Composition for preventing or treating muscle atrophy or muscle damage comprising CU-rich RNA

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle atrophy or muscle damage or a composition for promoting muscle differentiation, each composition comprising, as an active ingredient, CU-rich RNA derived from a ChRO1 gene, which is a long non-coding RNA, or an isoform thereof. The composition has the effects of maintaining the chromocenter structure of myocytes, reducing the expression of proteolytic factors, and increasing the expression of muscle activity-related factors, and thus can be more safely and effectively used for the prevention or treatment of muscle atrophy or muscle damage.

Description

Composition for the prevention or treatment of muscular dystrophy or muscle injury containing CU-RICH RNA

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of muscular atrophy or muscle damage or a composition for promoting muscle differentiation, comprising as an active ingredient CU-rich RNA derived from the ChRO1 gene, which is a long non-coding RNA, or a homomorphism thereof.

[0002]

[0003] Muscular dystrophy, characterized by a decline in skeletal muscle mass and function, is a myopathy caused by various factors. Muscles atrophy and muscle strength decline due to diseases of the muscles themselves, immobility resulting from limb fixation and bed rest, and malnutrition, as well as severe diseases such as aging, degenerative neurological disorders, acquired immunodeficiency syndrome (AIDS), chronic heart failure, sepsis, and cancer. Patients with muscular dystrophy experience a significant decline in quality of life due to limited movement caused by muscle loss, potentially reaching a point where they cannot even utilize the minimum muscles necessary for long-term survival. Furthermore, when muscular dystrophy accompanies severe diseases, the prognosis is poor, as it affects treatment methods and effectiveness and increases mortality rates. Therefore, while active treatment for muscular dystrophy alongside the disease is necessary, there are currently no effective preventive or therapeutic methods other than exercise and nutritional therapy. In particular, since exercise and nutritional therapy are difficult to apply to the elderly with muscular dystrophy and patients with severe diseases, the development of preventive and therapeutic substances is even more critical.

[0004] RNA therapeutics are treatments that use RNA (ribonucleic acid) molecules to treat or prevent diseases. Unlike DNA-based therapies, they are considered safer as there is no risk of insertion into the host cell genome. Furthermore, RNA therapeutics possess high selective specificity as they can act on target molecules that cannot be administered as small molecules. Unlike DNA-based therapies, which must penetrate the cytoplasm and nuclear membrane, RNA therapeutics have the advantage of being able to regulate the activity of intracellular proteins simply by entering the cytoplasm. In addition, they can be developed more rapidly compared to protein therapeutics, such as small molecules or monoclonal antibodies, resulting in relatively lower development costs.

[0005] Accordingly, the inventors completed the present invention by confirming that CU-rich RNA derived from the ChRO1 gene, which is a long noncoding RNA, or its isoform is effective in preventing or treating muscular dystrophy.

[0006]

[0007] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscular atrophy or muscle damage comprising CU-rich RNA as an active ingredient.

[0008] Another objective of the present invention is to provide a composition for promoting muscle differentiation comprising CU-rich RNA as an active ingredient.

[0009] Another objective of the present invention is to provide a method for preventing or treating muscular atrophy or muscle damage, comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0010] Another objective of the present invention is to provide a method for promoting muscle differentiation comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0011]

[0012] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of muscular atrophy or muscle damage comprising CU-rich RNA as an active ingredient.

[0013] In addition, the present invention provides a composition for promoting muscle differentiation comprising CU-rich RNA as an active ingredient.

[0014] In addition, the present invention provides a method for preventing or treating muscular atrophy or muscle damage, comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0015] In addition, the present invention provides a method for promoting muscle differentiation comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0016]

[0017] The composition of the present invention has the effect of maintaining the staining central structure of muscle cells, reducing the expression of proteolytic factors, and increasing the expression of muscle activity-related factors, so it can be usefully used for the prevention or treatment of muscular dystrophy or muscle damage safely and effectively compared to DNA therapeutics.

[0018]

[0019] Figure 1a shows the results of sequencing analysis of muscle-specifically expressed ChRO1 RNA (left: mouse ChRO1, mChRO1; and right: human ChRO1, hChRO1), 1b shows the results of ChIRP-seq data analysis of DNA sequences to which ChRO1 is bound in C2C12 myotube cells (GSE94498 for ChRO1; and GSE113248 for MyoDeRNA as a control), 1c shows the results of analyzing the sequences of mChRO1a and hChRO1a expressed in parts of exon1 and intron1 of ChRO1, 1d shows the results of structural prediction of mChRO1a and hChRO1a, and 1e shows the results of predicting the protein binding site of mChRO1a.

