Use of MRF4 expression inhibitor for promoting muscle development

Abstract

The present invention relates to the effect of promoting muscle development and growth through inhibition of the expression of MRF4. According to the present invention, MRF4 isolated and identified from skeletal muscles of Nile tilapia showed a strong association with MyoG and MEF2 proteins, and the expression of MyoG and MEF2 was found to significantly increase when the MRF4 gene was disrupted through CRISPR / Cas9 gene editing, and thus the present invention has the effect of being able to be used for the promotion of muscle development or the treatment of muscle diseases through the inhibition of the MRF4 gene.

Description

Uses of MRF4 expression inhibitors for promoting muscle development

[0001] The present invention relates to the effect of promoting muscle development and growth through the inhibition of MRF4 expression.

[0002] Muscles account for approximately 40% of the human body, and securing an adequate amount of muscle mass is essential for maintaining functional capabilities and preventing metabolic diseases. Muscles are broadly classified into smooth muscle, cardiac muscle, and skeletal muscle; skeletal muscle occupies a significant portion of the entire body and facilitates skeletal movement. As the largest organ in the human body, skeletal muscle accounts for 40–50% of total body weight and plays a crucial role in various metabolic functions, including energy homeostasis and heat generation. Human muscle mass decreases by more than 1% annually starting from age 40, and by age 80, it has declined to 50% of its peak mass; muscle loss in old age is recognized as the most significant factor in the decline of overall physical function.

[0003] Muscle diseases follow a course in which the weakening of skeletal muscles gradually leads to impaired walking and mobility functions, making activities of daily living (ADL) difficult and ultimately rendering independent living impossible. Furthermore, they cause cardiopulmonary dysfunction and induce other complications. Among these, cachexia is a syndrome commonly associated with chronic diseases such as cancer, tuberculosis, AIDS, and chronic obstructive pulmonary disease. It refers to a catabolic state of metabolism accompanied by persistent loss of appetite and weight loss, resulting in malnutrition, metabolic imbalance, and a decrease in muscle or fat. Unlike patients with other chronic diseases, cancer patients are characterized by the presence of not only cachexia but also the side effects of various anticancer therapies used to treat cancer (Fearon K. et al., Lancet Oncol 2011;12(5):489-495). Cachexia occurs in 50-80% of patients with gastrointestinal cancer and lung cancer, and the mortality rate due to cachexia reaches 20-30%. Cancer cachexia is characterized by weight loss resulting from muscle atrophy caused by increased catabolic responses stemming from inflammatory and metabolic changes triggered by various cytokines; it occurs when muscle wasting leads to a weight loss of more than 5% within 12 months or less. These changes lower the response rate to chemotherapy or radiation therapy, hinder the progression of effective anticancer treatment, degrade the patient's quality of life, and shorten survival. Muscle loss is one of the most significant characteristics of cachexia and is known to be caused by increased protein catabolism and decreased protein synthesis resulting from the overactivation of various cytokines. Cachexia and sarcopenia share significant overlap as symptoms involving muscle loss. While the majority of patients with cachexia also have sarcopenia, not all patients exhibiting muscle loss display symptoms of cachexia. Clinically speaking, muscle loss (sarcopenia) can be described as a prodromal symptom of cachexia.Inflammatory cytokines acting on cachexia affect insulin and testosterone, which regulate muscle metabolism, thereby causing abnormalities in muscle protein synthesis (Ryu, Seung-Wan, J. Clin. Nutr. 2017;9:2-6). Sarcopenia, one of the characteristics of cachexia, is a condition in which motor neurons that induce skeletal muscle contraction degenerate, preventing contraction, or the expression of proteins involved in muscle contraction within the skeletal muscle decreases or changes, preventing normal contraction; over the long term, these motor neurons or skeletal muscles transform into fibrous tissue. Sarcopenia is caused by various factors such as aging, hormonal imbalances, nutritional deficiencies, lack of physical activity, inflammation, and degenerative diseases; among these, cancer and chemotherapy are known to be major causes. Currently, exercise, protein, and calorie supplementation are known to be helpful for sarcopenia; however, since these are not significantly beneficial for the majority of patients and the elderly, there is an urgent need for a treatment for sarcopenia. However, drugs currently used for sarcopenia that demonstrate a direct effect on improving muscle loss or increasing muscle mass are still at the clinical trial stage, and there are currently no drugs that have received final FDA approval. Therefore, although there are efforts to develop selective androgen receptor modulators, activin receptor antagonists, and fast skeletal muscle troponin inhibitors as treatments for sarcopenia, they are currently at the stage of attempting early clinical trials. Currently, the treatment methods for the aforementioned sarcopenia mainly involve suppressing muscular atrophy caused by the degeneration or progressive mutation of muscle cells, which is a type of sarcopenia.For example, WO 2007 / 088123 discloses a treatment for muscular dystrophy containing a nitroxy derivative as an active ingredient, and WO 2006 / 081997 discloses a treatment for muscular dystrophy containing atralic acid or a derivative thereof as an active ingredient. However, these treatments containing compounds as active ingredients act not only on skeletal muscles affected by muscular dystrophy but also on visceral muscles or cardiac muscles unrelated to muscular dystrophy, which can cause various side effects of varying severity, and thus are not used for practical treatment. Meanwhile, hormone preparations have significantly fewer side effects than compound preparations, and because hormone preparations are biocompatible due to their characteristics, the development of drugs for treating muscular dystrophy or sarcopenia using hormone preparations is accelerating. In addition, muscle atrophy is caused by factors such as damage to muscle tissue due to the absence of mechanical stimulation, such as reduced muscle use; destruction of muscle due to direct injury or physical factors; impaired regenerative capacity of muscle cells due to aging; and impaired muscle use due to damage to nerves that control muscle function (Booth FW., J Appl Physiol Respir Environ Exerc Physiol., 1982).In general, disuse atrophy occurs when muscles in the affected area and surrounding regions are not used for a long period due to a disability or accident, leading to a loss of muscle strength and progressive muscle atrophy. It can also develop in the form of myasthenia gravis caused by a disease of the muscle itself, muscular dystrophy (progressive muscular dystrophy, myotonic muscular dystrophy, Duchenne type, Becker type, limb-girdle type, facioscapulohumeral type), spinal muscular atrophy (sphinobular amyotrophy) caused by inflammation of the muscle itself or damage to the nerves innervating the muscle (Weradnig-Hoffmann type, Kugelberg's disease, Welander's disease), amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), and sphinobular muscular atrophy (Kennedy's disease).

