HK40111887A - Anti-sense oligonucleotides and uses thereof - Google Patents

Description

Antisense oligonucleotides and their uses

Cross-reference to related applications

This application claims and enjoys priority and all rights to U.S. Provisional Patent Application No. 63/294,835, filed December 29, 2021, the contents of which are incorporated herein by reference.

Background of the Invention

1. Field of Invention

This invention relates to a treatment plan for a disease, specifically, a treatment of the disease by means of antisense oligonucleotides, which can reduce the expression level of protein 5 (TXNDC5) messenger RNA (mRNA) containing the thioredoxin domain.

2. Prior Art

TXNDC5 (thioredoxin domain-containing protein 5) is a disulfide isomerase that catalyzes thioredoxin activity and enables it to function as an endoplasmic reticulum escort protein. Upregulation of TXNDC5 expression is known to be associated with various diseases, including cancer, diabetes, arthritis, neurodegenerative diseases, organ fibrosis-related diseases (e.g., pulmonary fibrosis, renal fibrosis, liver fibrosis, or myocardial fibrosis), and vitiligo.

With the development of posttranscriptional gene silencing technology, antisense oligonucleotides (ASOs) have been used as tools to eliminate the expression of specific genes in various organisms. Scientists can now systematically silence functional genes along cellular signaling pathways to characterize protein-protein interactions, making gene silencing a novel approach for drug development. After long-term research and experimentation, the inventors of this application discovered several short oligonucleotide molecules that target TXNDC5 mRNA or pre-mRNA and reduce TXNDC5 RNA levels, thus lowering TXNDC5 protein expression. Antisense nucleotides act on their targets through various mechanisms, including RNase H degradation of mRNA, steric hindrance of ribosomal subunit binding, alteration of mRNA maturation, inhibition of 5'-cap formation, and transcription arrest. Therefore, these newly discovered short nucleic acid molecules can be used to develop drugs to treat diseases related to TXNDC5 protein overexpression, thereby alleviating symptoms in patients requiring such treatment.

Summary of the Invention

This invention relates to a single-stranded nucleic acid molecule for the treatment or prevention of diseases, particularly a single-stranded nucleic acid molecule for the treatment or prevention of diseases associated with TXNDC5 upregulation.

Therefore, the first aspect of this invention relates to a single-stranded antisense oligonucleotide (ASO) molecule that can reduce the expression of TXNDC5 mRNA. The ASO molecule is approximately 16-21 nucleotides in length and contains a deoxyribonucleic acid sequence with 80% sequence similarity to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

According to the preferred embodiment of this disclosure, the ASO molecule contains a deoxyribonucleic acid sequence having 90% sequence similarity to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

According to a preferred embodiment of this disclosure, the ASO molecule comprises at least one locked nucleic acid (LNA) molecule, a 2'-sugar modification, modified internucleotide linkages, or a combination thereof. According to some embodiments, the ASO molecule comprises six locked nucleic acid molecules. According to other embodiments, the ASO molecule comprises ten 2'-sugar modifications.

On the other hand, a second aspect of the present invention relates to a method for treating an individual suffering from a disease caused by upregulation of TXNDC5. The method comprises administering to the individual a pharmaceutically effective amount of the ASO molecule of the present invention to inhibit transcription of the TXNDC5 gene.

According to the preferred embodiment of this disclosure, the ASO molecule of the present invention is a single-stranded oligonucleotide capable of reducing the expression level of TXNDC5 mRNA. The ASO molecule is approximately 16-21 nucleotides in length and contains a deoxyribonucleic acid sequence having 80% sequence similarity to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

According to a preferred embodiment of this disclosure, the ASO molecule comprises at least one locked nucleic acid (LNA) molecule, a 2'-sugar modification, modified internucleotide links, or a combination thereof. According to some embodiments, the ASO molecule comprises six locked nucleic acid molecules. According to other embodiments, the ASO molecule comprises ten 2'-sugar modifications.

According to a preferred embodiment of this disclosure, the diseases caused by upregulation of TXNDC5 are selected from aging, arthritis (e.g., rheumatoid arthritis), cancer, diabetes (e.g., type II diabetes), neurodegenerative diseases, fibrosis, atherosclerosis, vitiligo, or viral infections. According to a preferred embodiment, the individual suffers from an organ fibrosis disease, such as pulmonary fibrosis, renal fibrosis, liver fibrosis, or myocardial fibrosis.

Another aspect of this disclosure relates to pharmaceutical compositions for treating, preventing, or alleviating diseases associated with overexpression of the TXNDC5 gene. These pharmaceutical compositions comprise the ASO molecule of the present invention and a pharmaceutically acceptable carrier.

After reading the following embodiments, those skilled in the art will easily understand the basic spirit and other inventive objectives of the present invention, as well as the technical means and implementation methods adopted by the present invention.

Attached Figure Description

To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described below:

Figures 1A-1I illustrate the results of monitoring the effects of the ASO-MOEs or ASO-LNAs molecules of the present invention on the expression patterns of TXNDC5 and fibrosis-related proteins with or without TGF-β induction using Western blotting according to certain embodiments of the present invention. The ASOs molecules of the present invention are (A) DCB11111128235, (B) DCB11111128255, (C) DCB11111128252, (D) DCB11111128266, (E) DCB11111128265, (F) DCB11111128238, (G) DCB11111128279, (H) DCB11111128280 and (I) DCB11111128281.

Figures 2A-2C illustrate the effect of bleomycin (BLM) on lung fibrosis in mice according to an embodiment of the present invention, followed by administration of the ASO-MOEs molecules of the present invention to improve lung function, wherein (A) is the lung compliance factor, (B) is the lung resistance factor, and (C) is the lung elasticity factor.

Figure 3 illustrates the pressure-volume curve measured in the lungs of BLM-induced fibrotic mice according to an embodiment of the present invention; and

Figure 4 illustrates the effect of ASO-MOEs molecules on reducing the fibrotic area in mice after BLM-induced fibrosis according to one embodiment of the present invention.

