Composition for promoting myogenesis and manufacturing method therefor

Abstract

The present application relates to a method and composition for inducing myogenesis.

Description

Composition for promoting muscle differentiation and method for producing the same

[0001] The present application relates to a composition for promoting (inducing) muscle differentiation for inducing muscle differentiation (myogenesis) of myoblasts or muscle satellite cells (muscle satellite cells; myosatellite cells).

[0002] The present application relates to an effective method for myogenesis of myoblasts or muscle satellite cells.

[0003] The present application relates to a culture medium for inducing myogenesis of myoblasts or muscle satellite cells.

[0004] The present application relates to various uses of the composition or culture medium for promoting muscle differentiation.

[0005] Satellite cells (muscle stem cells) exist within the myocyte membrane surrounding muscle cells. Satellite cells normally exist in a state of quiescence, but once they emerge from this state, they undergo cell division to create myoblasts.

[0006] Myoblasts develop into myotubes through fusion, etc., and myotube cells gather together to form myofibers.

[0007] In this way, muscle cells have the characteristic of differentiating into muscles through various stages of division and fusion.

[0008] Due to the unique characteristics of the process in which myoblasts differentiate into myotube cells and the process in which myotube cells gather to form muscle fibers, the method for differentiating myoblasts into myotube cells is different from the method for differentiating other somatic cells, and it is also known to be difficult to differentiate.

[0009] For example, when differentiating myoblasts into myotubes, horse serum is sometimes used instead of the fetal bovine serum contained in the culture medium. Specifically, horse serum is used at a concentration of approximately 2-5%.

[0010] Additionally, it has been reported that when myoblasts are differentiated into myotubes using low concentrations of serum, the differentiation efficiency becomes very low.

[0011] Therefore, a new method that can efficiently promote (induce) muscle differentiation is required.

[0012] One object of the present application is to provide an effective muscle differentiation method capable of inducing and promoting myogenesis of myoblasts or muscle satellite cells.

[0013] Another object of the present application is to provide a composition for promoting muscle differentiation for inducing myogenesis of myoblasts or muscle satellite cells.

[0014] Another object of the present application is to provide a culture medium for promoting muscle differentiation.

[0015] Another object of the present application is to provide a composition for inducing myogenesis of myoblasts or muscle satellite cells or a method for preparing a culture medium for promoting myogenesis.

[0016] In order to solve the above-mentioned problem, the present application provides a method for inducing muscle differentiation (myogenesis) using a culture solution or secretion of genetically engineered cells.

[0017] According to this application, the following effects occur.

[0018] According to the present application, an effective method for myogenesis of myoblasts or muscle satellite cells can be provided.

[0019] According to the present application, a composition for promoting muscle differentiation for inducing myogenesis of myoblasts or muscle satellite cells can be provided.

[0020] According to the present application, a culture medium for promoting muscle differentiation and a method for producing the same can be provided.

[0021] The present invention can be usefully used in industrial fields that seek to utilize the efficient differentiation effect into muscle cells, such as the production of cultured meat (synthetic meat), the production of functional cosmetics, and the production of functional products for muscle development.

[0022] Figure 1 is a schematic diagram of the MSTN gene and myostatin protein.

[0023] Figure 2 is a schematic diagram showing myostatin processing through several stages.

[0024] Figure 3 shows the results of confirming the differentiation effect of each cell into myotube cells in wild-type myoblasts (denoted as Wild type in the figure) and myoblasts containing the 12BP deleted MSTN gene (denoted as MSTN KO in the figure).

[0025] Figure 4 shows the results of confirming the paracrine effect of wild-type myoblasts and myoblasts containing the MSTN gene with 12BP deletion. The experiment was conducted using a transwell plate with a transwell insert inserted into a cell culture plate. In Figure 4(a), the upper layer (Top) with respect to the transwell insert refers to the membrane region of the transwell insert, and the lower layer (Bottom) refers to the inside of the cell culture plate. For convenience, when referring to the upper layer of the transwell in the following, it will be interpreted as the upper layer (Top) of Figure 4(a), and when referring to the lower layer of the transwell, it will be interpreted as the lower layer (Bottom) of Figure 4(a).

[0026] Figure 4(a) is a schematic diagram of an experimental design using the above transwell plate.

[0027] Wild-type myoblasts were cultured on the bottom of the transwell in all groups: the control group (indicated as Blank in Fig. 4(a)), the WT co-culture group, and the MSTN KO co-culture group.

[0028] In the upper layer of the transwell of the control group (indicated as Blank in Fig. 4(a)), only the basic medium was added without culturing cells, in the upper layer of the transwell of the WT co-culture group, wild-type myoblasts were cultured, and in the upper layer of the transwell of the MSTN KO co-culture group, myoblasts containing the MSTN gene with a 12BP deletion were cultured. Fig. 4(b) shows the results of observing differentiation into myotube cells by culturing cells based on the experimental design of Fig. 4(a). Blank in Fig. 4(b) corresponds to Blank in Fig. 4(a), +WT in Fig. 4(b) corresponds to the WT co-culture in Fig. 4(a), and +MSTN KO in Fig. 4(b) corresponds to the +MSTN KO co-culture in Fig. 4(a).

[0029] Figure 5 shows the results of confirming the level of differentiation of wild-type myoblasts into myotubes and myofibers in each case, for (i) when wild-type myoblasts were cultured in a mixed medium containing DMEM and PBS containing 2% horse serum and 100 U / ml penicillin / streptomycin (denoted as PBS in the figure) and (ii) when wild-type muscle satellite cells were cultured in a mixed medium containing DMEM containing 2% horse serum and 100 U / ml penicillin / streptomycin and a conditioned medium of fibroblasts containing the 12BP deleted MSTN gene (denoted as MSTN KO in the figure).

[0030] Figure 6 shows the results of examining whether mutations occurred in each cell after treating CHO cells with four different sgRNAs and Cas9 proteins. 1 in Figure 6 represents the group treated with sgRNA No. 1 in Table 2. 2 in Figure 6 represents the group treated with sgRNA No. 2 in Table 2. 3 in Figure 6 represents the group treated with sgRNA No. 3 in Table 2. 4 in Figure 6 represents the group treated with sgRNA No. 4 in Table 2.

[0031] Figure 7 shows the results of genomic DNA PCR performed on 10 types of CHO single cells into which sgRNAs 2 or 3 in Table 2 were introduced.

[0032] Figure 8 shows the results of western blot analysis performed after concentrating the culture medium of the wild-type CHO cell line (WT in the figure) and the culture medium of the #5 single cell line (#5 in the figure), respectively, and treating them with anti-MSTN antibodies.

[0033] Figure 9 shows the sequences included in three vectors. The first vector refers to a Piggybac transposon vector system containing a nucleic acid sequence linked to a CAG promoter (SEQ ID NO: 38) and a 12BP deleted MSTN gene (SEQ ID NO: 17). The second vector refers to a Sleeping beauty vector system containing a nucleic acid sequence linked to a ceMP#1 promoter (SEQ ID NO: 40) and a 12BP deleted MSTN gene (SEQ ID NO: 17). The third vector refers to a Tol2 transposon vector system containing a nucleic acid sequence linked to an EF1a promoter (SEQ ID NO: 39) and a 12BP deleted MSTN gene (SEQ ID NO: 17).

[0034] Figure 10 shows the results of a genomic DNA PCR experiment (B of Figure 10) for the three vectors of Figure 9. Information on the primers used is shown in A of Figure 10.

[0035] Figure 11 shows the results of PCR (B in Figure 11) performed to confirm vector introduction into a CHO cell line into which the Piggybac transposon vector of Figure 9 was inserted. At this time, the location confirmed through PCR is the same as A in Figure 11.

[0036] Figure 12 shows the results of PCR (B in Figure 12) performed to confirm vector introduction into a CHO cell line into which the Piggybac transposon vector of Figure 9 was inserted. At this time, the location confirmed through PCR is the same as A in Figure 12.