[0004] Meanwhile, the differentiation and formation of muscle cells are regulated by muscle regulatory factors (MRFs), and the MRF gene family consists of MyoD (myoblast determination protein), MRF5 (myogenic regulatory factor 5 or myogenic factor 5, myf5), MRF4 (myogenic regulatory factor 4 or myogenic factor 6, myf6), and MyoG (myogenin) (Hernandez-Hernandez, JM; Garcia-Gonzalez, EG; Brun, CE; Rudnicki, MA The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration. Semin. Cell Dev. Biol. 2017, 72, 10-18. doi: 10.1016 / j.semcdb.2017.11.010.). Among these, MyoD initiates the expression of muscle-specific genes and induces mesenchymal stem cells to differentiate into muscle cell lineages.

[0005] The objective of the present invention is to provide a composition for promoting muscle development.

[0006] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle diseases.

[0007] In addition, the objective of the present invention is to provide a food composition for increasing muscle mass or inhibiting muscle loss.

[0008] In addition, the objective of the present invention is to provide a feed composition for promoting muscle development.

[0009] To solve the above problem, the present invention provides a composition for promoting muscle development comprising an expression inhibitor of the MRF4 (Myogenic regulator factor 4) gene as an active ingredient.

[0010] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0011] In addition, the present invention provides a food composition for muscle growth or muscle loss inhibition comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0012] In addition, the present invention provides a feed composition for promoting muscle development comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0013] According to the present invention, MRF4 isolated and identified from the skeletal muscle of Nile tilapia showed a strong association with MyoG and MEF2 proteins, and it was confirmed that the expression of MyoG and MEF2 significantly increased upon the genetic destruction of MRF4 through CRISPR / Cas9 gene editing; thus, it has the effect of being utilized for promoting muscle development or treating muscle diseases through the genetic suppression of MRF4.

[0014] Figure 1 shows the total nucleotides and amino acid sequences of the Nile tilapia MRF4 (NT-MRF4) gene:

[0015] Red bold text: Start codon (ATG) and stop codon (TGA);

[0016] Underlined: Polyadenylation signal (AATAA);

[0017] Green shading: myogenic basic domain;

[0018] Purple shading: HLH (helix-loop-helix) domain;

[0019] Yellow box indicated: Nuclear localization signal (NLS);

[0020] Blue shading indicated: serine-rich areas; and

[0021] Red box marked: N-glycosylation sites.