[0020] Figure 2 shows the results of cloning several fragments of ChRO1a, transcribing them into IVT, and then performing in vitro phase separation experiments using the HP1a protein.

[0021] Figure 3a shows the results of observing the heterochromatin foci pattern with and without 1,6-HD treatment in C2C12 myoblasts capable of overexpressing CU-rich 413nt RNA (CUR 413nt), and Figure 3b shows the results of confirming the intracellular locations of chromatin proteins through immunostaining in C2C12 myoblasts capable of overexpressing CU-rich 413nt RNA (CUR 413nt).

[0022] Figure 4a is an experimental schedule in which C2C12 myoblasts capable of overexpressing CU-rich 413nt RNA (CUR 413nt) were differentiated into myotubes and then treated with Dox and Dex for 2 days for analysis; 4b is the result of analyzing changes in heterochromatin aggregation patterns and the number of foci through DAPI staining; 4c is the result of confirming the expression levels of the proteolytic factors ATROGIN1 and MuRF1; 4d is the result of analyzing the thickness of myotubes; 4e is the result of performing RNA-seq analysis on muscle samples from adult males (RNA-seq data: PRJNA450495); and 4f is the result of analyzing the correlation between the expression of ChRO1 and genes.

[0023] Figure 5a is the result of analyzing the sequence of human ChRO1a, 5b is the result of predicting the structure of human ChRO1a, 5c is a schematic diagram of an RNA molecule consisting of a CU-rich sequence and a sticker sequence capable of complementary binding to the ends, 5d is a CU-rich RNA molecule derived from the human ChRO1a sequence modified according to the RNA molecule schematic diagram, and 5e is the result of predicting the structure of the constructed CU-rich RNA molecule.

[0024]

[0025] The present invention will be described in detail below.

[0026] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0027] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0028]

[0029] The present invention provides a pharmaceutical composition for the prevention or treatment of muscular atrophy or muscle damage comprising CU-rich RNA as an active ingredient.

[0030] In the present invention, the CU-rich RNA may be derived from the ChRO1 gene, which is a long noncoding RNA, or an isoform thereof. The ChRO1 gene is described in Korean Registered Patent No. 10-2066459, and an isoform of the ChRO1 gene may include the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 2. SEQ ID NO. 1 is the human ChRO1a sequence (1-3016), and SEQ ID NO. 2 is the mouse ChRO1a sequence (1-1338).

[0031] In the present invention, the CU-rich RNA may comprise a CU dinucleotide repeat sequence and complementary sticker sequences at both ends of the repeat sequence.

[0032] In the present invention, the CU dinucleotide repeat sequence may be a sequence of length 100 to 900 nucleotides (nt), and preferably may be a sequence of length 200 to 850 nucleotides (nt), length 200 to 800 nucleotides (nt), length 250 to 800 nucleotides (nt), length 250 to 750 nucleotides (nt), length 250 to 700 nucleotides (nt), or length 250 to 600 nucleotides (nt). More preferably, the CU dinucleotide repeat sequence may include the base sequence of SEQ ID NO. 5 or SEQ ID NO. 10, but is not limited thereto.

[0033] In the present invention, the complementary sticker sequence may be a sequence of length 9 to 12 nucleotides (nt), and preferably, the complementary sticker sequence may include the base sequences of SEQ ID NO. 6 and SEQ ID NO. 7 or the base sequences of SEQ ID NO. 11 and SEQ ID NO. 12, but is not limited thereto, and may be constructed from sequences capable of complementary binding.

[0034] In the present invention, the CU dinucleotide repeat sequence and the complementary sticker sequence may be directly connected or connected by a linker sequence. The linker sequence may include the sequence of SEQ ID NO. 13 or SEQ ID NO. 14, but is not limited thereto, and any sequence that does not interfere with the formation of a loop structure of the CU dinucleotide repeat sequence without interfering with the complementary sticker sequence is possible.

[0035] In the present invention, the CU-rich RNA may form a loop structure.