[0037] Figure 13 shows a TFF device set up to filter the culture medium.

[0038] Figure 14 shows the results of confirming the level of differentiation into myotube cells after treating bovine, rat, and mouse myoblast cell lines with extracts of a culture medium of a wild-type CHO cell line (WT) or a CHO cell line containing a 12BP deleted MSTN gene produced in Preparation Example 4.

[0039] Figure 15 shows the results of confirming the level of differentiation into myotube cells after treating i) a culture medium of a wild-type CHO cell line (WT) (WT in Media of Figure 15); ii) a culture medium of a CHO cell line containing a 12BP deleted MSTN gene prepared in Preparation Example 4 (-12BP MSTN in Media of Figure 15); iii) a sample obtained by separating a culture medium of a wild-type CHO cell line (WT in TFF of Figure 15); and iv) a sample obtained by separating a culture medium of a CHO cell line containing a 12BP deleted MSTN gene prepared in Preparation Example 4 (-12BP MSTN in TFF of Figure 15) to a C2C12 (mouse myoblast cell line) cell line.

[0040] Figure 16 is the result of analyzing the fusion index to quantify the results of Figure 15.

[0041] Figure 17 shows the results of a western blot performed to confirm the expression level of a muscle differentiation marker in a C2C12 cell line treated with a sample separated by a TFF device from a culture medium of a wild-type CHO cell line (WT) or a sample separated by a TFF device from a culture medium of a CHO cell line containing a 12BP deleted MSTN gene manufactured in Manufacturing Example 4.

[0042] Figures 18 and 19 show the results of confirming the muscle differentiation efficacy after treating C2C12 (mouse myoblast cell line) cell line with i) a sample obtained by separating the culture medium of a wild-type CHO cell line (WT) using a TFF device (WT in Figures 18 and 19); ii) an extract of the culture medium of a CHO cell line into which a PB vector and a Tol2 vector were inserted (CAG+EF1a in Figures 18 and 19); and iii) an extract of the culture medium of a CHO cell line into which a PB vector, a Tol2 vector, and a SB vector were inserted (CAG+EF1a+ceM243 in Figures 18 and 19).

[0043] Hereinafter, the best mode for carrying out the invention is exemplified. This includes some, but not all, implementations of the invention disclosed herein. The embodiments described in this paragraph are merely exemplary, and the implementations described in this paragraph should not be construed as the "best mode for carrying out the invention." Those skilled in the art will likely envision numerous variations and more desirable implementations of the examples described in this paragraph, and such variations should also be considered to be included within the best mode for carrying out the invention.

[0044] In this specification, a method for inducing muscle differentiation is provided.

[0045] In one embodiment, the method may include:

[0046] Genetically engineered cells are cultured in basal medium; and

[0047] The conditioned medium obtained by culturing the genetically engineered cells is applied to target cells to be differentiated into muscle cells.

[0048] In one embodiment, the genetically engineered cell comprises a mutated MSTN gene in its genome, wherein the mutated MSTN gene may be a nucleic acid encoding a MSTN protein variant in which specific four amino acids are deleted from the wild-type MSTN protein.

[0049] In one embodiment, the MSTN protein variant may be any one sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 29.

[0050] In one embodiment, the method may further comprise removing cellular components from a conditioned medium obtained by culturing the cells prior to treating the target cells.

[0051] In one embodiment, after removing the cell component, the method may further include mixing the basic medium to prepare a mixed medium.

[0052] In one embodiment, the sequence of the mutated MSTN gene may be any one sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0053] In one embodiment, the cell containing the mutated MSTN gene in its genome may be a somatic cell, a progenitor cell, or a stem cell.

[0054] In one example, the cell containing the mutated MSTN gene in its genome may be selected from among a muscle satellite cell (myosatellite cell), a myoblast, a fibroblast, and a muscle cell.

[0055] In one example, the cell containing the mutated MSTN gene in its genome may be of bovine origin.

[0056] In one example, the cell containing the mutated MSTN gene in its genome may be a CHO cell.

[0057] In one embodiment, the target cell may be a muscle satellite cell (myosatellite cell) or a myoblast.

[0058] In one embodiment, the target cell may be of mammalian origin.

[0059] In one embodiment, the mammal may be selected from a human, a cow, a pig, a dog, and a cat.

[0060] In one embodiment, it may be selected from Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), Ham's F-12 (Ham's Nutrients Mixture F12), and Opti-MEM.

[0061] In one embodiment, the basic medium further comprises serum or antibiotics, wherein the serum is selected from among fetal bovine serum (FBS), bovine calf serum (BCS), horse serum (HS), human serum, and newborn calf serum (NCS), and the antibiotics are selected from among penicillin, streptomycin, and vancomycin.

[0062] In one embodiment, the culture may be cultured for 3 to 15 days.

[0063] In one embodiment, the conditioned medium may include exosomes, cytokines, hormones, or neurotransmitters secreted from cells.

[0064] In one embodiment, the removal of the cellular component may be performed by a method selected from among centrifugation, filtration, precipitation, and column use.

[0065] In the present specification, a composition for inducing muscle differentiation is provided, which includes a conditioned medium obtained by the method for inducing muscle differentiation.

[0066] In one embodiment, the composition can induce myogenic differentiation of target cells into myotubes and muscle fibers.

[0067] In one embodiment, the composition for inducing muscle differentiation further comprises a basic medium,

[0068] At this time, the above conditional badge and basic badge may be included in a ratio of 10%:90%, 20%:80%, 25%:75%, 30%:70%, 40%:60%, 50%:50%, 60%:40%, 70%:30%, 75%:25%, 80%:20% or 90%:10%.

[0069] In one embodiment, the target cell may be a muscle satellite cell (myosatellite cell) or a myoblast.

[0070] In the present specification, a functional cosmetic composition comprising the composition for inducing muscle differentiation is provided.

[0071] In this specification, a feed composition comprising the composition for inducing muscle differentiation is provided.

[0072] In this specification, a composition for producing muscle tissue from cultured meat containing the composition for inducing muscle differentiation is provided.

[0073] Hereinafter, the present invention will be described in more detail through specific implementations and examples with reference to the attached drawings. It should be noted that the attached drawings include some, but not all, implementations of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementations described herein. These implementations should be considered as provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementations of the invention disclosed herein. Therefore, the invention disclosed herein is not limited to the specific implementations described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0074]

[0075] Definition of terms and technical overview

[0076] muscle differentiation (myogenesis)

[0077] In this application, the term "myogenic differentiation" refers to a series of processes in which muscle satellite cells (myosatellite cells) or / and myoblasts differentiate into myotubes, myofibers, mature myofibers, etc. Furthermore, the term also includes the meaning of "myogenesis" which comprehensively refers to the process in which muscle cells are formed and muscle tissue is formed and developed. Therefore, in this specification, the terms "muscle differentiation" and "myogenicity" are used interchangeably.

[0078]

[0079] The term "promoting (inducing) muscle differentiation" in this application means providing an appropriate environment to muscle satellite cells and / or myoblasts so that differentiation into myotubes or muscle fibers can occur or be promoted efficiently. The term may be used interchangeably with activation of muscle differentiation.

[0080] For example, the above “providing an appropriate environment” may include adding or adding a substance that promotes muscle differentiation of muscle satellite cells or / and myoblasts to the culture medium of muscle satellite cells or / and myoblasts.

[0081] As another example, the above “providing an appropriate environment” may include culturing muscle satellite cells or / and myoblasts together with cells that secrete substances that promote muscle differentiation of muscle satellite cells or / and myoblasts (co-culture).

[0082]

[0083] Structure of wild-type myostatin gene and protein

[0084] The myostatin protein consists of approximately 375 to 376 amino acids and is encoded by the MSTN gene, which consists of three exons and two introns.