[0022] Figure 2 is a multiple sequence alignment analysis of the MRF4 amino acid sequences of seven vertebrate species:

[0023] Blue underline: Preserved HLH domain;

[0024] Pink underline: S-rich area;

[0025] Black underline: Nuclear localization signal; and

[0026] Black solid circle mark: 4 preserved cysteine ​​residues.

[0027] Figure 3 is a figure showing the protein-protein interaction network of the NT-MRF4 protein (A) and the predicted score for the functional partner protein of NT-NRF4 (B).

[0028] Figure 4 is a figure analyzing the relative mRNA expression levels of NT-MRF4 according to various tissues (A), developmental stage (B), and feeding status (C) of Nile tilapia.

[0029] Figure 5 is a figure showing the genomic structure of the NT-MRF4 gene:

[0030] Yellow-orange box: Exon;

[0031] Green bar: Intron;

[0032] Uppercase black text: Exon sequence;

[0033] Lowercase green text: intron;

[0034] Underlined blue text: sgRNA target site; and

[0035] Red text: PAM sequence.

[0036] Figure 6 shows the results of the generation and mutation analysis of Nile tilapia in which the NT-MRF4 gene was disrupted / inactivated using CRISPR / cas9 gene editing technology:

[0037] WT: Wild type;

[0038] -1: A mutation with a 1bp nucleotide deletion;

[0039] -15: A mutation in which a 15bp nucleotide is deleted;

[0040] Red text: Modified amino acid sequence;

[0041] A: Schematic diagram of the CRISPR / cas9 genome editing system;

[0042] B: MRF4 gene-edited (GE) mutants obtained from Nile tilapia; and

[0043] C: Predicted amino acid sequence of MRF4 in GE and WT Nile tilapia.

[0044] Figure 7 is a figure analyzing changes in mRNA expression of MRF and MEF2 (B) genes in GE and WT Nile tilapia.

[0045] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0046] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0047] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0048] In one aspect, the present invention relates to a composition for promoting muscle development comprising an expression inhibitor of the MRF4 (Myogenic regulator factor 4) gene as an active ingredient.

[0049] In one embodiment, the composition may have the effect of inhibiting muscle damage, improving muscle function, promoting the regeneration of damaged muscles, or improving muscle strength.

[0050] In one embodiment, the expression inhibitor may be shRNA (Short hairpin RNA), siRNA (Short interfering RNA), miRNA (Micro RNA), antisense oligonucleotide, aptamer, PNA (peptide nucleic acid), LNA (locked nucleic acid), ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA, and the gRNA may be sgRNA.

[0051] In one embodiment, the sgRNA may target exon 1 of the MRF4 gene and may include a polynucleotide complementary to the nucleotide sequence of SEQ ID NO. 1, which is a target region / sequence present in exon 1 of the MRF4 gene, or a polynucleotide encoding the same.

[0052] In one embodiment, the expression inhibitor may comprise a site-specific nuclease or a CRISPR / cas9-system and at least one gRNA.

[0053] In one embodiment, the expression inhibitor may induce a modification of the intrinsic MRF4 gene through a mutation, and the mutation may be a frameshift mutation or a deletion mutation.

[0054] In one embodiment, the expression inhibitor may induce a nucleotide deletion of 1 bp or more in the intrinsic MRF4 gene, thereby inducing disruption or inactivation of the MRF4 gene.

[0055] In one embodiment, the composition can increase the expression of MEF2b (myocyte enhancer factor 2b), MEF2a, MEF2d, or MyoG (myogenin).

[0056] In one embodiment, the expression inhibitor may hybridize to a specific DNA region of the MRF4 gene.

[0057] The gene expression inhibition composition of the present invention may be introduced into a cell or organism in the form of a recombinant vector comprising shRNA, siRNA, miRNA, antisense oligonucleotide, or aptamer, or in the form of a ribonucleoprotein comprising a mixture of Cas9 protein and sgRNA or a complex formed therefrom.

