumatoid arthritis. The subject may have undergone a medical treatment before undergoing the method and / or composition of the present invention. In the case of cancer, the medical treatment refers to surgery, chemotherapy or radiation therapy that is commonly applied to patients with tumors, and therefore, to augment the anti-tumor effect of gene therapy, a subject previously diagnosed with cancer may also undergo other anti-tumor therapies before, simultaneously with or after undergoing the method and / or composition of the present invention. In one example, the subject has pulmonary fibrosis and has been treated with pirfenidone or nintedanib prior to receiving the methods and / or compositions of the invention.

[0051] The following examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in the practice of the invention. These examples should not be construed in any way as limiting the scope of the invention in any manner. EXAMPLES

[0052] Materials and Methods cell culture Primary adult human lung fibroblasts (HPF-a) (ScienCell, CA, USA) were cultured in fibroblast medium supplemented with 2% fetal bovine serum (FBS), 1% fibroblast growth supplement (FGS) and 1% penicillin / streptomycin solution and maintained at 37°C in a humidified environment containing 95% O2 / 5% CO2.

[0053] Generation of this ASO Human TXNDC5 mRNA was used as the target sequence for the preparation of the ASO of the present disclosure. Specifically, the ASO of the present disclosure was prepared using the target sequence at positions 337-356, 670-689, 675-694, 862-881, 879-898, 1003-1022, 1007-1026, 1278-1297, 2864-2883, 2865-2884, 2868-2887 and 2873-2892, respectively.

[0054] All of the ASOs disclosed herein were obtained from Eurogentec or synthesized by using an AKTA OligoPilot10Plus synthesizer. The ASOs were purified by reversed-phase HPLC or IEXHPLC. The purity of the ASOs was analyzed by UPLC. The characterization of the ASOs was analyzed by MOLDI-TOF or LC-HRMS.

[0055] Additionally, ASOs were used independently to prepare modified ASOs containing locked nucleic acid (LNA) molecules (hereafter, "ASO-LNA") or 2'-O-methoxyethyl sugar (hereafter, "ASO-MOE") modifications. Each modified ASO was synthesized at a 1 nmol scale on a MOSS Expedite instrument platform following the procedures described in the instrument manual.

[0056] Transfecting HPF-a cells with the ASO HPF-a cells were cultured at 1 × 10 5The cells were maintained and cultured in 6-well plates at a density of 1000 cells / well and transfected with the ASO using TransIT-X2 (Mirus Bio, USA). Specifically, the ASO-containing plasmid and TransIT-X2 were mixed in serum-free Opti-MEM (Thermo Fisher Scientific, USA) and added to HPF-a cell culture medium at a final concentration of 0.4-60 nM ASO and 3.3 μL TransIT-X2 per mL of medium, and further incubated for 24 h. Transfection of human TXNDC5 mRNA ASO in the cells was confirmed by detection of human TXNDC5 mRNA gene expression, either at the RNA level by quantitative real-time PCR (qRT-PCR) analysis or at the protein level by immunoblot assay.

[0057] Quantitative RT-PCR (qRT-PCR) Total RNA from cells transfected with the ASO was isolated using the Direct-zol™ RNA MiniPrep kit according to the manufacturer's instructions (ZYMO Research, USA). A quantity of 100 ng of DNase-treated total RNA was used as template for first-strand DNA synthesis in a 20 μL reaction with 4×TaqMan™ Fast1-step master mix (Thermo Fisher Scientific, USA) containing TaqMan™ assay probe sets (hTXNDC5: Hs01046710_m1 (FAM); hGAPDH: Hs03929097_g1 (VIC)), and first-strand DNA synthesis was performed in an Applied Biosystems 7500Fast instrument running the following program: 50° C. for 5 min, 95° C. for 20 s, 95° C. for 15 s followed by 60° C. for 1 min, repeated 40 times. The expression level of each individual transcript was normalized to the control gene GAPDH and expressed relative to the mean expression value of the control samples.