[0036] In the present invention, the CU-rich RNA may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 8, and SEQ ID NO. 9. Specific sequences are as shown in Tables 1 to 4 below.

[0037]

[0038] Human ChRO1a Sequence (1-654): Sequence Number

[0039] CU-rich RNA: Sequence number 4AGAGGGGCUGU(sequence number 6)UGUCUCCCCUCCGUCUCUCUCUCCCU CUCUCCCUCUCUCUCUCUCUCUCUCUCUCUCCUCCUCUCUCUCUCUCUCUCUCUCUCUCUCCGUCUCUCUCUCCGUCUCCCUCUCUCUCUCUCUCUCCGUCUCUCUCUCUCCCUCUCUCCGUCUCUCUCUCUCCGU CUCUCUCUCUCCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCCGUCUCUCUCUCUCUCCGUCUCUCUCUCUCUCCCUCU (서열번호 5)ACAGCCCCUCU(서열번호 7)

[0040] Mouse ChRO1a Sequence (1-654): SEQ ID NO: 8ACAGACACCAAGGCCCAGGUGGAGAGGGUCCUGCUGCCGCUGGGGCCCAAGGGCUCGGUGUCUCCACCUGUCUGCUUGCUUCCUUCCUCUCUCUCUCUCUUCCUUACCCCCAUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUGUGUAUCU

[0041] CU-rich RNA: SEQ ID NO: 9AGACACAAAGGC(SEQ ID NO: 11)UCCAUACCCCCAUCCUCUCCUUCCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUGUGUGUGUGUGUCUUUGUAUGUCUCUCUCUCUCUCUCUCUCUCUCUGUGUAUCUCUCUCUGUAUGUGACUCUCUCUCUCUCUCUCUGUGUAUCUAUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU (SEQ ID NO: 10)GCCUUUGUGU (SEQ ID NO: 12)

[0042]

[0043] In the present invention, a recombinant vector containing the CU-rich RNA can be prepared and produced by transcribing RNA through IVT (In vitro Transcription), and the method of production is not particularly limited and can be produced by a technique known to a person skilled in the art.

[0044] In the present invention, the muscle atrophy may be a disease caused by a decline in muscle function, muscle wasting, or muscle degeneration.

[0045] The above-mentioned muscle wasting or muscle degeneration occurs due to genetic factors, acquired factors, aging, etc., and muscle wasting is characterized by the progressive loss of muscle mass and the weakening and degeneration of muscles, particularly skeletal or voluntary muscles and cardiac muscles.

[0046] In the present invention, the muscle injury may be selected from the group consisting of muscle strain, muscle rupture, muscle tearing, contusion, distortion, rotator cuff syndrome, and myositis, but is not limited thereto.

[0047] The above physical destruction occurs due to causes such as trauma, excessive temperature, myotoxins, local ischemia, inflammation, and exercise, and is characterized by damage to skeletal or voluntary muscles and cardiac muscles.

[0048] The aforementioned "muscle regeneration" refers to the rapid recovery of muscles when they have been physically or chemically damaged, and comprehensively designates the process and period until the damaged muscles can perform normal functions.

[0049] More specifically, the term "muscle" comprehensively refers to tendons, muscles, and ligaments, and "muscle function" refers to the ability to exert force through muscle contraction. It includes muscle strength, which is the ability of a muscle to exert maximum contractile force to overcome resistance; muscle endurance, which is the ability to repeat contraction and relaxation for a given weight or how many times; and explosiveness, which is the ability to exert strong force within a short period of time. The aforementioned muscle function is governed by the liver and is proportional to muscle mass.

[0050] The term "prevention" in this invention refers to any act of suppressing or delaying a disease by administering a composition according to this invention. "Treatment" refers to any act of improving or beneficially altering the symptoms of a disease by administering a composition according to this invention.

[0051] The pharmaceutical composition according to the present invention may contain the active ingredient of the present invention alone or be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may additionally contain excipients or diluents. In the above, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness.

[0052] Pharmaceutically acceptable carriers may additionally include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Additionally, they may include various drug delivery materials used for oral administration of peptide preparations. Furthermore, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose and glycol, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may additionally include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be referenced in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0053] The composition of the present invention may be administered to mammals, including humans, by any method. For example, it may be administered orally or parenterally. Parenteral administration methods may include, but are not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, rectal, mucosal delivery, or administration in the form of ophthalmic drops, and preferably may be mucosal delivery, transdermal, local, or ophthalmic delivery.