[0058] The cas9 of the CRISPR / cas9 and gRNA complex of the present invention mediates the cleavage of target DNA when the correct protospacer-adjacent motif (PAM) (GGG at the 3' end of the nucleotide of SEQ ID NO. 1) is also present at the 3' end of the protospacer. For protospacer targeting, immediately following the sequence, there must be a protospacer-adjacent motif (PAM), which is a short sequence recognized by the cas9 nuclease required for DNA cleavage.

[0059] The gRNA of the present invention provides targeting for a CRISPR / cas9-based system. The gRNA is a fusion of two non-coding RNAs: crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by exchanging a sequence encoding a protospacer that confers targeting specificity through complementary base pairing with the desired DNA target. The terms "target region," "target sequence," or "protospacer," used interchangeably in the present invention, refer to a region of a target gene targeted by a CRISPR / cas9-based system. Following the target sequence or protospacer, a PAM sequence is present at the 3' end of the protospacer.

[0060] The term "hybridization" as used in the present invention refers to a reaction in which one or more nucleic acids react to form a complex, and the complex is stabilized through hydrogen bonding between the bases of the nucleic acid residues.

[0061] The term "expression" as used in the present invention refers to the process in which a nucleic acid is transcribed from a DNA template (e.g., mRNA or other RNA transcript) and / or the subsequent process in which the transcribed mRNA is translated into a peptide, polypeptide, or protein.

[0062] As used in the present invention, the terms "frameshift" or "frameshift mutation" refer to a type of gene mutation in which the addition or deletion of one or more nucleotides causes a change in the reading frame of a codon in mRNA. A change in the reading frame can induce a change in the amino acid sequence during protein translation, such as a missense mutation or an early stop codon.

[0063] The term "disrupt" as used in the present invention refers to a mutant gene having a mutation that causes an early stop codon. The disrupted gene product is terminally truncated compared to the undisrupted full-length gene product.

[0064] The terms “stop codon” or “stop codon” as used in the present invention refer to nonsense mutations in DNA sequences that produce a stop codon at a location not typically found in wild-type genes. An early stop codon can cause a protein to be truncated at the end or shortened compared to the full-length version of the protein.

[0065] The term “skeletal muscle” as used in the present invention refers to a type of striated muscle that is under the control of the somatic nervous system and attached to bones by bundles of collagen fibers known as tendons. Skeletal muscle consists of individual components known as myocytes or “muscle cells” and sometimes colloquially referred to as “muscle fibers.” Myocytes are formed from the fusion of developing myoblasts (a type of embryonic progenitor cell that gives rise to muscle cells) in a process known as myogenesis.

[0066] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0067] In one embodiment, the muscle disease may be one or more selected from the group consisting of muscular atrophy, myotonic dystrophy, myopathy, muscle degeneration, myasthenia, muscular injury, dystrophinopathy, atony, myopathy, muscular dystrophy, cachexia, and sarcopenia.

[0068] In one embodiment, the expression inhibitor may be shRNA (Short hairpin RNA), siRNA (Short interfering RNA), miRNA (Micro RNA), antisense oligonucleotide, aptamer, PNA (peptide nucleic acid), LNA (locked nucleic acid), ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA, and the gRNA may be sgRNA.

[0069] In one embodiment, the sgRNA may target exon 1 of the MRF4 gene and may include a polynucleotide complementary to the nucleotide sequence of SEQ ID NO. 1, which is a target region / sequence present in exon 1 of the MRF4 gene, or a polynucleotide encoding the same.

[0070] In one embodiment, the expression inhibitor may comprise a site-specific nuclease or a CRISPR / cas9-system and at least one gRNA.

[0071] In one embodiment, the expression inhibitor may induce a modification of the intrinsic MRF4 gene through a mutation, and the mutation may be a frameshift mutation or a deletion mutation.

[0072] In one embodiment, the expression inhibitor may induce a nucleotide deletion of 1 bp or more in the intrinsic MRF4 gene, thereby inducing disruption or inactivation of the MRF4 gene.

[0073] In one embodiment, the composition can increase the expression of MEF2b (myocyte enhancer factor 2b), MEF2a, MEF2d, or MyoG (myogenin).

[0074] In one embodiment, the expression inhibitor may hybridize to a specific DNA region of the MRF4 gene.

[0075] The gene expression inhibition composition of the present invention may be introduced into a cell or organism in the form of a recombinant vector comprising shRNA, siRNA, miRNA, antisense oligonucleotide, or aptamer, or in the form of a ribonucleic acid protein comprising a mixture of Cas9 protein and sgRNA or a complex thereof.