[0058] Immunoblot assay After 24 h of transfection of HPF-a cells, the cells were replaced with serum-free medium and treated with 10 ng / ml TGFβ1 (Pepro Tech, USA) for 48 h. HPF-a cells were homogenized with 2× sample buffer (BioRad Laboratories, USA) and subsequently boiled at 95°C for 10 min. Protein samples were fractionated on 10% SDS-PAGE gels, transferred to PVDF membranes, and blocked with blocking buffer (Visual Protein, Taiwan, BP01-1L). The membrane was incubated overnight at 4°C with primary antibodies against COL1A1 (1:500, OriGene, USA, TA309096, for human species), fibronectin (1:2000, BD Biosciences, USA, 610077), TXNDC5 (1:15000, Proteintech, USA, 19834-1-AP), αSMA (1:1000, Abcam, UK, ab5694), and β-actin (1:1000, Millipore, Germany, MAB1501). Blots were developed with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5000, Cell signaling Technology, USA, 7076, 7074) and SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Fisher Scientific, USA, 34080, 34094). Protein band detection was performed using a ChemiDoc MP system (BioRad Laboratories, USA). Protein band intensity quantification analysis was performed using ImageLab software version 5.2.1.

[0059] Bleomycin-induced pulmonary fibrosis animal model Male C57BL / 6 mice, 8-9 weeks old, were quarantined for 1 week after purchase. Mice were housed in an individually ventilated cage system with 5 mice per cage at constant temperature and humidity. The room was kept on a 12-h light / 12-h dark cycle (lights on at 07:00, lights off at 19:00) with a room temperature of 22 ± 2°C and humidity of 55 ± 10%. Animals were allowed free access to rodent pellet chow and water. Animal experiments were performed in accordance with the ethical regulations of the National Institute of Health (NIH) Guidance for the Care and Use of Laboratory Animals.

[0060] To induce pulmonary fibrosis, mice were intratracheally instilled with bleomycin (dissolved in sterile saline) at a dose of 3U / Kg body weight. Mice in the placebo group were instilled with the same volume of sterile saline alone. Seven days after bleomycin induction, mice were randomly divided into 5 groups and administered vehicle solution, nintedanib, the modified ASO of SEQ ID NO: 73 (i.e., DCB1111128235), SEQ ID NO: 14 (i.e., DCB1111128279), or SEQ ID NO: 82 (i.e., DCB1111128281) on the same test day. Each group consisted of 6 mice. Nintedanib was dissolved in PBS containing 10% dimethylformamide (DMF) in a final volume to a final concentration of 6 mg / mL and administered at 60 mg / kg body weight (mpk) by oral gavage (PO) once a day (QD) for 14 days. Each of the modified ASOs (i.e., DCB1111128235, DCB1111128279, or DCB1111128281) was dissolved in TruboFect Transfection Reagent (Thermo Scientific, Massachusetts, USA) and administered at 0.2 mg / kg body weight (mpk) by intratracheal instillation twice a week (BIW) for 2 weeks. Mice in the vehicle group were injected with the same volume of TruboFect Transfection Reagent alone and served as the control group. The study was terminated 21 days after disease induction, lung function was determined, and lung tissue was harvested and appropriately stored until analysis.

[0061] Pulmonary function tests Pulmonary function was assessed using the flexiVent system (Scireq, Montreal, Quebec, Canada). Mice were tracheotomized and ventilated with a respiratory rate of 150 breaths / min, a tidal volume of 10 ml / kg, and a positive end-expiratory pressure of 2–3 cm H2O. Deep inflation perturbations were used to estimate inspiratory volume. Pressure-volume loops were generated by a constant increasing pressure followed by a periodic decreasing pressure. Other pulmonary function parameters, including air resistance, compliance, and elastance, were measured using a SnapShot-150. Compliance reflects the ability of the lung to expand and contract; air resistance reflects the change in intrapulmonary pressure required to generate a unit flow of gas through the lung's airways and is the pressure difference between the mouth and the alveoli of the lung divided by the air flow; and elastance reflects the pressure required to inflate the lung.

[0062] Pathological evaluation Left lungs from mice were fixed in buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin or picrosirius red (Abcam, Cambridge, UK). Measurement of fibrotic area by picrosirius red staining was quantified using ImageJ software.

[0063] Example 1 Inhibition of TXNDC5 mRNA transcription by the ASO HPF-a cells were treated with the indicated ASOs and TXNDC5 mRNA expression was measured by qRT-PCR according to the procedure described in the section "Materials and Methods." The results are summarized in Table 3, and the TXNDC5 mRNA expression inhibitory activity of the indicated ASOs above 50% at 30 nM is ranked as follows: +++ is less than 50% TXNDC5 mRNA level and ++ is 70%-50% TXNDC5 mRNA level. Table 3. In vitro inhibition of human TXNDC5 mRNA via this ASO [Table 3] JPEG2025501269000007.jpg182144

[0064] According to the data summarized in Table 3, after treatment, of all ASOs prepared according to the procedures described in "Materials and Methods," 26 of them were effective in suppressing TXNDC5 expression by more than 50%, and 41 of them moderately suppressed TXNDC5 mRNA transcription with TXNDC5 mRNA levels ranging from 70% to 50%, as listed in Table 3. The remaining ASOs (i.e., ASOs other than the 69 ASOs listed in Table 3) were only able to mildly suppress TXNDC5 mRNA transcription with TXNDC5 mRNA levels maintained at greater than 70% after treatment (data not shown).