Invention Description

The following detailed description of the invention is intended to illustrate how the invention can be implemented, but does not imply that the invention can only be implemented in the manner described. The detailed description aims to explain the function of the embodiments and the steps and sequence of operation of those embodiments, but different implementation methods can also be used to achieve the same or equivalent functions as the foregoing embodiments.

1. Definition

For convenience, the terminology used in this invention is compiled herein. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meaning as understood and commonly used by those skilled in the art to which this invention pertains.

The term "nucleic acid" in this document refers to a molecule composed of two or more nucleotides covalently linked together, including DNA, RNA, and various variants or analogs of said DNA or RNA. The terms "nucleic acid" and "polynucleotide" are used interchangeably in this document. "Oligonucleotide" refers to a molecule composed of fewer than 25 nucleotides (e.g., 20 nucleotides).

In this document, antisense oligonucleotides (ASOs) refer to single-stranded DNA or single-stranded RNA that are complementary to pre-mRNA or mRNA sequences and can reduce RNA content, thereby reducing protein content. According to a preferred embodiment of the present invention, the ASOs are complementary to the nucleotide sequences of TXNDC5 mRNA (NCBI reference sequence: NM_030810.4) 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, thereby downregulating the expression level of TXNDC5 mRNA.

As stated above, in this article, "nucleic acid sequence" refers to the order of the nucleotides that make up a nucleic acid. In this article, all nucleic acid sequences have 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 is the 3' end.

"Locked nucleosides (LNAs)" refers to nucleic acid molecules in which a portion of the nucleotides are locked nucleosides (i.e., bicyclic nucleotides or analogues). LNA nucleotides have a bridging bond between the 2'-oxygen and 4'-carbon on the pentose sugar, causing the pentose sugar to remain in the 3'-endo configuration, which is commonly found in A-type double helix structures. Descriptions of these LNA monomers can be found in the publications of WO 2001/25248, WO 2003/006475, or WO 2003/095467, which are incorporated herein by reference.

"Complementary" refers to polynucleotides (i.e., mononucleotide sequences) that are linked together by the base-pairing rule. For example, the "A-G-T" sequence and the "T-C-A" sequence are complementary. In heterozygosity, when two mononucleotides exist in an antiparallel configuration, they are called "complementary".

The "sequence similarity percentage" (%) mentioned here refers to the percentage of nucleotide residues in a candidate sequence that are completely identical to those in a reference nucleic acid sequence. During the above alignment, the candidate nucleotide fragment and the specific polynucleotide fragment can be placed side-by-side, with gaps introduced if necessary, to achieve the highest possible sequence similarity. When calculating similarity, conserved substitutions of nucleotide residues are considered distinct residues. Various methods exist in the related field for performing this side-by-side alignment, such as publicly available software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can select appropriate parameters and calculation methods to obtain the optimal arrangement when performing the alignment. In this specification, the sequence comparison between two nucleic acid sequences is performed using the nucleotide-nucleotide BLAST analysis database Blastn provided by the National Center for Biotechnology Information (NCBI). The nucleotide sequence similarity (also referred to in this specification as the percentage (%) of nucleotide sequence similarity between nucleic acid sequence A and nucleic acid sequence B) of candidate nucleic acid sequence A compared to reference nucleic acid sequence B is calculated as follows:

Where X is the number of identical nucleotides obtained by arranging sequences A and B using the BLAST sequence alignment procedure, and Y is the total number of nucleotides in the shorter of sequences A and B.

As used herein, the term "treatment" refers to preventative, curative, or palliative treatment. The term "treatment" as used herein means administering the ASO of this invention to an individual who has a medical condition, symptoms of that medical condition, a disease or abnormality associated with that medical condition, or a precursor to that medical condition, so as to partially or completely reduce, slow, eliminate, delay its development, inhibit its progression, reduce its severity, and/or reduce the probability of the occurrence of one or more features or symptoms of a particular disease, abnormality, and/or condition. Treatment may also be administered to individuals who do not exhibit a particular disease, abnormality, and/or condition at all, or who only exhibit early signs of a particular disease, abnormality, and/or condition, so as to reduce the risk of developing the disease associated with that particular disease, abnormality, and/or condition in individuals exhibiting pathological conditions associated with that particular disease, abnormality, and/or condition. A reduction in one or more of the aforementioned symptoms or clinical indicators indicates that the treatment is "effective"; or, if the progression of the disease is delayed or stopped, the treatment is also considered "effective." In other words, treatment includes not only improving disease symptoms or their indicators, but also stopping or delaying the progression or worsening of symptoms compared to the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reducing one or more symptoms, eliminating disease severity, stabilizing the disease state, delaying disease progression, alleviating or slowing the disease state, and relieving disease.

As used herein, "effective amount" refers to the amount of an ingredient that achieves the desired response. "Pharmaceutically effective amount" refers to the amount of a pharmaceutical agent (e.g., the ASO of this invention) used to achieve the desired therapeutic effect. Specific pharmaceutically effective amounts can vary depending on the condition to be treated, the patient's physiological characteristics (e.g., weight, age, or sex), the species of mammal to be treated, the duration of treatment, concurrent therapies, and the formulation used. Pharmaceutically effective amount also refers to the amount of any compound or composition whose pharmaceutically beneficial effects far outweigh its toxic or adverse effects.

In this document, "individual" or "patient" means a human or non-human animal with dysregulated TXNDC5 expression (specifically, an upregulated TXNDC5 expression relative to a healthy individual) and to whom the methods of this invention are applicable. Unless otherwise specified, "individual" or "patient" herein encompasses both male and female animals. Examples of non-human animals include all vertebrates, such as mammals (e.g., primates, dogs, rodents (e.g., mice or rats), cats, sheep, horses, or pigs); and non-mammalian animals, such as birds, amphibians, etc.

Unless otherwise specified, scientific or technical terms used herein have the same meaning as understood by one of ordinary skill in the art. Unless otherwise specified, the singular form of a noun encompasses its plural form. As stated herein and in the claims, the singular form "a, an" includes its plural form.