[0076] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of muscle disease by administering a pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of muscle disease by administering a composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases to which the composition of the present invention is effective, and to judge the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0077] In the present invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating muscle diseases, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0078] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, the type of muscle disease, the cause and severity of the muscle disease, the drug's activity, sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0079] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0080] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, and an oral or injectable formulation is more preferred.

[0081] As used in the present invention, the term "administration" means providing a specific substance to an individual or patient by any appropriate method. Depending on the intended method, it may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally. The dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of transporting the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectables can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), non-aqueous solvents, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0082] The term "individual" as used in the present invention may refer to any animal, including vertebrates, that has developed or may develop the muscle disease, such as fish, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual. The pharmaceutical composition of the present invention may be administered in conjunction with existing therapeutic agents.

[0083] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0084] In one aspect, the present invention relates to a food composition for the prevention or improvement of muscle diseases comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0085] In one aspect, the present invention relates to a food composition for muscle growth or muscle loss inhibition comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0086] In one embodiment, the expression inhibitor may be shRNA, siRNA, miRNA, antisense oligonucleotide, aptamer, PNA, LNA, ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA.

[0087] When the composition of the present invention is used as a food composition, the composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The composition may include food-grade dietary additives in addition to the active ingredient, and the amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0088] The term "food auxiliary additive" as used in the present invention refers to a component that can be added to food as an auxiliary component, and can be appropriately selected and used by a person skilled in the art as it is added to manufacture health functional foods of each formulation. Examples of food auxiliary additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food auxiliary additives of the present invention are not limited by the above examples.

[0089] The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that possess functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the field of the art, and during such manufacturing, raw materials and ingredients commonly added in the field of the art may be added. Furthermore, the formulation of the health functional food may be manufactured without restriction as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations. Unlike general pharmaceuticals, it uses food as a raw material and has the advantage of not causing side effects that may occur with long-term use of pharmaceuticals. Due to its excellent portability, the health functional food of the present invention can be consumed as an adjuvant to enhance the effects of anticancer drugs.

[0090] In addition, there are no restrictions on the types of health foods to which the composition of the present invention can be used. Furthermore, a composition containing the Magnolia bark extract of the present invention as an active ingredient may be prepared by mixing other appropriate auxiliary ingredients and known additives that may be contained in health functional foods, depending on the choice of a person skilled in the art. Examples of foods to which it can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it may be prepared by adding it to juices, teas, jellies, and juices prepared using the extract according to the present invention as a main ingredient.

[0091] In one aspect, the present invention relates to a feed composition for promoting muscle development comprising an expression inhibitor of the MRF4 gene as an active ingredient.

[0092] In one embodiment, the composition may be a feed additive.

[0093] In one embodiment, the composition may be for increasing muscle mass in farmed fish.

[0094] In one embodiment, the composition may be administered to the larval stage of cultured fish.

[0095] In one embodiment, the expression inhibitor may be shRNA, siRNA, miRNA, antisense oligonucleotide, aptamer, PNA, LNA, ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA.

[0096] In one embodiment, the composition can increase the expression of MEF2b, MEF2a, MEF2d, or MyoG.

[0097] In one embodiment, the farmed fish may be tilapia, rockfish, halibut (fish belonging to the family Paralichthyidae of bony fish, including Paralichthys olivaceus and Pseudorhombus pentophthalmus), red sea bream, black sea bream, rockfish, sea bass, mullet, sole, triggerfish, yellowtail, mackerel, gizzard shad, grouper, trout, croaker, yellowtail scad, pufferfish, or jack mackerel.

[0098] The feed composition of the present invention may be prepared by including, in addition to the MRF4 gene expression inhibitor, ingredients commonly used in aquaculture fish feed for purposes such as promoting growth, promoting digestion, improving feed efficiency, enhancing immunity, and improving disease resistance. Examples of such ingredients include plant-based ingredients, animal-based ingredients, and mineral-based ingredients.

[0099] In addition to the above-mentioned plant-based, animal-based, and mineral-based components, the feed composition of the present invention may further include binders, preservatives, vitamins, enzymes, microorganisms, flavorings, etc., for purposes such as enhancing immunity, improving feed preservation, promoting uniform mixing of feed components, and facilitating formulation. These components will be included in the feed composition of the present invention in appropriate amounts (0.0001% to 10% by weight) depending on their intended use.