[0054] The pharmaceutical composition of the present invention can be formulated into an oral or parenteral administration formulation according to the administration route described above, and preferably into a parenteral administration formulation. For example, in the case of a parenteral administration formulation, it can be formulated in the form of an injection, a cream, a lotion, a topical ointment, an oil, a moisturizer, a gel, an aerosol, a nasal inhaler, and an eye drop by methods known in the art. These formulations are described in the literature (Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995), which is a prescription generally known in all pharmaceutical chemistry.

[0055] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or administered via a fractionated treatment protocol involving multiple doses administered over a long period. The content of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg per kg of patient body weight per day, most preferably 0.1 μg to 500 mg. However, since the effective dose for a patient is determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, a person of ordinary knowledge in the art would be able to determine an appropriate effective dose of the composition of the present invention in light of these factors. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0056] In addition, the present invention provides a composition for promoting muscle differentiation comprising CU-rich RNA as an active ingredient.

[0057] In addition, the present invention provides a method for promoting muscle differentiation comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0058] In addition, the present invention provides a method for preventing or treating muscular atrophy or muscle damage, comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

[0059] It is preferable to apply the above therapeutic effective dose differently depending on various factors and similar factors well known in the pharmaceutical field, including the specific composition (such as the type and degree of the response to be achieved and whether other agents are used in some cases), the individual's age, body weight, general health status, gender, diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used together or concurrently with the specific composition. Therefore, it is preferable to determine the effective dose of the composition suitable for the purpose of the present invention by taking into consideration the aforementioned matters.

[0060] The above-mentioned individual is applicable to any mammal, and said mammal includes not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

[0061]

[0062] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0063]

[0064] Example 1. Sequence analysis of ChRO1, a long non-coding RNA

[0065] Through prior research described in Korean Patent No. 10-2066459, the inventors confirmed that ChRO1, a long noncoding RNA, is expressed specifically in muscles and is a sequence that regulates muscle differentiation and development. Subsequently, to identify a key substance that can be utilized for the prevention or treatment of muscular dystrophy, the sequence of ChRO1 was analyzed using the Reploc program.

[0066] As a result, it was confirmed that both mouse and human sequences had high repeat sequence scores in ChRO1 intron 1, and through analysis of repeating k-mers, it was confirmed that CU (or UC) dinucleotides were significantly present in intron 1 (Fig. 1a). In addition, through analysis of ChRO1 ChIRP-seq data, it was confirmed that ChRO1 specifically binds to satellite DNA and DNA containing simple repeats that are similar to the repeat sequences of RNA (Fig. 1b). These sequences are also present in the chromosomal center where ChRO1 functions, and since it is known that similar repeat sequences bind to each other, it was predicted that the repeating CU sequence would be key to the function of ChRO1.

[0067] Next, analysis of the repetitive sequence of ChRO1a, an isoform containing parts of ChRO1 exon 1 and intron 1, confirmed the particularly repetitive presence of CU dinucleotides (Fig. 1c). Furthermore, structural prediction of the ChRO1a sequence using the RNAfold program revealed the possibility that the region containing the CU repeat sequence forms a loop structure without participating in base pairing (Fig. 1d). Additionally, since RNA loop structures can act as binding platforms for proteins, protein binding prediction analysis was performed. As a result, it was confirmed that the CU-rich region of the ChRO1a sequence has a very high probability of binding to a protein (Fig. 1e).

[0068]

[0069] Example 2. Identification of the core sequence of ChRO1a RNA

[0070] We conducted an in-depth analysis of the ChRO1a RNA sequence to identify the core sequence responsible for alleviating myopathy. First, to locate the core sequence of mouse ChRO1a, the protein was divided into several fragments and cloned. The RNA was transcribed via IVT (In vitro Transcription), and in vitro phase separation experiments were performed using the HP1a protein, which is known to be important for heterochromatin phase separation. During this process, changes in the size of droplets containing the HP1a protein were observed after each RNA fragment was added.