While the numerical ranges and parameters used to define the broader scope of this invention are approximate values, the relevant values in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual testing methods. Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "approximately" means that the actual value falls within the acceptable standard error of the average, as determined by those skilled in the art to which this invention pertains. Except for experimental examples, or unless explicitly stated otherwise, it is understood that all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, and the like) are modified with "approximately". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying claims are approximate values and are subject to change as needed. At a minimum, these numerical parameters should be understood as the indicated significant digits and values obtained by applying general rounding. Here, a range of values is expressed as a distance from one endpoint to another or between two endpoints; unless otherwise stated, all ranges of values herein include the endpoints.

2. The present invention relates to antisense oligonucleotides (ASOs).

This disclosure relates to the use of ASO molecules to treat diseases associated with TXNDC5 upregulation. Therefore, broadly speaking, this invention is a single-stranded deoxyribonucleic acid (SDNA) complementary to at least a portion of a target gene (e.g., TXNDC5 mRNA), which, when transfected into host cells, can inhibit the expression of the target gene's mRNA.

This single-stranded deoxyribonucleic acid, or the ASO molecule of the present invention, is approximately 16-21 nucleotides in length and contains a deoxyribonucleic acid sequence having at least 80% sequence similarity to the sequence numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. According to the preferred embodiment (page 8 of 29, specification), the ASO molecule of the present invention contains a deoxyribonucleic acid sequence having at least 90% sequence similarity to the sequence numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In a particular embodiment, the ASO molecule of the present invention contains a deoxyribonucleic acid sequence having 100% sequence similarity to the sequence numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. The ASO molecule of the present invention has 16-21 nucleotides, for example, about 16, 17, 18, 19, 20, or 21 nucleotides. In some instances, the ASO molecule of the present invention has 20 nucleotides; in other instances, the ASO molecule of the present invention has 16 nucleotides.

3. Modified ASO

Alternatively, the ASO of the present invention can also be modified by introducing substituents into the bases or sugar rings of its backbone, or by changing the internucleotide linkages, sugar groups, or bases. In some embodiments, the nucleic acid of the ASO molecule of the present invention is composed of DNA and one or more LNA molecules. In other embodiments, the nucleic acid of the ASO molecule of the present invention is composed of DNA and one or more 2'-position modified sugars (e.g., 2'-O-methoxyethyl substitution). Modified ASOs are usually derived from their natural form of ASO and have preferred properties, for example, they are more easily absorbed by cells, have a higher affinity for target nucleic acids, are more stable in the presence of nucleases, or have higher inhibitory activity.

(i) Modified internucleotide linkage

Natural nucleotide linkages in RNA or DNA refer to phosphodiester bonds from the 3' to 5' ends. Oligonucleotides with such modified nucleotide linkages include those that retain phosphorus atoms and those that do not. Representative examples of phosphorus-containing nucleotide linkages include, but are not limited to, phosphodiester, phosphotriester, methyl phosphonate, phosphoramide, and thiophosphate. Methods for producing sulfur-containing or sulfur-free linkages are well known in the field.

In some embodiments, the ASO of the present invention contains one or more modified internucleotide links, such as one or more phosphate thioester links.

(ii) Modified sugars

The pentose sugar of the ASO nucleotide of the present invention may also be selectively modified. In some embodiments, the ASO of the present invention comprises a chemically modified ribofuransoe ring moiety. Examples of this chemically modified ribofuransoe ring include, but are not limited to, the addition of substituents (e.g., adding substituents at the 5'- or 2'- positions), bridging atoms on the ring that are not covalently bound to the same position to form locked nucleic acids (LNAs), and replacing oxygen atoms on the carbon ring with S, N(R), or C( _Ra )( _Rb ) ₂ , wherein R, _Ra , or _Rb are _C1-12 alkyl groups, protecting groups, or combinations thereof.

Examples of chemically modified sugars include, but are not limited to, nucleotides substituted with 2'-fluorine or 5'-methyl, or sugars in which the oxygen atom on the pentose ring is replaced with a sulfur atom and a substituent is added at the 2'-position.

According to some embodiments, chemically modified sugars also include substitution at the 5'-position of the locked nucleic acid (LNA) molecule. Examples of LNAs include, but are not limited to, nucleotides in which bridging bonds are formed between ring atoms at the 4'- and 2'- positions of the ribose ring. In a specific embodiment, the ASO of the present invention comprises one or more LNA molecules, wherein the bridging bonds comprise any of the following general 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' or 4' _- CH( _CH2OCH3 )-O-2';4'- _CH2 -N( _OCH3 )-2', 4'- _CH2 -ON( _CH3 )-2', 4'- _CH2 -NR-O-2', 4'- _CH2 -C( _CH3 )-2' or 4'-CH2- _C (= _CH2 )-2'; wherein R is H, C _1-12 alkyl groups, or protecting groups. Each of the above LNA molecules may include various stereochemical configurations of sugars, such as α-L-ribofranose or β-L-ribofranose.

In certain embodiments, nucleotides are chemically modified by replacing ribose with a sugar surrogate. Such modifications include, but are not limited to, replacing ribose with sugar surrogate systems (sometimes also called DNA analogs), such as morpholine rings, cyclohexenylalkyl rings, cyclohexane rings, or tetrahydropyran rings as shown below:

Many other bicyclic or tricyclic sugar substitutes, well known to those skilled in the art, can also be used to modify nucleotides and incorporate them into the ASO molecule of this invention.