[0065] Example 2 Inhibition of TXNDC5 mRNA and TGF-β-induced fibrosis-related proteins by the modified ASO In this example, ASOs with 2'-O-methoxyethyl modified sugars (i.e., ASO-MOE) or LNA molecules (i.e., ASO-LNA) were derived from the ASOs in Table 1 according to the procedures described in the "Materials and Methods" section, and their respective effects on the transcriptional expression levels of TXNDC5 mRNA and transforming growth factor beta (TGF-β)-induced fibrosis-associated proteins were examined. The results are summarized in Table 4 and Figure 1.

[0066] As the data in Table 4 show, all ASO-MOEs had IC values ​​below 60 nM. 50 DCB1111112238 (SEQ ID NO: 75), DCB1111112240 (SEQ ID NO: 76) and DCB1111112277 (SEQ ID NO: 79) were able to successfully inhibit the transcription of TXNDC5 mRNA, with IC values ​​of less than 10 nM. 50 Regarding ASO-LNA, the IC of ASO-LNA was 1.21 in suppressing the transcription of TXNDC5 mRNA. 50 were generally more potent than the corresponding ASO-MOEs, such that the potency of the ASO-MOEs was smaller than that of the ASO-MOEs (+++ vs. ++ for SEQ ID NO:3 (i.e., DCB1111128003) and SEQ ID NO:4 (i.e., DCB1111128004), respectively). Table 4. In vitro inhibition of TXNDC5 mRNA by the present ASO-MOE or ASO-LNA [Table 4] IC of TXNDC5 mRNA expression inhibitory activity 50 IC++ below 10nM 50 ++ IC of 10nM to 20nM 50 Level, + IC of 20nM to 60nM 50 are rated as levels

[0067] TGF-β is known to induce the expression of fibrosis-associated proteins, therefore, the expression of TXNDC5 and fibrosis-associated proteins including fibronectin, type I collagen, and α-smooth muscle actin (α-SMA) in the presence or absence of ASO-MOE was measured by immunoblot analysis, respectively. The results are shown in Figure 1.

[0068] It is evident from the data in Figures 1A-1I that TGF-β (10 ng / mL) enhanced the expression of fibrosis-associated proteins, including fibronectin, type I collagen, and α-SMA, and this enhanced protein expression was significantly suppressed in a dose-dependent manner by the present ASO-MOEs, including DCB11111128235 (Figure 1A), DCB11111128255 (Figure 1B), DCB1111128252 (Figure 1C), DCB1111128266 (Figure 1D), DCB1111128265 (Figure 1E), DCB1111128238 (Figure 1F), DCB1111128279 (Figure 1G), DCB1111128280 (Figure 1H), and DCB1111128281 (Figure 1I).

[0069] Example 3 The ASO-MOE reduced the progression of pulmonary fibrosis To determine the in vivo function of the ASO, pulmonary fibrosis was induced by intratracheal instillation of bleomycin (BLM, 3U / Kg body weight) according to the procedure described in the Materials and Methods section. Fibrotic mice were then randomly divided into 5 groups (6 mice / group), with the animals in the vehicle group being instilled with PBS (intratracheal, 2 weeks), the animals in the ASO group being instilled with the ASO-MOE (SEQ ID NO: 73 or 14) or scrambled ASO (SEQ ID NO: 82) (all by intratracheal route of administration, 0.2mg / Kg) on ​​days 7, 10, 14 and 17, while the animals in the nintedanib group were instilled with a daily dose of nintedanib (60mg / Kg, 14 days). Healthy animals (i.e., non-fibrotic mice) in the placebo group received no treatment. Pulmonary function was assessed using the FlexiVent system to determine various factors on the specified days, including compliance (a factor reflecting the ability of the lung to expand and contract), air resistance (a factor reflecting the change in intrapulmonary pressure required to generate a unit flow rate of gas through the airways of the lung, which is the pressure difference between the mouth and the alveoli of the lung divided by the air flow rate), and elastance (a factor reflecting the pressure required to inflate the lungs), and animals were sacrificed on day 21 to take lung samples for identification of fibrotic areas. The results are shown in Figures 2, 3, and 4.