[0071] As a result, full-length ChRO1a and 1-824nt CU-rich RNA improved droplet size with high efficiency, confirming that the CU-rich portion can have the greatest influence on the phase separation of HP1a (Fig. 2). However, when comparing RNAs of similar length to exclude the influence of RNA length, it was observed that 1-413nt CU-rich RNA further improved the HP1a phase separation droplet size compared to 825-1338nt RNA in which no CU repeat sequence was observed (Fig. 2).

[0072] From the above results, it was confirmed that ChRO1a-derived CU-rich RNA having a myopathy alleviation function can be produced as an RNA molecule containing a CU-rich region of 100 to 900 nucleotides (nt) in length, preferably 200 to 850 nucleotides (nt) in length.

[0073]

[0074] Example 3. Function of staining median formation in muscle cells by CU-rich core sequence

[0075] Based on the results of the above example, an experiment was performed to determine whether 1-413nt CU-rich RNA (hereinafter, CU-rich 413nt RNA) can contribute to the regulation of intracellular chromatin structure and the formation of stained foci. Briefly, mouse C2C12 myoblasts capable of overexpressing CU-rich 413nt RNA by Dox (Doxycycline) were prepared, and the myoblasts were treated with Dox. The heterochromatin foci pattern was observed through DAPI staining with or without the presence of 1,6-HD (hexanediol) treatment.

[0076] As a result, when CU-rich 413nt RNA was overexpressed by treatment with Dox, it was confirmed that heterochromatins existing as small foci aggregated to form staining central crypts (Fig. 3a). On the other hand, when treated with 1,6-HD, a phase separation inhibitor, it was observed that the staining central crypts formed by CU-rich 413nt RNA dispersed back into small foci. Thus, it was confirmed that CU-rich RNA has molecular activity that promotes protein phase separation.

[0077] Next, immunostaining was performed with respective antibodies to confirm the intracellular localization of chromatin proteins in C2C12 myoblasts induced with CU-rich 413nt RNA expression. Confocal microscopy revealed that HP1g and DAXX, which are specifically localized with heterochromatin in myotubes, were located in the stained center due to the overexpression of CU-rich 413nt RNA (Fig. 3b).

[0078] From the above results, it was confirmed that the CU-rich RNA of the present invention can enable the formation of staining media in muscle cells.

[0079]

[0080] Example 4. Efficacy of preventing or treating muscular dystrophy by a CU-rich core sequence

[0081] Experiments were conducted at the cell and organism levels to determine whether CU-rich 413nt RNA could contribute to the reconstruction of chromatin structure as well as the prevention or treatment of muscular dystrophy.

[0082] First, C2C12 myoblasts cultured in DMEM medium containing 10% FBS were differentiated into myotubes by culturing them in DMEM medium supplemented with 2% HS for 4 days. Muscle atrophy was induced in the differentiated C2C12 myotubes by treating them with the steroid drug dexamethasone, and simultaneously, overexpression of CU-rich 413nt RNA was induced by treating them with Dox (Doxycycline) for 2 days. Subsequently, heterochromatin aggregation patterns and changes in the number of foci were observed using DAPI staining. As a result, it was confirmed that heterochromatin foci were scattered in cells treated with dexamethasone without overexpression of CU-rich 413nt RNA, whereas the structure of the stained central filaments was maintained in cells where overexpression of CU-rich 413nt RNA was induced along with treatment with dexamethasone (Fig. 4b).

[0083] In addition, RNA was extracted with Tizol under the above experimental conditions to synthesize cDNA, and the gene expression levels of proteolytic factors ATROGIN1 and MuRF1, which are used as indicators of muscular dystrophy, were confirmed using qRT-PCR. As a result, the expression of proteolytic factors ATROGIN1 and MuRF1 was significantly increased in cells treated with dexamethasone, whereas the expression of proteolytic factors was observed to decrease in cells in which overexpression of CU-rich 413nt RNA was induced along with treatment with dexamethasone (Fig. 4c).

[0084] In addition, an experiment was performed to measure the diameter of myotube cells using the Image J program after immunohistochemistry with the muscle fiber protein Myh. As a result, it was confirmed that myotube cells overexpressing CU-rich 413nt RNA did not lose their thickness and maintained the thickness of healthy myotube cells even after treatment with dexamethasone (Fig. 4d).