[0085] The amino acids that make up the above myostatin protein may vary somewhat depending on the species. For example, bovine myostatin protein is composed of 375 amino acids, porcine myostatin protein is composed of 375 amino acids, mouse myostatin protein is composed of 376 amino acids, and human myostatin protein is composed of 375 amino acids. For example, bovine myostatin protein may be composed of the amino acid sequence of sequence number 34.

[0086] In this specification, as an example, a schematic diagram of a bovine wild-type MSTN gene and a wild-type myostatin protein is disclosed in Fig. 1.

[0087] Myostatin protein is largely composed of three regions: the signal domain (or signal sequence domain), the propeptide domain (prodomain), and the mature domain (mature domain).

[0088] The signal domain is located at the N-terminal (NH2) side of the myostatin protein, the maturation domain is located at the C-terminal (COOH) side, and a propeptide domain exists between the signal domain and the maturation domain that connects them. In other words, the myostatin protein has the structure of [NH2-signal domain-propeptide domain-mature domain-COOH].

[0089] In one example, in the bovine MSTN protein, the signal domain consists of about 24 amino acids, the pro-peptide domain of about 240 amino acids, and the mature domain of about 111 amino acids.

[0090] The MSTN gene sequence encoding the above myostatin protein includes a CDS (coding sequence) sequence. The CDS sequence is the nucleotide sequence of SEQ ID NO: 35.

[0091] Hereinafter, as an example of one implementation of the present application, a more specific description will be given focusing on the wild-type MSTN gene present in the cow genome.

[0092] Exon 1 of the MSTN gene is approximately 740 bp and encodes the signal domain and part of the propeptide domain of the myostatin protein.

[0093] For example, exon 1 of the MSTN gene may consist of the nucleotide sequence of SEQ ID NO: 1.

[0094] Exon 2 of the MSTN gene is approximately 374 bp and encodes part of the propeptide region of the myostatin protein.

[0095] For example, exon 2 of the MSTN gene may be composed of the nucleotide sequence of sequence number 2.

[0096] Exon 3 of the MSTN gene is approximately 1883 bp and encodes part of the propeptide region and the mature region of the myostatin protein.

[0097] For example, exon 3 of the MSTN gene may be composed of the nucleotide sequence of sequence number 3.

[0098] That is, the signal region of the myostatin protein is coded by a part of the sequence of exon 1 of the MSTN gene, the propeptide region is coded by another part of the sequence of exon 1, exon 2, and exon 3, and the mature region is coded by another part of the sequence of exon 3.

[0099] More specifically, exon 1 of the MSTN gene corresponds to about the 1st to about the 124th amino acid of the myostatin protein; exon 2 of the MSTN gene corresponds to about the 125th to about the 248th amino acid of the myostatin protein; and exon 3 of the MSTN gene corresponds to about the 249th to about the 375th amino acid of the myostatin protein.

[0100] The mutated MSTN gene of the present invention differs from the exon 2 sequence of the wild-type MSTN gene. For example, exon 2 of the mutated MSTN gene of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 4.

[0101]

[0102] Myostatin protein activation process

[0103] Myostatin protein is activated in the body through post-translational modifications. These post-translational modifications are described with reference to Figure 2.

[0104] The above post-translational modification process includes a step (step 1) in which two promyostatins form a dimer, a step (step 2) in which the signal peptide is removed and cleavage occurs at the RSRR (Arg-Ser-Arg-Arg) position to form latent myostatin or a latent complex, and a step (step 3) in which the connection between the propeptide domain and the mature domain is completely severed to form an active myostatin ligand. Each step is described in detail below (Fig. 2).

[0105]

[0106] Step 1: Promyostatin formation

[0107] Myostatin is first produced within cells as an inactive precursor protein, promyostatin, which is then produced in the form of a homodimer with disulfide bonds.

[0108] At this time, the region existing on the N-terminal side is called the pro-domain or pro-peptide, and the region existing on the C-terminal side is called the active-domain or mature region.

[0109]

[0110] Step 2: Formation of latent myostatin (or latent complex)

[0111] After the above promyostatin moves to the endoplasmic reticulum, the signal peptide having about 24 aa (amino acids) is removed through the first cleavage (① in Figure 2).

[0112] Afterwards, additional cleavage occurs at the Arg-Ser-Arg-Arg (RSRR) site near the 266th amino acid of promyostatin by furin convertase of the furin family within the Golgi (② in Figure 2).

[0113] By this process, NH2-terminal (each about 27.7 kDa) and COOH-terminal (each about 12.4 kDa) fragments are generated.

[0114] The COOH-terminal fragment remains noncovalently bound to the NH2-terminal fragment (propeptide region), and the COOH-terminal fragments form disulfide bonds with each other, still existing as a dimer overall. This dimeric myostatin is called latent myostatin or latent complex, and latent myostatin cannot bind to the receptor.

[0115]

[0116] Step 3: Formation of active myostatin

[0117] In the extracellular space, latent myostatin is activated when the propeptide (prodomain) region is cleaved by metalloproteinases such as BMP-1 (Bone morphogenetic protein 1), TLL-1 (Tolloid-like 1), and TLL-2 (Tolloid-like 2) (③ in Figure 2).

[0118] Active myostatin (mature myostatin) formed through this process is secreted and participates in muscle production (myogenesis).

[0119]

[0120] Thus, after the three-step process described above, myostatin is secreted outside the cell and regulates gene transcription within the target cell (see Scand J Med Sci Sports 2008: 18: 123-131).

[0121] In blood, myostatin can exist in various forms. In serum, myostatin is known to exist primarily as a latent myostatin complex, while in plasma, it exists as active myostatin. The active myostatin is known to circulate primarily bound to proteins such as follistatin, Follistatin-Like 3 (FSTL3), GDF-associated serum protein-1 (GASP1), GDF-associated serum protein-2 (GASP2), and decorin. Therefore, the muscle production efficiency of myostatin can be affected by the regulation of its binding to follistatin and others.

[0122] Activated myostatin (mature domain) secreted outside the cell binds to the activin receptor type II (ActRIIB) protein, a serine / threonine kinase, and activates signaling within the target cell. Simply put, when myostatin binds to the activin receptor type II protein, it is phosphorylated, and the signal generated by this is transmitted to the activin receptor type I protein. The signal is transmitted to the receptor-regulated proteins Smad2 (SMAD Family Member 2) and Smad3 (SMAD Family Member 3), and Smad2 and Smad3 interact with Smad4 (SMAD Family Member 4) to influence the transcription of target genes within the nucleus. One example of the target gene is MyoD. MyoD is known to interact with and bind to muscle gene promoters and to be involved in myoblast proliferation. Within cells, myostatin inhibits the expression of MyoD and stimulates the expression of cyclin-dependent kinase (CDK) inhibitors such as p21, thereby inhibiting myoblast growth.

[0123]

[0124] Conventional methods for promoting muscle differentiation using myostatin protein

[0125] Researchers in the field have been using the aforementioned myostatin protein activation process to create transgenic animals with developed muscles. In other words, attempts have been made to promote muscle differentiation in animals by suppressing the expression or function of myostatin protein.

[0126] In particular, researchers have focused on activated myostatin, which is composed of the mature domain generated during the myostatin activation process. This is because latent myostatin (Latent Complex) cannot bind to receptors, and the 'mature domain of myostatin (activated myostatin)' formed by detaching from the latent myostatin regulates the expression of transcription factors such as MyoD. In other words, in order to promote muscle generation (differentiation) by activating MyoD, etc., methods have been used to disable the function of the mature domain of myostatin or to inhibit the production of active myostatin. To achieve this purpose, researchers have previously targeted exon 3 of the MSTN gene, which encodes the mature domain of the myostatin protein, to inhibit the expression or production of active myostatin. For example, some studies have reported that the muscle mass of transgenic animals was significantly increased by targeting and knocking out exon 3 of MSTN using TALEN and CRISRP / Cas technologies (Biosci Rep. 2018 Dec 21; 38(6); doi: 10.1042 / BSR20180742).