Therefore, the ASO of the present invention can be composed entirely of DNA molecules or can be composed of DNA molecules and at least one modified nucleotide. In some embodiments, the ASO of the present invention is composed of DNA molecules and at least one LNA molecule (e.g., a 2'-O-, 4'-C methylene bicyclic nucleotide monomer). One advantage of adding LNA to nucleic acids is that it can increase the stability of the nucleic acids. Therefore, the ASO of the present invention includes incorporating LNA molecules into standard DNA oligonucleotides, thereby improving the stability of the resulting nucleotides, for example, increasing the resistance of the ASO to enzymes (endonucleotidases or exonucleotidases), and thus increasing its half-life in biological samples. Generally, based on the total number of nucleotides in the single-stranded nucleic acid, the single-stranded ASO of the present invention may contain at least about 5%, 10%, 15%, 20%, 25%, or 30% LNA molecules; preferably about 40% LNA monomers; more preferably about 60% LNA monomers. In one embodiment of the invention, the ASO of the present invention is composed entirely of DNA and contains a deoxyribonucleic acid sequence having at least 90% sequence similarity to any of the sequences numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, for example, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity. In another embodiment of the invention, the ASO of the present invention is composed of DNA and LNA, wherein the single-stranded ASO is composed of DNA and at least 30% LNA. In one example, the single-stranded ASO of the present invention contains at least one LNA. In another example, the single-stranded ASO of the present invention contains six LNAs.

Alternatively, the single-stranded ASO of the present invention may also consist of DNA and at least one modifying sugar modified at the 2'-position, such as a sugar modified with 2'-O-methoxyethyl (2'-O-MOE). Generally, in terms of the total number of nucleotides in the single-stranded nucleic acid, the single-stranded ASO of the present invention may contain at least about 5%, 10%, 15%, 20%, 25%, or 30% of modifying sugars modified at the 2'-position. According to a specific embodiment, in terms of the total number of nucleotides in the single-stranded nucleic acid, the single-stranded ASO of the present invention may contain at least about 25%, 30%, 40%, 50%, or 60% of modifying sugars modified at the 2'-position; preferably, it contains about 40% of modifying sugars modified at the 2'-position; more preferably, it contains about 50% of modifying sugars modified at the 2'-position. According to one embodiment, the single-stranded ASO of the present invention is composed entirely of DNA molecules, comprising a deoxyribonucleic acid sequence having at least 90% sequence similarity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity) to any of the sequences numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another embodiment, the single-stranded ASO of the present invention is composed of DNA and one or more modifying sugars modified at the 2'-position, wherein the single-stranded ASO is composed of DNA and at least 20% of the modifying sugars modified at the 2'-position. In one example, the single-stranded ASO of the present invention contains only one 2'-O-MOE modifying sugar. In another example, the single-stranded ASO of the present invention contains ten 2'-O-MOE modifying sugars.

(iii) Modified bases

The single-stranded ASO of this invention may also contain chemically modified bases to enhance its affinity for target nucleic acids. The purpose of modifying or substituting bases is to differentiate their structure from native or unmodified bases, while maintaining the same or at least equivalent function. Both native and chemically modified bases must be capable of participating in hydrogen bonding. The purpose of base modification is to improve their stability against nucleases, their binding affinity to antisense compounds, or to impart other advantageous biological properties. Chemically modified bases include native or synthetic bases, such as 5-methylcytosine (5-me-C). Substituent bases include substituted 5-methylcytosine, which is particularly useful for enhancing the binding of antisense oligonucleotides to target nucleic acids.

Therefore, the single-stranded ASO of the present invention can be composed of a DNA molecule and at least one chemically modified base, such as 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine or guanine; 2-propyl and other alkyl derivatives of adenine or guanine; 2-thiouracil; 2-thiothymine; 5-halouracil; 5-halocytosine; 5-propynyluracil and other alkynyl derivatives of cytosine and pyrimidine bases; 6-azourazine; 6-azycytosine. ; 6-Azothymidine; 5-Uracil; 4-Thiouracil; 8-amino, 8-thio, 8-thionyl, 8-hydroxy, and other substituents at the 8th position of adenine or guanine; uracil or cytosine substituted at the 5-position, such as 5-halogen (especially 5-bromo), 5-trifluoromethyl, and other substituents; 7-methylguanine; 7-methyladenine; 2-fluoroadenine; 2-aminoadenine; 8-Azoguanine; 8-Azoadenine; 7-Denitroguanine; 7-Denitroadenine; 3-Denitroguanine and 3-Denitroadenine. Generally, based on the total number of bases contained in each nucleic acid, the single-strand ASO of the present invention may contain at least about 5%, 10%, 15%, 20%, 25%, or 30% of modified bases. According to a specific embodiment, based on the total number of nucleotides in the single-stranded nucleic acid, the single-stranded ASO of the present invention may contain at least about 25%, 30%, 40%, 50%, or 60% of modified bases; preferably about 40% of modified bases; more preferably about 50% of modified bases. According to one embodiment, the single-stranded ASO of the present invention is composed entirely of a DNA molecule, containing a deoxyribonucleic acid sequence having at least 90% sequence similarity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity) to any of the sequences numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another embodiment of the present invention, the single-stranded ASO of the present invention is composed of DNA and 5-methylcytosine (e.g., at least about 20% 5-methylcytosine). In some embodiments, the single-stranded ASO of the present invention contains only one 5-methylcytosine. In other embodiments, the single-stranded ASO of the present invention contains ten 5-methylcytosines.

4. Method for manufacturing the ASO of this invention

Currently, there are various methods to produce the ASO of this invention, with or without chemical modification, for use with silencing genes, including chemical synthesis or by cleaving long double-stranded DNA with group III DNases. These methods all involve first preparing nucleic acid molecules in vitro, and then introducing them into cells by lipid transfection, electroporation, or other methods.

The ASO of this invention is first prepared in vitro and/or chemically modified using known methods. For example, the single-stranded nucleic acid of this invention can be prepared using polymerization techniques well-known in the field of nucleic acid chemistry. Generally, it can be prepared using the standard oligomerization cycle of phosphorous amide, but it can also be prepared using chemical methods such as H-phosphonates or triphosphates. The ASO of this invention can be delivered directly or in vivo via a delivery carrier (e.g., liposomes). The ASO of this invention can also be formulated into pharmaceutically acceptable formulations with other suitable carriers and/or diluents. Methods for delivering nucleic acids are well-known in the relevant art, including, but not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other carriers (e.g., biodegradable polymers, hydrogels, or cyclodextrins).