[0085] Next, to confirm the preventive or therapeutic efficacy of CU-rich RNA on muscular dystrophy in individuals, RNA-seq data were analyzed using muscle samples from 18 males aged 60–79 years who were bedridden for 5 days to induce muscular dystrophy. As a result, it was confirmed that the response to muscular dystrophy in response to bed rest varied significantly among individuals, and this was correlated with changes in ChRO1 expression (Fig. 4e). In particular, genes involved in protein degradation showed a negative correlation with ChRO1 expression, while genes involved in muscle activity, such as transcriptional activity and calcium or sodium transport, showed a correlation with ChRO1 expression (Fig. 4f).

[0086] From the above results, it was confirmed that the CU-rich RNA of the present invention can be usefully used for the prevention or treatment of muscular dystrophy.

[0087]

[0088] Example 5. Construction of CU-rich RNA sequence derived from ChRO1a

[0089] Human and mouse ChRO1a RNA sequences were analyzed in depth to identify the core sequence of CU-rich RNA that could be used for the prevention or treatment of ChRO1a-derived muscular dystrophy. Human ChRO1a RNA forms three large loops, and it was confirmed that sequences capable of complementary binding exist at both ends of the CU-rich loops that do not participate in base-pairing (Figs. 5a and 5b). Through this analysis, it was confirmed that the structure predicted to be key for the prevention or treatment of muscular dystrophy is a CU-rich sequence containing 9-12 nucleotide complementary sticker sequences capable of complementary binding at both ends (Fig. 5c).

[0090] Based on the above analysis, an RNA sequence was constructed by modifying the terminal sequence to enable perfect complementary binding based on the loop 1 sequence of human ChRO1 and removing unnecessary sequences to borrow a minimal CU-rich sequence (Fig. 5d). Subsequently, through structural prediction, it was confirmed that the constructed CU-rich RNA sequence could form a CU-rich loop by binding with an 11 bp complementary sticker sequence (Fig. 5e).

[0091] The CU-rich RNA produced by the present invention can be usefully used for the prevention or treatment of muscular atrophy or muscle damage due to its superior efficacy and safety compared to DNA therapeutics.

[0092]

[0093] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0094] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of muscular atrophy or muscle damage comprising CU-rich RNA as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition in which the above CU-rich RNA is derived from the ChRO1 gene, which is a long noncoding RNA, or an isoform thereof.

3. In Paragraph 1, A pharmaceutical composition wherein the above CU-rich RNA comprises a CU dinucleotide repeat sequence and complementary sticker sequences at both ends of the repeat sequence.

4. In Paragraph 3, A pharmaceutical composition in which the above CU dinucleotide repeat sequence and complementary sticker sequence are directly connected or connected by a linker sequence.

5. In Paragraph 3, A pharmaceutical composition in which the above CU dinucleotide repeat sequence is a sequence of length 100 to 900 nucleotides (nt).

6. In Paragraph 3, A pharmaceutical composition wherein the above CU dinucleotide repeat sequence comprises the nucleotide sequence of SEQ ID NO. 5 or SEQ ID NO.

10.

7. In Paragraph 3, A pharmaceutical composition wherein the above complementary sticker sequence is a sequence of length 9 to 12 nucleotides (nt).

8. In Paragraph 3, A pharmaceutical composition wherein the above complementary sticker sequence comprises the nucleotide sequences of SEQ ID NO. 6 and SEQ ID NO. 7, or the nucleotide sequences of SEQ ID NO. 11 and SEQ ID NO.

12.

9. In Paragraph 1, A pharmaceutical composition in which the above CU-rich RNA forms a loop structure.

10. In Paragraph 1, A pharmaceutical composition wherein the above CU-rich RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 8, and SEQ ID NO.

9.

11. In Paragraph 1, A pharmaceutical composition in which the above-mentioned muscular atrophy is caused by decreased muscle function, muscle wasting, or muscle degeneration.

12. In Paragraph 1, A pharmaceutical composition wherein the above-mentioned muscle injury is selected from the group consisting of muscle strain, muscle rupture, muscle tearing, contusion, distortion, rotator cuff syndrome, and myositis.

13. A composition for promoting muscle differentiation containing CU-rich RNA as an active ingredient.

14. A method for the prevention or treatment of muscular dystrophy or muscle injury comprising the step of administering a pharmaceutically effective amount of CU-rich RNA to an individual.

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