[0127]

[0128] However, as mentioned above, the process of artificially differentiating specific cells into muscle cells is known to be much more difficult compared to the differentiation process into other types of target cells. This is expected to be related to the characteristics and growth method of muscle cells that go through the stages of forming myoblasts, myotubes, and muscle fibers.

[0129] In this current state of the art, the present inventors have confirmed that secretions or cultures of somatic cells having a specific MSTN mutation can very effectively achieve artificial differentiation into muscle cells.

[0130] Hereinafter, the present invention will be described in detail.

[0131]

[0132] Overview of compositions for promoting muscle differentiation

[0133] One aspect of the present application relates to a composition for promoting muscle differentiation.

[0134] The composition for promoting muscle differentiation comprises a secretion or culture of a "genetically engineered cell." The composition for promoting muscle differentiation may comprise an extract of a secretion or culture of a "genetically engineered cell."

[0135]

[0136] 1. Genetically engineered cells

[0137] generalization

[0138] The term "genetically engineered cell" can be interpreted to include both cases where the genomic DNA of the cell has been manipulated or transfected with a vector to express a gene or protein of interest.

[0139] The genetically engineered cell of the present application refers to a cell engineered to express a specific MSTN protein variant. The MSTN protein variant is an MSTN protein variant in which four specific amino acids, as described below, are deleted.

[0140] At this time, the MSTN protein variant can be expressed from an “artificially engineered genome”, and the genetically engineered cell of the present application comprises a nucleic acid sequence of the mutated MSTN gene.

[0141]

[0142] In one embodiment, the cell is a cell in which the MSTN gene in the genome has been knocked down or knocked out. In this case, the knockdown or knockout of the MSTN gene may be due to an artificial mutation occurring in the MSTN gene in the genome. For example, the genetically engineered cell of the present application may comprise a "mutated MSTN gene" comprising a specific mutation (deletion of a specific 12 bp sequence) in the genome. In another example, the genetically engineered cell of the present application may comprise an MSTN gene in which an out-of-frame deletion has occurred in the genome.

[0143] As another example, the genetically engineered cell of the present application may be a cell in which the MSTN gene in the genome has been knocked out and a vector containing a nucleic acid sequence including a specific mutation (deletion of a specific 12 bp sequence) has been introduced.

[0144] As another example, the genetically engineered cell of the present application may have an endogenous MSTN gene knocked out in the genome and an exogenous mutated MSTN gene inserted into the genome.

[0145]

[0146] Sequence of the mutated MSTN gene

[0147] In the present application, the "mutated MSTN gene" contained in the genetically engineered cell is one in which a specific 12 bp sequence is deleted from the nucleic acid sequence of the wild-type MSTN gene.

[0148] This mutation corresponds to an in-frame deletion, in which three nucleotides of the MSTN gene encoding the nucleic acid sequence are deleted. Since this in-frame deletion does not induce a frameshift mutation, it has the characteristic of not disrupting the amino acid sequence frame of the myostatin protein encoded by the MSTN gene. Therefore, cells containing the mutated MSTN gene of the present application can express a modified MSTN protein (MSTN variant).

[0149] In particular, the in-frame deletion (specific 12 bp deletion) of the mutated MSTN gene of the present application occurs in exon 2 of the wild-type MSTN gene. For example, the in-frame deletion may be a deletion of about 466 nt to 477 nt of exon 2 of the wild-type bovine MSTN gene; or a deletion of about 469 nt to 480 nt. The specific 12 bp deleted nucleotide sequence is 5'-CTGTGGATATAT-3' (SEQ ID NO: 7) or 5'-TGGATATATCTG-3' (SEQ ID NO: 8).

[0150] In this specification, the MSTN gene having the specific mutation described above is sometimes abbreviated as “12bp deleted MSTN gene” or “-12bp MSTN gene.”

[0151]

[0152] The sequence of the mutated MSTN protein disclosed in the present application may be any one amino acid sequence selected from the following:

[0153] Sequence number 13, Sequence number 19, Sequence number 24, and Sequence number 29.

[0154] SEQ ID NO: 13 is the amino acid sequence of the bovine wild-type MSTN protein with the amino acid sequence of SEQ ID NO: 9 deleted. SEQ ID NO: 19 is the amino acid sequence of the human wild-type MSTN protein with the amino acid sequence of SEQ ID NO: 9 deleted. SEQ ID NO: 24 is the amino acid sequence of the canine wild-type MSTN protein with the amino acid sequence of SEQ ID NO: 9 deleted. SEQ ID NO: 29 is the amino acid sequence of the feline wild-type MSTN protein with the amino acid sequence of SEQ ID NO: 9 deleted.

[0155]

[0156] The sequence of the mutated MSTN gene disclosed in the present application may be a nucleic acid sequence encoding the mutated MSTN protein. For example, the sequence of the mutated MSTN gene may be a nucleic acid encoding any one of the following amino acid sequences:

[0157] Sequence number 13, Sequence number 19, Sequence number 24, and Sequence number 29.

[0158]

[0159] The sequence of the mutated MSTN gene disclosed in the present application may be any one of the following nucleic acid sequences:

[0160] SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0161] SEQ ID NO: 12 is a nucleic acid sequence in which a specific 12 bp sequence of SEQ ID NO: 7 or 8 is deleted from the wild-type bovine MSTN gene. SEQ ID NO: 14 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 13. SEQ ID NO: 15 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 13, and is a codon optimized sequence for expression in bovine cells. SEQ ID NO: 16 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 13, and is a codon optimized sequence for expression in human cells. SEQ ID NO: 17 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 13, and is a codon optimized sequence for expression in canine cells. Sequence number 18 is a nucleic acid sequence encoding a mutated MSTN protein represented by sequence number 13, and is a codon optimized sequence for expression in cat cells.

[0162] SEQ ID NO: 20 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 19. SEQ ID NO: 21 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 19, and is a codon-optimized sequence for expression in human cells. SEQ ID NO: 22 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 19, and is a codon-optimized sequence for expression in canine cells. SEQ ID NO: 23 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 19, and is a codon-optimized sequence for expression in feline cells.

[0163] SEQ ID NO: 25 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 24. SEQ ID NO: 26 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 24, and is a codon-optimized sequence for expression in human cells. SEQ ID NO: 27 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 24, and is a codon-optimized sequence for expression in canine cells. SEQ ID NO: 28 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 24, and is a codon-optimized sequence for expression in feline cells.

[0164] SEQ ID NO: 30 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 29. SEQ ID NO: 31 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 29, and is a codon-optimized sequence for expression in human cells. SEQ ID NO: 32 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 29, and is a codon-optimized sequence for expression in canine cells. SEQ ID NO: 33 is a nucleic acid sequence encoding a mutant MSTN protein represented by SEQ ID NO: 29, and is a codon-optimized sequence for expression in feline cells.

[0165]

[0166] The mutated MSTN gene of the present invention may be operably linked to a promoter. The promoter linked to the mutated MSTN gene may be an endogenous promoter or an exogenous promoter. The exogenous promoter may be a known promoter, such as the CAG promoter or the EF1a promoter. The nucleic acid sequence of the exogenous promoter may be SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

[0167]

[0168] Amino acid sequence encoded by the mutated MSTN gene

[0169] The genetically engineered cell disclosed in the present application comprises a nucleic acid sequence of a mutated MSTN gene comprising the specific 12 bp deletion (in-frame deletion), thereby expressing an MSTN protein variant having a specific 4 amino acid deletion.

[0170] The nucleic acid sequence of the MSTN gene, in which a specific 12 bp sequence is deleted from the exon 2 sequence of the wild-type MSTN gene, encodes a protein variant in which four specific amino acid sequences in the propeptide region of the myostatin protein are deleted.