Table 1 lists all the ASO molecules of this invention. Modified ASOs (i.e., ASO-LNAs or ASO-MOEs) are obtained by modifying the corresponding ASO molecules in Table 1 with at least one LNA or 2'-O-MOE modifying sugar. Table 2 lists all the modified ASO molecules of this invention.

Table 1. Deoxyribonucleic acid sequence of the ASO of this invention

Table 2. Deoxyribonucleic acid sequences of ASO-LNA or ASO-MOE of the present invention

Therefore, a further aspect of the present invention relates to the use of the aforementioned single-stranded ASO to manufacture drugs for the treatment of diseases associated with upregulation of the TXNDC5 gene, 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, leukoplakia, or viral infections. Examples of cancers that can be treated with the ASO of the present invention include, but are not limited to, breast cancer, cervical cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, multiple myeloma, non-small cell lung cancer, pancreatic cancer, prostate cancer, kidney cancer, or uterine cancer. Examples of neurodegenerative diseases that can be treated with the ASO of the present invention include, but are not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, or Prion disease. In a preferred embodiment, the present invention ASO is used to manufacture a medicament for treating fibrotic diseases (including pulmonary fibrosis, renal fibrosis, liver fibrosis, or myocardial fibrosis).

Therefore, the present invention also provides a pharmaceutical composition for treating or preventing diseases caused by upregulation of the TXNDC5 gene. The pharmaceutical composition comprises at least one monosense ASO of the present invention as its active ingredient, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition further comprises another agent suitable for promoting the treatment of the aforementioned diseases, such as an antidiabetic drug for treating diabetes, a chemotherapy agent for treating cancer, a nonsteroidal anti-inflammatory drug (NSAID) for treating arthritis, or an antifibrotic drug (e.g., nintedanib or pirfenidone for treating pulmonary fibrosis).

The nucleic acids of this invention can be suspended in a suitable dispersing matrix, such as water, PBS, physiological saline, oil, or fatty acids. The prepared pharmaceutical composition can be administered via non-gastrointestinal routes, inhalation, topical application, rectal administration, intranasal administration, buccal administration, or vaginal administration. The term "non-gastrointestinal administration" herein encompasses techniques such as subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intracisional, intraspinal, intrahepatic, intralesional, and intracranial injection or perfusion. Preferably, the pharmaceutical composition is administered via intramuscular, intraperitoneal, or intravascular injection; more preferably, the pharmaceutical composition is administered via intramuscular injection. In one example, the pharmaceutical composition is injected intramuscularly into one site on an individual's limb (hand or leg). The appropriate body site for injection is selected based on factors such as the nucleic acid to be administered, individual physical conditions including age, sex, weight, and/or current and previous medical history. Experienced physicians can select suitable injection sites based on these factors without excessive experimentation. The sterile form of the pharmaceutical compositions of this invention can be a solution or an oily suspension. Such suspension formulations can be formulated using techniques well known in the art, with appropriate dispersants or wetting agents and suspending agents. Sterile injectable formulations can be sterile injectable solutions or suspensions dissolved in non-toxic, acceptable non-gastrointestinal diluents or solvents (e.g., 1,3-butanediol). Acceptable carriers or solvents that can be used in this invention include water, Ringer's solution, phosphate buffer, and isotonic sodium chloride solution (i.e., physiological saline). Furthermore, sterile fixed oils have conventionally been used as solvents or suspension media. Therefore, any blended fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and their glyceride derivatives can be used to prepare injectables, as can pharmaceutically acceptable natural oils (e.g., olive oil, castor oil, and especially polyoxyethylated oils). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxypsy cellulose or similar dispersants, which are commonly used to formulate pharmaceutical dosage forms such as emulsions and suspensions. For formulation purposes, other commonly used surfactants, such as Tween, Spans, and other emulsifiers or bioavailability enhancers, may also be used to manufacture pharmaceutical solids, liquids, or other dosage forms. The dosage form and dosage are determined based on factors such as the route of administration, the nature of the formulation itself, the individual's disease condition, weight, body surface area, age or sex, whether other medications are being used, and the physician's judgment. An appropriate dose is approximately 0.15 mg to 1.5 mg of nucleic acid per kilogram of body weight, such as approximately 0.15, 0.20, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mg of nucleic acid per kilogram of body weight; preferably, approximately 0.3 mg to 1.2 mg of nucleic acid per kilogram of body weight, such as approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or 1.2 mg of nucleic acid per kilogram of body weight; even more preferably, approximately 0.5 mg to 1.0 mg of nucleic acid per kilogram of body weight, such as approximately 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg of nucleic acid per kilogram of body weight. The dosage is expected to vary slightly depending on the route of administration. Those skilled in the art will be able to easily assess various relevant factors based on the information provided herein and determine the appropriate dosage for the intended use.

This invention also relates to a method for treating an individual expressing upregulated TXNDC5 gene, the method comprising administering the individual the single-stranded deoxyribonucleic acid of this invention or the composition of this invention, and further comprising administering the individual additional drugs, such as chemotherapy drugs for treating cancer. The term "individual" herein refers to a human or a non-human animal. In a preferred embodiment, the individual is a human. In one example, the individual has been diagnosed with pulmonary fibrosis. In another example, the individual has been diagnosed with cancer. In yet another example, the individual has been diagnosed with rheumatoid arthritis. The individual has received other medical treatment prior to receiving treatment with the method and/or composition of this invention. For example, in the case of cancer, if the individual has previously had cancer, the medical treatment received could be surgery, chemotherapy, or radiation therapy for cancer. Therefore, to enhance the anti-cancer effect of gene therapy, the individual previously diagnosed with cancer may receive other anti-cancer medical treatments before, during, or after receiving treatment with the method and/or composition of this invention. In one instance, the individual was diagnosed with pulmonary fibrosis and had previously received pirfenidone or nintedanib prior to treatment with the methods and/or compositions of the present invention.