[0100] The feed composition of the present invention can be formulated in the form of a liquid (particularly in the form of a swine blood culture stock solution or its concentrate), powder, granule, pill, or pellet.

[0101] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0102] Example 1. Analysis of sequence and domain characteristics of MRF4

[0103] 1-1. cDNA and amino acid sequence analysis of Nile tilapia MRF4

[0104] Skeletal muscle tissues of Nile tilapia were collected, RNA was extracted using the ISOSPIN Cell 108 & Tissue RNA kit (Nippon Gene, Tokyo, Japan), and First-strand cDNA and 5' and 3' RACE (rapid amplification of cDNA ends) were synthesized using the Superscript III First-strand cDNA synthesis kit (Invitrogen, USA) and the SMARTer®111 RACE 5' / 3' kit (Takara Bio Inc., Shiga, Japan). A subfragment of the NT-MRF4 (Nile tilapia Myogenic regulator factor 4) gene was cloned via reverse transcription polymerase chain reaction (RT-PCR) using the Phusion® High-Fidelity DNA Polymerase Kit (New England Biolabs Inc., Ipswich, MA, USA). Then, 5'-RACE and 3'-RACE primers were designed from this subfragment, and the 5'-RACE and 3'-RACE fragments were amplified using the SMARTer®RACE 5' / 3' Kit. The RACE PCR products were linked to a linearized pRACE vector, and Stellar Competent cells (Takara Bio Inc., Shiga, Japan) were transformed. Positive clones were selected and sequenced at Macrogen (Seoul, South Korea). The 5'-RACE fragment and the 3'-RACE fragment were combined, and the overlapping region of the partial fragments was trimmed to obtain the full-length NT-MRF4 cDNA sequence (GenBank Accession No. PQ497691). The full-length nucleotide sequence of NT-MRF4 (GenBank Accession No.The structure of PQ497691 was analyzed using ORFfinder, the EMBOSS Transeq online tool, ProtParam, the NCBI Conserved Domain database, SMART (Simple Modular Architecture Research Tool), Motif scan, and the NLStradamus web server. The results confirmed that a poly-A tail is present at 1146 bp, the 5' untranslated region (5'-UTR) is 123 bp, and the 3'-UTR is 345 bp (Fig. 1). The polyadenylation signal (AATAAA) was estimated to be located 262 bp downstream of the stop codon. The open reading frame (ORF) of NT-MRF4 is 678 bp and is estimated to encode a protein of 225 amino acids. The amino acid sequence of NT-MRF4 was analyzed to consist of a myogenic basic domain located at 2-95 aa, an HLH (helix-loop-helix) domain located at 96-147 aa, and a serine-rich region located at 201-224 aa. Additionally, the NT-MRF4 protein contained a nuclear localization signal at the 90-111 aa position (Fig. 1). The molecular weight (MW) of NT-MRF4 was estimated to be 24.91 kDa and the isoelectric point (pI) was estimated to be 5.83.

[0105] 1-2. Comparative Analysis of MRF4 in Chordates

[0106] MRF4 sequences of other chordate species (NCBI database) were aligned using the online multiple sequence alignment program ClustalO, and then visualized and edited using Jalview software v. 2.11.

[0107] As a result, the MRF4 protein in vertebrates was found to contain a conserved HLF domain, nuclear localization signaling and a conserved serine-rich region, and four cysteine ​​residues were found to be conserved in the myogenic basic domain (Fig. 2).

[0108] 1-3. Predictive Analysis of Protein-Protein Interactions

[0109] Other proteins interacting with NT-MRF4 were identified using the Predict protein and Protter server.

[0110] As a result, 10 proteins showed functional association with NT-MRF4, with MEF2b (myocyte enhancer factor 2b) showing the highest interaction score of 0.722, followed by MyoG (myogenin) at 0.664 (Fig. 3).