[0171] For example, the region of the protein encoded by exon 2 of the bovine MSTN gene corresponds to the amino acid sequence from about 125th to about 248th positions of the wild-type bovine myostatin protein, and the protein variant of the present application may have specific 4 amino acids deleted (removed) from the amino acid sequence from positions 125 to 248 of the wild-type MSTN protein.

[0172] In one specific example, the MSTN protein variant disclosed in the present application may be one in which the amino acid sequence constituting the wild-type bovine MSTN protein (SEQ ID NO: 34) is deleted, that is, four amino acid sequences consisting of "leucine, tryptophan, isoleucine, tyrosine (LWIY: SEQ ID NO: 9)" in the order from the N-terminus to the C-terminus. This may be expressed by an MSTN gene having a DNA deletion mutation of SEQ ID NO: 7 in the DNA sequence of the wild-type MSTN gene. In this case, the sequence of the MSTN protein variant may be SEQ ID NO: 13.

[0173] In another specific example, the MSTN protein variant disclosed in the present application may be one in which the amino acid sequence constituting the wild-type bovine MSTN protein (SEQ ID NO: 34) is deleted, that is, four amino acid sequences consisting of "tryptophan, isoleucine, tyrosine, leucine (WIYL: SEQ ID NO: 10)" in the order from the N-terminus to the C-terminus. This may be expressed by an MSTN gene having the DNA deletion mutation of SEQ ID NO: 8 in the DNA sequence of the wild-type MSTN gene. In this case, the sequence of the MSTN protein variant may be SEQ ID NO: 13.

[0174] In another specific example, the MSTN protein variant disclosed in the present application may be one in which the amino acid sequence of SEQ ID NO: 9 is deleted from the amino acid sequence constituting the human wild-type MSTN protein. In this case, the sequence of the MSTN protein variant may be SEQ ID NO: 19.

[0175] In another specific example, the MSTN protein variant disclosed in the present application may be one in which the amino acid sequence of SEQ ID NO: 9 is deleted from the amino acid sequence constituting the wild-type MSTN protein. In this case, the sequence of the MSTN protein variant may be SEQ ID NO: 24.

[0176] In another specific example, the MSTN protein variant disclosed in the present application may be one in which the amino acid sequence of SEQ ID NO: 9 is deleted from the amino acid sequence constituting the wild-type MSTN protein of a cat. In this case, the sequence of the MSTN protein variant may be SEQ ID NO: 29.

[0177]

[0178] Embodiment of a genetically engineered cell disclosed in this application

[0179] In one embodiment, the genetically engineered cell may comprise a sequence of a mutated MSTN gene.

[0180] In one embodiment, the genetically engineered cell may comprise a sequence of a mutated MSTN gene in its genome.

[0181] In one embodiment, the genetically engineered cell may have an artificial mutation in the endogenous MSTN gene and may additionally comprise the sequence of the mutated MSTN gene.

[0182] For example, the sequence of the mutated MSTN gene included in the genetically engineered cell may be artificially inserted into the genome as an exogenous sequence. As another example, the sequence of the mutated MSTN gene included in the genetically engineered cell may be included in an expression vector as an exogenous sequence.

[0183] In one embodiment, the genetically engineered cell may have an out-of-frame deletion in the MSTN gene in the genome of the cell, and the sequence of the MSTN gene with the out-of-frame deletion may be SEQ ID NO: 45 or SEQ ID NO: 46.

[0184] In one embodiment, the genetically engineered cell may have an out-of-frame deletion in the MSTN gene in the genome of the cell and an exogenous mutated MSTN gene inserted into the genome.

[0185]

[0186] In one embodiment, the genetically engineered cell may be a mammalian cell. The mammal may be a human, cow, pig, dog, cat, mouse, horse, goat, hamster, or the like.

[0187] For example, the genetically engineered cell may be a bovine cell. As a specific example, the genetically engineered cell may be a bovine muscle satellite cell, a bovine myoblast, a bovine muscle cell, or a bovine fibroblast.

[0188] For another example, the genetically engineered cell may be a human-derived cell. Specifically, the genetically engineered cell may be a human-derived muscle satellite cell, a human-derived myoblast, a human-derived muscle cell, or a human-derived fibroblast.

[0189] For another example, the genetically engineered cell may be a canine-derived cell. Specifically, the genetically engineered cell may be a canine-derived muscle satellite cell, a canine-derived myoblast, a canine-derived muscle cell, or a canine-derived fibroblast.

[0190] For another example, the genetically engineered cell may be a cat-derived cell. Specifically, the genetically engineered cell may be a cat-derived muscle satellite cell, a cat-derived myoblast, a cat-derived muscle cell, or a cat-derived fibroblast.

[0191] For another example, the genetically engineered cell may be a hamster-derived cell. Specifically, the genetically engineered cell may be a hamster-derived muscle satellite cell, a hamster-derived myoblast, a hamster-derived muscle cell, or a hamster-derived fibroblast. Specifically, the genetically engineered cell may be a CHO (Chinese Hamster Ovary) cell.

[0192]

[0193] In one embodiment, the genetically engineered cell may be a somatic cell, a stem cell, or a progenitor cell.

[0194] The above somatic cells are mature cells that constitute various tissues and organs of an adult body, and may be muscle cells, fibroblasts, keratinocytes, mucosal cells, adipocytes, epithelial cells, vascular endothelial cells, bone marrow cells, nerve cells, etc. In one embodiment, the somatic cells may be muscle cells or fibroblasts.

[0195] The above progenitor cells are cells that are in the process of differentiating into a specific type of cell, although they have not yet achieved their final form or function. In one embodiment of the present invention, myoblasts and muscle satellite cells can be used.

[0196] The above stem cells are undifferentiated cells that can differentiate into various types of tissues, and can be broadly classified into embryonic stem cells derived from fertilized eggs and adult stem cells derived from various somatic cells. Adult stem cells can be obtained by isolation from peripheral blood, fat, bone marrow, placenta, etc.

[0197]

[0198] Specific examples of MSTN gene nucleic acid sequences

[0199] In one embodiment, the genetically engineered cell of the present invention comprises a mutated MSTN gene nucleic acid sequence represented by SEQ ID NO: 12 in its genome.

[0200] In one specific example, the nucleic acid sequence of the MSTN gene included in the genetically engineered cell of the present invention may include the sequences of SEQ ID NOs: 1, 3, 4, 5, and 6. At this time, SEQ ID NO: 1 is the nucleic acid sequence of wild-type exon 1 of the bovine MSTN gene, SEQ ID NO: 5 is the nucleic acid sequence of wild-type intron 1 of the bovine MSTN gene, SEQ ID NO: 4 is the nucleic acid sequence of mutated exon 2 in which the sequence of 12BP is deleted, SEQ ID NO: 6 is the nucleic acid sequence of wild-type intron 2 of the bovine MSTN gene, and SEQ ID NO: 3 is the nucleic acid sequence of wild-type exon 3 of the bovine MSTN gene.

[0201]

[0202] In the examples described above, within the scope of achieving the purpose of the present invention, a silent mutation may be included in at least one of the nucleic acid sequences constituting exon 1, exon 3, intron 1, and intron 2 of the MSTN gene.

[0203]

[0204] Specific examples of genetically engineered cells

[0205] In one embodiment, the genetically engineered cell of the present invention may be a bovine cell comprising a mutated MSTN gene in its genome. Specifically, the genetically engineered cell may be a bovine cell having a deletion in the endogenous MSTN gene in its genome, wherein the deleted endogenous MSTN gene comprises the nucleic acid sequence of SEQ ID NO: 4.

[0206] In one embodiment, the genetically engineered cell of the present invention may be a CHO cell in which the endogenous MSTN gene is knocked out or knocked down in the genome and comprises an exogenous mutated MSTN gene. Specifically, the genetically engineered cell may be a CHO cell in which the endogenous MSTN gene is knocked out or knocked down in the genome and comprises a nucleic acid sequence of any one of SEQ ID NO: 45 and SEQ ID NO: 46. The exogenous mutated MSTN gene may be a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. Alternatively, the exogenous mutated MSTN gene may be a nucleic acid sequence of SEQ ID NO: 17.