The following embodiments are provided to illustrate the present invention and to facilitate implementation by those skilled in the art. The scope of the present invention is not limited to the provided embodiments.

Example

Materials and Methods

Cell culture

Adult lung fibroblasts (HFP-a) (ScienCell, CA, USA) were seeded in a fibroblast matrix supplemented with 2% fetal bovine serum (FBS), 1% fibroblast growth supplement (FGS), and 1% penicillin/streptomycin solution, and cultured at 37°C in a humidified environment containing 95% _O2 /5% _CO2 .

Producing the ASOs of this invention

The ASOs of this invention are produced using human TXNDC5 gene mRNA as the target sequence. Specifically, the ASOs of this invention are produced using sequences at positions 337 to 356, 670 to 689, 675 to 694, 862 to 881, 879 to 898, 1003 to 1022, 1007 to 1026, 1278 to 1297, 2864 to 2883, 2865 to 2884, 2868 to 2873, or 2873 to 2892 of the human TXNDC5 gene mRNA as target sequences.

All produced ASOs were either sourced from Eurogenetc or synthesized using an AKTA OligoPilot 10 Plus synthesizer, followed by separation using reverse-phase HPLC or TEX HPLC. UPLC was used to confirm the purity of the isolated ASOs, and MOLDI-TOF or LC-HRMS was used to analyze their molecular weight.

Furthermore, modified ASOs are manufactured using the produced ASOs, including ASOs containing locked nucleic acids (hereinafter referred to as "ASO-LNA") or ASOs containing 2'-O-methoxyethylated sugars (hereinafter referred to as "ASO-MOE"). Each modified ASO is synthesized in 1 nmol amounts on the MOSS Expedite instrument platform, according to the manufacturer's manual.

HFP-a cells transfected with the ASOs of this invention

HFP-a cells were cultured in 6-well plates at a density of 1 x ^10⁵ cells/well and transfected with the ASOs of this invention using TransIT-X2 (Mirus Bio, USA). Specifically, plastids containing the ASOs of this invention were mixed with TransIT-X2 in plasma-free Opti-MEM (Thermo Fisher Scientific, USA), and then added to the culture medium containing HFP-a cells at a final ASO concentration of 0.4-60 nM and a TransIT-X2 volume of 3.3 μL, and cultured for 24 hours. The success of transfection with the ASOs of human TXNDC5 gene mRNA was confirmed by detecting the mRNA expression level of the TXNDC5 gene at the gene level using quantitative real-time PCR (qRT-PCT) or by detecting human TXNDC5 protein at the protein level using immunoassay.

Quantitative real-time PCR (qRT-PCR)

Following the user manual, RNA was isolated from cells transfected with the ASO of this invention using the Direct-Zol ^™ RNA MiniPrep kit (ZYMO Research, USA) in accordance with the above method. 100 ng of DNase-treated total RNA was used as a template for the synthesis of the first DNA strand. The reaction was carried out in 20 μL of 4-fold TaqMan ^™ Fast single-step mixing stock solution (Thermo Fisher Scientific, USA) containing the TaqMan ^™ analytical probe set (hTXNDC5:Hs01046710_m1(FAM); hGAPDH:Hs03929097_g1(VIC)). The reaction was performed in an Applied Biosystem 7500 Fast Instrument using the following program: 50°C for 5 minutes; 95°C for 20 minutes; 40 cycles of 95°C for 15 seconds; followed by 60°C for 1 minute. The expression levels of each individual transcript were normalized relative to the control gene GAPDH and expressed as a fold increase relative to the average expression level of the control sample.

Immunoblotting analysis

Twenty-four hours after transfection, HFP-a cells were replaced with serum-free growth medium and treated with TGFβ1 (10 ng/mL) (PeproTech, USA) for 48 hours. HFP-a cells were homogenized using 2x sample buffer (BioRad Laboratories, USA) and then boiled at 95°C for 10 minutes. Proteins were separated on 10% SDS-PAGE gelatin and transferred to a PVDF membrane, then blocked with a masking buffer (Visual Protein, BP01-1L). PVDF membranes were incubated overnight at 4°C with primary antibodies against anti-COL1A1 (1:500, OriGene, USA, TA309060, for human use), anti-fibronectin (1:2000, BD Biosciences, USA, 610077), anti-TXNDC5 (1:15000, Proteintech, USA, 19834-1-AP), anti-αSMA (1:1000, Abcam, UK, ab5694), or β-myoprotein (1:1000, Milpore, Germany, MAB1501). Ink dots were developed using HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5000, Cell Signaling Technology, USA, 7076, 7074) and either SuperSignal West Pico or Femto Chemiluminescent substrate (Thermo Fisher Scientific, USA, 34080, 34094). Protein bands were detected using a ChemiDoc MP system (BioRad Laboratories, USA), and the intensity of the protein bands was quantitatively analyzed using ImageLab software (version 5.2.1).

Bleomycin-induced pulmonary fibrosis animal model

Purchased C57BL/6 male mice (8-9 weeks old) were quarantined for one week before being used in this experiment. Mice were housed in cages of five each, in temperature- and humidity-controlled, well-ventilated environments. The room was set with 12 hours of light/12 hours of darkness (daylight hours from 7:00 AM to 7:00 PM), and the temperature was maintained at 22 ± 2°C, with humidity at 55 ± 10%. Animals had free access to food and water. This animal experiment was conducted in accordance with guidelines for the care and use of laboratory animals.

Bleomycin (3 U/kg body weight) was injected intratracheally into the trachea of animals to induce pulmonary fibrosis. The control group received the same volume of sterile saline. Seven days later, the mice with induced pulmonary fibrosis were randomly divided into five groups of six animals each. On the same day, each group was administered a carrier solution, nintedanib, or the ASOs of this invention (serial numbers: 73 (DCB1111128235), 14 (DCB1111128279), or 82 (DCB1111128281), respectively). Nintedanib was dissolved in PBS containing 10% DMF to a final concentration of 6 mg/mL and administered daily via feeding tube (PO) at a dose of 60 mg/kg body weight (mpk) for 14 consecutive days. Each of these inventions has page 22 of 29 (Invention Specification).