[0111]

[0112] Example 2. Analysis of NT-MRF4 expression

[0113] 2-1. Analysis of NT-MRF4 Expression Patterns According to Tissue Type

[0114] mRNA expression of NT-MRF4 in various tissues of Nile tilapia was analyzed by qRT-PCR (Quantitative Real-Time PCR). Specifically, skeletal muscle (MUS), brain (BRN), gill (GIL), heart (HRT), stomach (STM), intestine (INT), liver (LIV), kidney (KID), spleen (SPL), and gonads (GND) were collected from Nile tilapia. The collected tissues were washed with 1X PBS, total RNA was extracted, and then synthesized into cDNA. Subsequently, NT-MRF4 mRNA expression in each tissue was analyzed by qRT-PCR using the LightCycler®96 System (Roche, Mannheim, Germany) with a primer set specific to NT-MRF4 and the 2ХqPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems Ltd., London, UK). qRT-PCR data were analyzed using LightCycler®96 System software, and relative gene expression levels were calculated using EF1a (GenBank accession no. AB075952), a housekeeping gene of Nile tilapia, as an internal reference. ΔΔCT It was calculated using the method, and the relative expression levels by tissue were compared by setting the gonads to 1.

[0115] As a result, the expression of NT-MRF was found to be increased by more than 7 times in muscle (MUS) tissue compared to other tissues (Fig. 4A).

[0116] 2-2. Analysis of NT-MRF4 Expression According to Developmental Process

[0117] To confirm changes in mRNA expression of NT-MRF4 according to the developmental stages of Nile tilapia, tissue samples were collected from Nile tilapia at the fertilized egg (FtE), 1 day post-fertilization (1-F), 2 days post-fertilization (2-F), 1 day post-hatching (1-H), 10 days post-hatching (10-H), 30 days post-hatching (30-H), 60 days post-hatching (60-H), and adult stages, and qRT-PCR analysis was performed as in Example 2-1 above.

[0118] As a result, the mRNA expression level of NT-MRF4 during the developmental stages was found to be significantly increased in the larval stages (days after fertilization, dph) compared to the embryonic stages (days after fertilization, dpf) (Fig. 4B).

[0119] 2-3. Analysis of NT-MRF4 Expression According to Starvation Conditions

[0120] To determine the change in mRNA expression of NT-MRF4 according to the starvation status of Nile tilapia, Nile tilapia were divided into a control group (control, CNT), a group with no feeding for 7 days (7-day starvation, 7D-S), a group with no feeding for 14 days (14-day starvation, 14D-S), and a group that was re-fed (refed, ReF). Skeletal muscle samples were collected from the control group on day 1 of the experiment, from 7D-S on day 7 of feeding suspension, from 14D-S on day 14 of feeding suspension, and from ReF on day 1 of re-feeding, and qRT-PCR analysis was performed as in Example 2-1 above.

[0121] As a result, the mRNA expression levels of NT-MRF4 were found to be significantly reduced in the starving 7D-S and 14D-S groups compared to the control and re-fed groups (Fig. 4C).

[0122]

[0123] Example 3. Analysis of changes in expression of muscle development-related genes following gene editing of NT-MRF4

[0124] 3-1. sgRNA Design

[0125] After obtaining the genomic sequence of NT-MRF4 from the NCBI database, single-guide RNAs (sgRNAs) (Table 1, Fig. 5, and Fig. 6A) for two CRISPR / cas9 target sites (Fig. 5) located within the coding region of exon 1 of NT-MRF4 were designed using the CRISPRscan online tool (https: / www.crisprscan.org / ) and synthesized at Bioneer (Daejeon, South Korea) (Fig. 5 and Table 1).

[0126]

[0127] 3-2. Generation and Analysis of NT-MRF4 Mutant Prototypes Using CRISPR / cas9

[0128] Eggs and sperm were collected from Nile tilapia and artificially fertilized. A total of 500 single-cell embryos were collected and microinjected using a microinjection device (WPI, USA) with a complex of sgRNA (final concentration: 250 ng / mL) and Cas9 protein (final concentration: 500 ng / mL) synthesized in Example 3-1, and cultured to 90 dpf (Fig. 6A). After hatching, no significant difference in aberration or mortality was observed between the control group (WT) and the gene-edited microinjection group (GE), so the doses of the sgRNA and Cas9 complex used were considered suitable for the experiment.

[0129] 3-3. Mutation Analysis

[0130] Fin samples were collected from 3-month-old Nile tilapia of the GE group and WT group of Example 3-2, and genomic DNA was extracted using the AccuPrep Genomic DNA Extraction kit (Bioneer, Daejeon, South Korea). Then, a 402 bp region containing the sgRNA target site was amplified using primers specific to it, sequenced, and mutation analysis was performed using the ICE analysis tool on the Synthego web server.