[0207] In one embodiment, the genetically engineered cell of the present invention may be a CHO cell in which the endogenous MSTN gene is knocked out or knocked down in the genome and comprises an exogenous mutated MSTN gene. Specifically, the genetically engineered cell may be a CHO cell in which the endogenous MSTN gene is knocked out or knocked down in the genome and comprises a nucleic acid sequence of either SEQ ID NO: 45 or SEQ ID NO: 46. The exogenous mutated MSTN gene may be a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24. Alternatively, the exogenous mutated MSTN gene may be a nucleic acid sequence of SEQ ID NO: 27.

[0208]

[0209] Secretions or cultures of genetically modified cells

[0210] In one embodiment, the composition for promoting muscle differentiation of the present application comprises a secretion of the genetically engineered cell or a culture obtained by culturing the genetically engineered cell. In another embodiment, the composition for promoting muscle differentiation of the present application comprises an extract of the secretion of the genetically engineered cell or an extract of the culture obtained by culturing the genetically engineered cell.

[0211] The secretory substance (secretory substance) of the genetically engineered cell is a substance secreted by the cell while performing various metabolic functions (cell metabolism) or signaling functions (cellular signaling), and includes both substances that the cell produces and secretes on its own or substances that are secreted in response to external stimuli.

[0212] The culture of the genetically engineered cells is a result obtained after culturing the above-mentioned cells under predetermined conditions for a certain period of time, and may include both the materials constituting the cell culture medium and the secretions of the cells.

[0213] The cultivation of genetically engineered cells is described in detail in <Culture medium for promoting muscle differentiation> described below.

[0214] The secretory material or culture of the genetically engineered cell of the present invention may include substances produced by various metabolic and signaling activities of the cell, which are caused by a mutated MSTN gene with a specific 12-bp sequence deleted from the cell's genome. For example, the substances may include exosomes, cytokines, hormones, neurotransmitters, etc. that promote muscle differentiation.

[0215] In addition, the extract of the secretion (culture) of the genetically engineered cells of the present application refers to a material obtained by isolating / extracting a specific substance or cell organelle from the above-mentioned secretion (culture) through an additional process.

[0216] The extract of the secretion of the genetically engineered cells or the extract of the culture obtained by culturing the genetically engineered cells may be extracted using a filter, a 2-layer system, a TFF pump system (Cytiva, Minimate EVO system), etc.

[0217]

[0218] Paracrine effect - induction and promotion of muscle differentiation

[0219] The muscle differentiation promotion effect (function) disclosed in the present application is an effect possessed by the secretion or culture of the genetically engineered cells described above, and is achieved by providing an environment in which the target cells can differentiate well into muscle cells.

[0220]

[0221] As previously described, previous studies have shown that suppressing the expression of normal mature myostatin protein in animals leads to overdevelopment of animal muscles.

[0222] However, the method of the present application shows that the genetically engineered cells have a specific mutation introduced into the region encoding the pro-peptide of the myostatin protein in the genome, and by expressing a specific myostatin mutant according to the mutation, differentiation of the target cells into muscle cells is effectively promoted.

[0223] The genetically engineered cells described above have a paracrine effect by secreting various substances in response to the microenvironment.

[0224] The above "paracrine effect" refers to a phenomenon in which compounds produced in cells or tissues affect surrounding cells or tissues. In this application, it refers to a function that induces or promotes differentiation into muscle cells. In other words, the muscle differentiation promoting effect (function) disclosed in this application is derived from the paracrine effect of cells containing a specific mutated MSTN gene.

[0225]

[0226] order

[0227] The sequence numbers corresponding to the sequence of nucleic acids or the sequence of proteins that can be included in genetically engineered cells are as shown in Table 1 below.

[0228] [Table 1]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253] Below, a culture medium containing a secretion or culture of the genetically engineered cells of the present application having a muscle differentiation promoting effect is described.

[0254]

[0255] 2. Culture media for promoting muscle differentiation

[0256] Overview of culture media for promoting muscle differentiation

[0257] Another aspect of the present application relates to a culture medium for promoting muscle differentiation.

[0258] The above culture medium for promoting muscle differentiation is

[0259] i) It may be a conditioned media obtained by culturing the genetically engineered cells described above in a basic medium, or

[0260] ii) It may be a mixed medium that mixes the above-mentioned conditioned medium and the basic medium.

[0261]

[0262] In this specification, a medium containing a secreted substance of the cell or a culture medium (culture medium) containing the secreted substance obtained by culturing the genetically engineered cell is referred to as a conditioned medium. That is, the conditioned medium contains various secreted substances secreted by the genetically engineered cell.

[0263]

[0264] base media (basic media)

[0265] The base media (basic media) used in the above conditioned medium or mixed medium refers to a composition containing nutrients for proliferation or growth of cells, and the base media may be in the form of a solid, liquid, gel, or powder, but is not limited thereto.

[0266] The above basic medium may contain growth factors, vitamins, proteins, minerals, carbohydrates, dyes, etc. A person skilled in the art can select and use the basic medium appropriately depending on the cell type.

[0267] For example, the base medium may be Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), Ham's F-12 (Ham's Nutrients Mixture F12), Opti-MEM, etc. In one embodiment, DMEM was used as the base medium.

[0268]

[0269] The above basic medium may optionally contain serum, antibiotics, etc. as needed.

[0270] At this time, the serum may be serum derived from mammalian blood. For example, the serum may be fetal bovine serum (FBS), bovine calf serum (BCS), horse serum (HS), human serum, newborn calf serum (NCS), etc.

[0271] The above antibiotics may be, for example, penicillin, streptomycin, vancomycin, etc.

[0272] The above serum or / and antibiotics can be used at 0.1% to 50% of the total volume of the basic medium, but the user can select and use them appropriately depending on the type of cell and the purpose of the experiment.

[0273] As an example, a base medium may contain about 1% to 30% serum relative to the total volume of the medium.

[0274] As another example, the base medium may contain between about 1% and 30% of the total volume of the medium of antibiotics.

[0275] As another example, the base medium may contain about 1% to 30% serum and about 1% to 30% antibiotics by volume of the total medium.

[0276] In an optional embodiment, the base medium may be DMEM containing about 20% fetal bovine serum (FBS) and penicillin and streptomycin.

[0277] In any other embodiment, the base medium may be DMEM containing about 2% horse serum (HS) and penicillin and streptomycin.

[0278]

[0279] Conditioned media for promoting muscle differentiation

[0280] In one embodiment, the culture medium for promoting muscle differentiation of the present application refers to a conditioned medium (CM) for promoting muscle differentiation.

[0281] Conditioned medium refers to the culture medium after cells have been cultured in the aforementioned basic medium. In other words, it refers to a culture medium in which the initial composition of the medium (i.e., the composition before culturing cells) has been changed by culturing cells in the basic medium for cell growth, proliferation, etc.

[0282] Therefore, the above-mentioned muscle differentiation-promoting conditioned medium contains various secretory substances secreted by the genetically engineered cells through metabolic and / or signaling actions.

[0283] The conditioned medium for promoting muscle differentiation disclosed by the present application is a culture medium in which genetically engineered cells are cultured in a basic medium.

[0284] For example, the conditioned medium of the present application may be a culture medium in which fibroblasts, myoblasts, muscle satellite cells, or muscle cells containing a 12BP deleted MSTN gene are cultured in a basal medium for a certain period of time.

[0285] For another example, the conditioned medium of the present application may be obtained by culturing fibroblasts, myoblasts, muscle satellite cells, or muscle cells containing the MSTN gene with 12BP deletion in the genome in a basal medium for a certain period of time, and then removing cellular components and debris.