Modified ASOs (i.e., DCB1111128235, DCB1111128279, or DCB111112881) were prepared in TruboFect transfection reagent (Thermo Scientific, Mass, USA) and administered intratracheally twice weekly (BIW) at a dose of 0.2 mg/kg body weight (mpk) for two consecutive weeks. The vector group mice received the same volume of TruboFect transfection reagent intratracheally and served as the control group. Twenty-one days after disease induction, the experiment was terminated, and lung function was measured. Lung tissue was collected and stored until further analysis.

Lung function test

Lung function was assessed using the FlexVent system (Scireg, Montreal, QC, Canada). Mice were bronchized and ventilated at a rate of 150 breaths per minute, a tidal volume of approximately 10 ml/kg body weight, and a positive-end expiratory pressure of 2–3 cm of water. Deep inflation perturbation was used to assess respiratory capacity. Pressure-volume cycles were generated using constant pressure increase and normal pressure decrease. SnapShot-150 was used to measure other factors used to assess lung function, including air resistance, compliance, and elastance. Compliance reflects the lung's ability to stretch and expand; air resistance reflects the transpulmonary pressure change required to generate one unit of airflow through the trachea (the pressure between the mouth and alveoli divided by the airflow); and elastance reflects the pressure required to inflate the lung.

Pathological assessment

The left lung of mice was fixed in formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin or picrosirius red (Abcam, Cambridge, UK). The size of the fibrotic area was assessed by picrosirius red staining and Image J imaging software analysis.

Example 1: Using the ASO of the present invention to inhibit the transcription of TXNDC5 mRNA

HFP-a cells were treated with the specified ASO, and then the amount of expressed TXNDC5 mRNA was determined by qRT-PCR as described in "Materials and Methods". The results are shown in Table 3, where the inhibitory activity was graded according to the degree to which the ASO inhibited TXNDC5 mRNA expression by more than 50% at a concentration of 30 nM: +++ indicates that the amount of TXNDC5 mRNA remaining is less than 50%, and ++ indicates that the amount of TXNDC5 mRNA remaining is between 50% and 70%.

Table 3. Efficacy of the ASOs of this invention in inhibiting human TXNDC5 mRNA in vitro.

Based on the data in Table 3, among the ASOs prepared according to the steps described in the "Materials and Methods" section of this invention, 26 ASOs effectively inhibited TXNDC5 expression by more than 50%, and 41 ASOs moderately inhibited TXNDC5 mRNA expression. Therefore, after treatment, the amount of TXNDC5 mRNA remaining was between 70% and 50%. The remaining ASOs (i.e., ASOs other than the 69 ASOs listed in Table 3) only slightly inhibited TXNDC5 mRNA expression, thus the amount of TXNDC5 mRNA remaining was greater than 70% (data not shown).

Example 2: The ASOs of this invention inhibit TXNDC5 mRNA and TGF-β-induced pulmonary fibrosis.

In this embodiment, 2'-O-methoxyethyl (2'-O-MOE) modified sugars derived from the ASOs listed in Table 1 (i.e., ASO-MOEs) or locked nucleic acid molecules (i.e., ASO-LNAs) were used to inhibit the transcription of TXNDC5 mRNA and their effect on inhibiting TGF-β-induced pulmonary fibrosis. The results are summarized in Table 4 and Figure 1.

As shown in Table 4, all ASO-MOEs successfully inhibited TXNDC5 mRNA expression at concentrations with _IC50 values below 60 nM. Among them, DCB111112238 (sequence number: 75), DCB111112240 (sequence number: 76), and DCB111112277 (sequence number: 79) exhibited the strongest inhibitory activity, with _IC50 values less than 10 nM. As for ASO-LNAs, generally, their inhibitory activity on TXNDC5 mRNA was better than that of ASO-MOEs, and _{their IC50} values were lower (for sequence number 3 (or DCB1111128003) or sequence number 4 (or DCB1111128004), ASO-LNA activity was +++, while ASO-MOE activity was ++).

Table 4. Effects of the ASO-MOEs or ASO-LNAs of the present invention on the inhibition of human TXNDC5 mRNA in vitro.

_IC50 levels for inhibiting TXNDC5 mRNA activity: +++, _IC50 below 10 nM; ++, _IC50 between 10-20 nM.

Interval; +, IC ₅₀ is between 20-60 nM

It is known that TGF-β can induce the expression of pulmonary fibrosis-related proteins. Therefore, immunoblotting analysis was used to confirm the expression of TXNDC5 and pulmonary fibrosis-related proteins (including fibronectin, type I collagen, and α-actin) with or without the ASO-MOE of this invention. The results are shown in Figure 1.

As shown in Figures 1A-1I, TGF-β (10 ng/mL) increases the expression of various pulmonary fibrosis-related proteins, including fibronectin, type I collagen, and α-actin. However, the increased expression of these proteins can be inhibited by the ASO-MOEs of this invention, including DCB1111128235 (Figure 1A), DCB1111128255 (Figure 1B), DCB1111128252 (Figure 1C), DCB1111128266 (Figure 1D), DCB1111128265 (Figure 1E), DCB1111128238 (Figure 1F), DCB1111128279 (Figure 1G), DCB1111128280 (Figure 1H), and DCB1111128281 (Figure 1I), and this effect is dose-dependent.