[0070] See FIG. 2, which is a bar graph showing the changes in compliance, resistance and elastance with or without treatment with the present ASO-MOE or nintedanib. The data clearly show that when animals were treated with the present ASO-MOE, compliance was much higher than that of the vehicle control (FIG. 2A), and resistance and elastance were independently much lower than that of the vehicle control (FIGS. 2B and 2C). Furthermore, the effect of ASO-MOE (SEQ ID NO: 73 or 14) on pressure-volume loops was more effective than that of nintedanib (60 mg / Kg) (FIG. 3). These data showed that fibrotic mice treated with the present ASO-MOE (SEQ ID NO: 73 or 14) showed improved lung function (including compliance, resistance, elastance and pressure-volume loops) compared to those of the vehicle control group and the scrambled ASO group (DCB1111128281, i.e., SEQ ID NO: 82).

[0071] Regarding the effect of the present ASO-MOE in reducing BLM-induced fibrotic areas in lung tissue, it was found that the present ASO-MOE of sequence number 73 (DCB1111128235) was capable of reducing BLM-induced fibrotic areas, and its effect was more potent than that of nintedanib (Figure 4).

[0072] Taken together, the findings of the present invention support the proposition that diseases and / or disorders resulting from dysregulation of TXNDC5, such as aging, arthritis, cancer, diabetes, neurodegenerative diseases, pulmonary fibrosis, vitiligo and viral infections, may be treated by introducing antisense oligonucleotides, particularly antisense oligonucleotides that interfere with the expression of TXNDC5 mRNA, into a subject (e.g., a human patient) in need of such treatment.

[0073] It should be understood that the above description of the embodiments is given by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of the exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, those skilled in the art could make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. 1. A single-stranded antisense oligonucleotide (ASO) that inhibits translation of thioredoxin domain-containing protein 5 (TXNDC5) mRNA, the single-stranded ASO being approximately 16-21 nucleotides in length and comprising a deoxyribonucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

2. The single-stranded ASO of claim 1, comprising at least one locked nucleic acid (LNA) molecule, a 2'-sugar modification, a modified internucleotide linkage, a modified nucleobase, or a combination thereof.

3. The single-stranded ASO of claim 2, comprising at least one 2'-fluoro sugar, 2'-O-methyl sugar, or 2'-O-methoxyethyl sugar.

4. The single-stranded ASO of claim 2 comprising six LNA molecules.

5. The single-stranded ASO of claim 2 comprising 10 2'-O-methoxyethyl sugars.

6. The single-stranded ASO of claim 2 comprising at least one phosphorothioate internucleotide bond.

7. The single-stranded ASO of claim 2, comprising at least one 5-methylcytosine.

8. A method for treating a disease in a subject mediated through upregulation of thioredoxin domain-containing protein 5 (TXNDC5), comprising administering to the subject an effective amount of a single-chain ASO described in claim 1 to inhibit transcription of TXNDC5 mRNA.

9. 9. The method of claim 8, wherein the single stranded ASO comprises at least one locked nucleic acid (LNA) molecule, a 2'-sugar modification, a modified internucleotide linkage, a modified nucleobase, or a combination thereof.

10. 10. The method of claim 9, wherein the single-stranded ASO comprises at least one 2'-fluoro, 2'-O-methyl or 2'-O-methoxyethyl sugar.

11. The method of claim 10, wherein the single-stranded ASO comprises six LNA molecules.

12. The method of claim 10, wherein the single-stranded ASO comprises ten 2'-O-methoxyethyl sugars.

13. The method of claim 10, wherein the single-stranded ASO comprises at least one phosphorothioate internucleotide linkage.

14. The method of claim 10, wherein the single-stranded ASO comprises at least one 5-methylcytosine.

15. 9. The method of claim 8, wherein the disease is selected from the group consisting of aging, arthritis, cancer, diabetes, neurodegenerative diseases, fibrosis, atherosclerosis, vitiligo and viral infection.

16. 16. The method of claim 15, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, multiple myeloma, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cancer and uterine cancer.

17. 16. The method of claim 15, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease and prion diseases.

18. 16. The method of claim 15, wherein the fibrosis is selected from the group consisting of pulmonary fibrosis, renal fibrosis, hepatic fibrosis and myocardial fibrosis.

19. The method of claim 8 , wherein the subject is a human.

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