[0286] For another example, the conditioned medium of the present application may be a culture medium in which bovine cells containing a mutated MSTN gene in the genome are cultured for a certain period of time in a basic medium. Specifically, the genetically engineered cells may be bovine cells in which the endogenous MSTN gene in the genome has been deleted, and the deleted endogenous MSTN gene comprises the nucleic acid sequence of SEQ ID NO: 4.

[0287] For another example, the conditioned medium of the present application may be a culture medium in which CHO cells containing an exogenous mutated MSTN gene and an endogenous MSTN gene in the genome have been knocked out or knocked down are cultured for a certain period of time in a basic medium. Specifically, the genetically engineered cell may be a CHO cell in which the endogenous MSTN gene in the genome has been knocked out or knocked down and contains the nucleic acid sequence of either SEQ ID NO: 45 or SEQ ID NO: 46.

[0288]

[0289] Meanwhile, another aspect of the present application relates to a method for producing the conditioned medium for promoting muscle differentiation.

[0290] The method for making the above conditioned medium for promoting muscle differentiation is as follows:

[0291] It includes obtaining a culture solution by culturing genetically engineered cells in a basic medium for a certain period of time until they reach a predetermined confluency.

[0292] Optionally, the obtained culture medium may further include removing cell components and debris.

[0293] The genetically engineered cells described above may be referred to as described above.

[0294] For example, the genetically engineered cell may be a bovine fibroblast, myoblast, muscle satellite cell, or muscle cell. Or, it may be a CHO cell. In this case, the cell's genome contains a mutated MSTN gene with a 12BP sequence deleted.

[0295] The above basic badge can refer to the contents described above in <Basic Badge>.

[0296] For example, the basic medium for culturing genetically engineered cells containing a mutated MSTN gene with a 12BP sequence deleted may be Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), Ham's F-12 (Ham's Nutrients Mixture F12), Opti-MEM, etc. In this case, the basic medium may optionally further include serum, antibiotics, etc.

[0297]

[0298] Another aspect of the method of the present application may include a process of filtering the conditioned medium for promoting muscle differentiation.

[0299] The process of filtering the above-mentioned muscle differentiation promoting conditioned medium may include one or more of the following:

[0300] Centrifugation at about 1000 to 3000 rpm for about 1 to 60 minutes to separate floating cells from the conditioned medium;

[0301] Separate the supernatant of the conditioned medium using a filter of about 0.1 to 0.5 um; and

[0302] A filtrate is obtained from a conditioned medium or a portion of a conditioned medium using a tangential flow filtration (TFF) method.

[0303] Here, the tangential flow filtration method may use a TFF pump system (Cytiva, Minimate EVO system).

[0304] The extract of the genetically engineered cell secretion or the extract of the genetically engineered cell culture disclosed by the present application may be obtained by performing a process of filtering the conditioned medium for promoting muscle differentiation.

[0305]

[0306] Various culture conditions for culturing the genetically engineered cells using a basic medium are described.

[0307] The above culture conditions may vary depending on the type of genetically engineered cells, the condition of the cells, the proliferation rate, etc. For example, the culture conditions of the genetically engineered cells may include the culture period of the genetically engineered cells, the culture temperature, the number of cells, confluence, etc.

[0308] The number of genetically engineered cells can be, but is not limited to, about 1x10^3 / well (or 1 plate) to 1x10^10 / well (or 1 plate). In any example, the myoblasts comprising the 12BP deleted MSTN gene can be seeded at a number of 3x10^4 to 4x10^4 / well of 6 wells. In any other example, the myosatellite cells comprising the 12BP deleted MSTN gene can be seeded at a number of 3x10^4 to 4x10^4 / well of 6 wells. In any other example, the fibroblasts comprising the 12BP deleted MSTN gene can be seeded at a number of 2x10^5 to 3x10^6 / 75T plate. As another example, CHO cells containing the 12BP deleted MSTN gene can be seeded at a number of 2x10^5 to 6x10^6 / well.

[0309] The culture period of the genetically engineered cells may be about 1 to 15 days, and the medium may be replaced with fresh medium about every 2 to 5 days. In any example, the myoblasts comprising the MSTN gene with a 12BP deletion may be cultured for about 1 to 10 days, and the medium may be replaced with fresh medium about every 2 to 3 days. In any other example, the fibroblasts comprising the MSTN gene with a 12BP deletion may be cultured for about 1 to 7 days, and the medium may be replaced with fresh medium about every 2 to 5 days. In any other example, the myotubes comprising the MSTN gene with a 12BP deletion may be cultured for about 1 to 7 days, and the medium may be replaced with fresh medium about every 2 to 3 days. As another example, CHO cells containing a 12BP deleted MSTN gene can be cultured for about 1 to 7 days.

[0310] Genetically engineered cells can be cultured for a period of time to achieve a confluency of approximately 50% to 100%. For example, they may achieve a confluency of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0311] At this time, subculture can be performed at an appropriate confluency. Cell subculture can be performed approximately 1 to 50 times.

[0312] As an example, myoblasts comprising a 12bp deleted MSTN gene can be passaged when they reach about 70% confluency. As another example, myosatellite cells comprising a 12bp deleted MSTN gene can be passaged when they reach about 70% confluency. As another example, fibroblasts comprising a 12bp deleted MSTN gene can be passaged when they reach about 90% confluency.

[0313] The culture temperature of genetically engineered cells may be approximately 25 to 40°C. At this time, the culture temperature may be the incubation temperature in a device for culturing cells.

[0314] Additionally, the culture pH can be approximately 6 to 8. The pH can be controlled using carbon dioxide. For example, a cell culture incubator with a temperature of approximately 37°C and a carbon dioxide content of approximately 5% can be used.

[0315]

[0316] Meanwhile, removal of cells, debris, etc. within the conditioned medium can be performed appropriately using a known method known in the art.

[0317] For example, it may be a method using centrifugation, chromatography, filtration, precipitation, or a method using a column.

[0318] As a specific example, it can be performed using a filter, a 2-layer system, a TFF pump system (Cytiva, Minimate EVO system), etc.

[0319] The above two-layer system refers to a system that uses a container or device including a membrane, filter, etc. together with a cell culture vessel or device. In one specific example, the two-layer system may be a cell culture vessel including a transwell insert.

[0320] The conditioned medium for promoting muscle differentiation of the present application can be manufactured by the method described above, and the conditioned medium manufactured thereby has a very different composition of materials constituting the medium from the initial composition of the basic medium (i.e., the composition of various materials before culturing cells) due to various secreted substances of the cells.

[0321] More specific manufacturing examples are given below:

[0322] Example of manufacturing conditioned medium (1)

[0323] As an example, the method for making a conditioned medium for promoting muscle differentiation of the present application is as follows:

[0324] Genetically engineered cells containing a mutated MSTN gene with a deletion of the 12BP sequence are cultured in DMEM medium until a predetermined confluency is reached.

[0325] As a specific example,

[0326] It may include culturing myoblasts containing a mutated MSTN gene represented by sequence number 12 in DMEM supplemented with 100 U / ml of penicillin / streptomycin and 20% fetal bovine serum (FBS) until the cells reach a confluence of about 70% or more.

[0327] As another specific example,

[0328] It may include culturing muscle satellite cells containing a mutated MSTN gene represented by sequence number 12 in DMEM supplemented with 100 U / ml of penicillin / streptomycin and 20% fetal bovine serum (FBS) until the cells reach a confluence of about 70% or more.

[0329] As another specific example,

[0330] The method may further include culturing fibroblasts containing a mutated MSTN gene represented by sequence number 12 in DMEM supplemented with 100 U / ml of penicillin / streptomycin and 10% fetal bovine serum (FBS) until the cell confluence reaches about 90%, and then further culturing the cells in DMEM supplemented with 100 U / ml of penicillin / streptomycin until the cell confluence reaches about 100% for about 5 days.