Example 3: Using the ASO-MOEs of the present invention to reduce the progression of pulmonary fibrosis

To confirm the in vivo function of the ASOs of this invention, in this embodiment, pulmonary fibrosis was induced in test animals by intratracheal injection of bleomycin (BLM, 3U/kg body weight), the procedure of which is described in "Materials and Methods". The experimental animals were randomly divided into 5 groups (6 animals in each group). The control group was given sterile PBS (for 2 weeks). The experimental animal groups were given the ASO-MOE of this invention (sequence number: 73 (DCB1111128235) or sequence number: 14 (DCB1111128279)) or a mixed ASO (sequence number: 82 (DCB1111128281)) or a mixed ASO (sequence number: 82 (DCB1111128281)) at days 0, 7, 10, 14 and 17 of the experiment (all administered intratracheally, 0.2 mg/kg). The nintedanib group was given nintedanib (60 mg/kg, for 14 consecutive days). In addition, the healthy animals (i.e., those without pulmonary fibrosis) received no treatment. Lung function was assessed using the FlexiVent system, including measuring lung tissue compliance (a factor reflecting the lung tissue's ability to expand and extend), air resistance (a factor reflecting the change in transpulmonary pressure required to generate one unit of airflow through the lung air channels), and elastance (a factor reflecting the pressure required to inflate the lungs) at specified dates. On day 21 of the experiment, the animals were sacrificed, and their lung tissue was collected and the size of the fibrotic areas was measured. The results are shown in Figures 2-4.

Referring to Figure 2, which is a bar graph showing the changes in lung tissue compliance, air resistance, and elasticity in test animals after treatment with or without the ASO-MOE of the present invention or nintedanib, the data in Figure 2 clearly show that when treated with the ASO of the present invention, the lung tissue compliance of the test animals was higher than that of the carrier control group (Figure 2A), and the air resistance and elasticity were also lower than those of the carrier control group (Figures 2B and 2C). Furthermore, the ASO-MOE (sequence number: 73 or 14) was also more effective in improving pressure-volume circulation than nintedanib (60 mg/Kg) treatment (Figure 3). These results indicate that the ASO-MOE of the present invention (sequence number: 73 or 14) can significantly improve lung function in pulmonary fibrosis mice compared to mice treated with a mixture of ASO (sequence number: 82 or DCB1111128281) or a carrier.

Regarding the effect of the ASO-MOE of the present invention on reducing the area of BLM-induced pulmonary fibrosis, it was found that the ASO-MOE of the present invention (serial number: 73 or DCB1111128235) can reduce the area of BLM-induced pulmonary fibrosis, and its effect is superior to that of nintedanib (Figure 4).

In summary, the results of this invention support the possibility of treating diseases and/or conditions caused by TXNDC5 dysregulation (e.g., aging, arthritis, cancer, diabetes, neurodegenerative diseases, pulmonary fibrosis, atherosclerosis, vitiligo, or viral infections) by introducing antisense oligonucleotides, particularly antisense oligonucleotides that interfere with TXNDC5 mRNA expression, into an individual (e.g., a human).

While the above embodiments disclose specific examples of the present invention, they are not intended to limit the invention. Those skilled in the art can make informed decisions without departing from the original intent of the invention. (Page 28 of 29, Invention Specification)

In the context of the principles and spirit of the invention, various modifications and alterations may be made to it; therefore, the scope of protection of this invention shall be determined by the claims of the appended patent applications.

[Symbol Explanation]

[Biomaterial Storage]

Claims (19)

1. A single-stranded anti-sense oligonucleotide (ASO) that inhibits the transcription of protein 5 (TXNDC5) messenger RNA (mRNA) containing a thioredoxin domain, wherein the single-stranded ASO is approximately 16-21 nucleotides in length and contains a deoxyribonucleic acid sequence having 80% sequence similarity to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. 2. The single-stranded ASO of claim 1, wherein the single-stranded ASO comprises at least one locked nucleic acid (LNA) molecule, a modified 2'-sugar, a modified internucleotide link, a modified nucleobase, or a combination thereof. 3. The single-stranded ASO as claimed in claim 2, wherein the single-stranded ASO comprises at least one 2'-fluorinated sugar, 2'-O-methylated sugar, or 2'-O-methoxyethylated sugar. 4. The single-stranded ASO as claimed in claim 2, wherein the single-stranded ASO comprises six locked nucleic acid molecules. 5. The single-stranded ASO as claimed in claim 3, wherein the single-stranded ASO comprises ten 2'-O-methoxyethylated sugars. 6. The single-stranded ASO of claim 2, wherein the single-stranded ASO comprises at least one inter-linkage of phosphate thioester nucleotides. 7. The single-stranded ASO as claimed in claim 2, wherein the single-stranded ASO comprises at least one 5-methylcytosine. 8. A method of treating a disease caused by upregulation of thioredoxin domain-containing protein 5 (TXNDC5) in an individual, comprising administering to the individual an effective amount of the single-stranded ASO of claim 1 to inhibit transcription of TXNDC5 messenger RNA (mRNA). 9. The method of claim 8, wherein the single-stranded ASO comprises at least one locked nucleic acid (LNA) molecule, a modified 2'-sugar, a modified internucleotide link, a modified base, or a combination thereof. 10. The method of claim 9, wherein the single-stranded ASO comprises at least one 2'-fluorinated sugar, 2'-O-methylated sugar, or 2'-O-methoxyethylated sugar. 11. The method of claim 9, wherein the single-stranded ASO comprises six locked nucleic acid molecules. 12. The method of claim 10, wherein the single-stranded AS O comprises ten 2'-O-methoxyethylated sugars. 13. The method of claim 10, wherein the single-stranded ASO comprises at least one phosphate thioester nucleotide linker. 14. The method of claim 10, wherein the single-stranded ASO comprises at least one 5-methylcytosine. 15. The method of claim 8, wherein the disease is aging, arthritis, cancer, diabetes, neurodegenerative disease, fibrosis, atherosclerosis, vitiligo, or viral infection. 16. The method of claim 15, wherein the cancer is breast cancer, cervical cancer, rectal cancer, colorectal cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, multiple myeloma, non-small cell lung cancer, pancreatic cancer, prostate cancer, kidney cancer, or uterine cancer. 17. The method of claim 15, wherein the neurodegenerative disease is amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, or Prion disease. 18. The method of claim 15, wherein the fibrosis is pulmonary fibrosis, liver fibrosis, kidney fibrosis, or myocardial fibrosis. 19. The method of claim 8, wherein the individual is a human being.