[0131] As a result, sgRNA1 did not induce mutations, but sgRNA2 (sgRNA for sequence number 1) produced two types of deletions (-1 bp and -15 bp) in the F1 generation (Fig. 6B). A 15 bp nucleotide deletion (-15 bp) caused a deletion of 5 amino acids, and a 1 bp nucleotide deletion (-1 bp) induced a frameshift mutation, causing premature termination of transcription (Fig. 6C). This indicates that NT-MRF4 sgRNA2 can effectively edit the MRF4 gene, and the mutation types and frequencies showed that most deletions caused frameshifts, leading to the destruction of the gene structure.

[0132] 3-2. Analysis of Changes in MRF Gene Expression Caused by Mutation

[0133] The expression of MRF genes (MRF4, MyoG, MRF5, and MyoD) in skeletal muscle tissues of 90 dph WT and GE group Nile tilapia was analyzed by qRT-PCR.

[0134] As a result, compared to the WT group, the expression of NT-MRF4 was significantly reduced in the GE group, while the expression of MyoG was upregulated by about twofold, and the expression of MyoD (myoblast determination protein) was also significantly increased (Fig. 7A).

[0135] 3-3. Analysis of Changes in MEF2 Gene Expression

[0136] The expression of MEF2 genes (MEF2a, MEF2b, MEF2c, and MEF2d) in skeletal muscle tissues of 90 dph WT and GE group Nile tilapia was analyzed by qRT-PCR.

[0137] As a result, the mRNA expression levels of MEF2a, MEF2b, and MEF2d were found to be significantly increased in the GE group compared to the WT group (Fig. 7B).

Claims

1. A composition for promoting muscle development comprising an expression inhibitor of the MRF4 (Myogenic regulator factor 4) gene as an active ingredient.

2. A composition for promoting muscle development according to claim 1, wherein the expression inhibitor is shRNA (Short hairpin RNA), siRNA (Short interfering RNA), miRNA (Micro RNA), antisense oligonucleotide, aptamer, PNA (peptide nucleic acid), LNA (locked nucleic acid), ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA.

3. A composition for promoting muscle development according to claim 1, wherein the expression inhibitor induces a modification of the endogenous MRF4 gene through mutation.

4. A composition for promoting muscle development, wherein the mutation in claim 3 is a frameshift mutation or a deletion mutation.

5. A composition for promoting muscle development, wherein, in paragraph 3, the modification of the gene is gene disruption or gene inactivation.

6. A composition for promoting muscle development according to claim 1, which increases the expression of MEF2b (myocyte enhancer factor 2b), MEF2a, MEF2d, or MyoG (myogenin).

7. A pharmaceutical composition for the prevention or treatment of muscle diseases comprising an MRF4 gene expression inhibitor as an active ingredient.

8. A pharmaceutical composition for the prevention or treatment of a muscle disease according to claim 7, wherein the muscle disease is one or more selected from the group consisting of muscular atrophy, myotonic dystrophy, myopathy, muscle degeneration, myasthenia, muscular injury, dystrophinopathy, atony, myopathy, muscular dystrophy, cachexia, and sarcopenia.

9. A pharmaceutical composition for the prevention or treatment of muscle disease according to claim 7, wherein the expression inhibitor is shRNA, siRNA, miRNA, antisense oligonucleotide, aptamer, PNA, LNA, ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA.

10. A pharmaceutical composition for the prevention or treatment of muscle disease, wherein the composition of claim 7 increases the expression of MEF2b, MEF2a, MEF2d, or MyoG.

11. A food composition for muscle growth or muscle loss inhibition comprising an MRF4 gene expression inhibitor as an active ingredient.

12. A feed composition for promoting muscle development containing an expression inhibitor of the MRF4 gene as an active ingredient.

13. A feed composition for promoting muscle development, for increasing muscle mass in farmed fish, according to Clause 12.

14. A feed composition for promoting muscle development, administered to the larval stage of farmed fish according to Clause 12.

15. A feed composition for promoting muscle development according to claim 12, wherein the expression inhibitor is shRNA, siRNA, miRNA, antisense oligonucleotide, aptamer, PNA, LNA, ribozyme, or CRISPR / cas9 and gRNA that specifically binds to the MRF4 gene or its mRNA.

16. A feed composition for promoting muscle development that increases the expression of MEF2b, MEF2a, MEF2d, or MyoG according to claim 12.

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