[0331]

[0332] As another example, the method for making a conditioned medium for promoting muscle differentiation of the present application is as follows:

[0333] It may include culturing CHO cells containing a mutated MSTN gene represented by SEQ ID NO: 17 in Ham's F-12 (Ham's Nutrients Mixture F12) supplemented with 20% FBS until the cells reach a confluence of about 70% or more.

[0334]

[0335] Example of manufacturing conditioned medium (2)

[0336] As another example, the method for making a conditioned medium for promoting muscle differentiation of the present application is as follows:

[0337] Genetically engineered cells containing a mutated MSTN gene with a 12BP sequence deleted are cultured in DMEM medium until a predetermined confluence is reached to obtain a culture medium; and

[0338] Removing cell components and debris from the culture medium obtained above

[0339] Includes.

[0340]

[0341] As a specific example,

[0342] Myoblasts containing the mutated MSTN gene represented by SEQ ID NO: 12 were cultured in DMEM supplemented with 100 U / ml of penicillin / streptomycin and 20% fetal bovine serum (FBS) until the cell confluence reached approximately 70% or more;

[0343] The above cultured culture solution may include removing debris using a filtration system (e.g., layer system, filter, etc.).

[0344] As another specific example,

[0345] Muscle satellite cells containing the mutated MSTN gene represented by sequence number 12 were cultured in DMEM supplemented with 100 U / ml of penicillin / streptomycin and 20% fetal bovine serum (FBS) until the cell confluence reached approximately 70% or more;

[0346] The above cultured culture solution may be centrifuged to remove debris.

[0347] As another specific example,

[0348] Fibroblasts containing a mutated MSTN gene with a 12BP sequence deletion were cultured in DMEM containing 100 U / ml of penicillin / streptomycin and 10% fetal bovine serum (FBS) until the cell confluence reached approximately 90%, and then cultured for an additional 5 days in DMEM containing 100 U / ml of penicillin / streptomycin until the cell confluence reached approximately 100%;

[0349] The above cultured culture solution may include removing cells and debris using a TFF pump system.

[0350]

[0351]

[0352] As another example, the method for making a conditioned medium for promoting muscle differentiation of the present application is as follows:

[0353] CHO cells containing a mutated MSTN gene represented by sequence number 17 were cultured in Ham's F-12 (Ham's Nutrients Mixture F12) supplemented with 20% FBS until the cell confluence reached about 70% or more to obtain a culture medium; and

[0354] The obtained culture solution may be filtered using a centrifugation and / or filtration system (e.g., layer system, TFF pump system, etc.).

[0355] Impurities such as cells, germs, bacteria, and debris can be removed from the culture solution through the above centrifugation and / or filtration system.

[0356]

[0357] Complex media for promoting muscle differentiation (mixed media)

[0358] Another embodiment of the culture medium for promoting muscle differentiation disclosed by the present application refers to a mixed medium that mixes the above-mentioned conditioned medium and the basic medium.

[0359] That is, it is a medium in which the above-mentioned conditioned medium and basic medium are mixed in a predetermined ratio, and at this time, the conditioned medium and basic medium used in the mixed medium of the present application are as described above, respectively.

[0360]

[0361] The conditioned medium used in the mixed medium is a culture (culture solution) in which genetically engineered cells are cultured in a basal medium. In one embodiment, the genetically engineered cells may be fibroblasts, myoblasts, muscle satellite cells, or muscle cells containing the MSTN gene with a 12BP deletion.

[0362] The basic medium used in the mixed medium can be selected from, for example, Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), Ham's F-12 (Ham's Nutrients Mixture F12), Opti-MEM, etc., and in one embodiment, Dulbecco's Modified Eagle Medium (DMEM) can be used.

[0363] At this time, the basic medium used in the mixed medium may be the same as or different from the basic medium used to obtain the above-mentioned conditioned medium.

[0364]

[0365] The base medium used in the mixed medium may have the following additional components:

[0366] In one specific embodiment, the basal medium may contain about 1% to 30% of a serum selected from among Fetal Bovine Serum (FBS), Bovine Calf Serum (BCS), or Horse Serum (HS).

[0367] For example, the base medium may be DMEM containing about 1% to 20% of the serum. In some embodiments, the base medium may be DMEM containing 10% FBS. In some other embodiments, the base medium may be DMEM containing 20% ​​FBS. In some other embodiments, the base medium may be DMEM containing 2% HS.

[0368] For example, the base medium may be Ham's F-12 (Ham's Nutrients Mixture F12) containing about 1% to 20% of the serum. In some embodiments, the base medium may be Ham's F-12 containing 10% FBS. In some other embodiments, the base medium may be Ham's F-12 containing 20% ​​FBS.

[0369]

[0370] Additionally, the basic medium may further contain about 0.1% to 20% of an antibiotic selected from penicillin, streptomycin, and vancomycin.

[0371] For example, the base medium may be DMEM containing about 1 to 20% of the antibiotic. In some embodiments, the base medium may be DMEM containing 100 U / ml of penicillin / streptomycin.

[0372]

[0373] In another specific example, the base medium may be DMEM containing about 1% to 20% of the serum and about 1% to 20% of the antibiotic.

[0374] In any embodiment, the base medium may be DMEM containing 100 U / ml penicillin / streptomycin and 10% FBS.

[0375] In any embodiment, the base medium may be DMEM containing 100 U / ml penicillin / streptomycin and 20% FBS.

[0376] In any embodiment, the base medium may be DMEM containing 100 U / ml penicillin / streptomycin and 2% HS.

[0377]

[0378] The above-mentioned mixed medium for promoting muscle differentiation may be a mixture of a basic medium and a conditioned medium for promoting muscle differentiation at a specific mixing ratio. For example, the mixing ratio of the basic medium and the conditioned medium of the mixed medium for promoting muscle differentiation may be selected from among 10%:90%, 20%:80%, 25%:75%, 30%:70%, 40%:60%, 50%:50%, 60%:40%, 70%:30%, 75%:25%, 80%:20%, and 90%:10%. For another example, the mixing ratio of the basic medium and the conditioned medium of the mixed medium for promoting muscle differentiation may be selected from among 25%:75%, 50%:50%, 75%:25%, and 90%:10%. In one embodiment, the mixing ratio of the basic medium and the conditioned medium of the mixed medium for promoting muscle differentiation may be 90%:10%.

[0379]

[0380] Example of composition of mixed media

[0381] In one specific example, the mixed medium of the present application comprises, for the entire composition,

[0382] 90% of conditioned medium of fibroblasts containing the MSTN gene with 12BP deletion; and

[0383] It can be composed of 10% basal medium containing DMEM solution containing 2% horse serum and 100 U / ml of penicillin / streptomycin.

[0384] In another specific embodiment, the mixed medium of the present application comprises, for the entire composition,

[0385] 90% of conditioned medium of myoblasts containing the MSTN gene with 12BP deletion; and

[0386] It can be composed of 10% basal medium containing DMEM solution containing 20% ​​FBS and 100 U / ml penicillin / streptomycin.

[0387] In another specific example, the mixed medium of the present application comprises, for the entire composition,

[0388] 90% of conditioned medium of muscle satellite cells containing the MSTN gene with 12BP deletion; and

[0389] It can be composed of 10% basal medium containing DMEM solution containing 20% ​​FBS and 100 U / ml penicillin / streptomycin.

[0390]

[0391] Another aspect of the present application relates to a method for preparing a mixed medium for promoting muscle differentiation.

[0392] The method for producing a mixed medium for promoting muscle differentiation of the present application includes mixing a conditioned medium for promoting muscle differentiation and a basic medium.

[0393] The above-mentioned conditioned medium and basic medium for promoting muscle differentiation are the same as those described above.

[0394] The above mixing can be performed using a method known in the art.

[0395] In one specific example, mixing can be performed by a method using a conventional stirrer, for example, a magnetic stirrer.

[0396]

[0397] At this time, the basic medium and the conditioned medium for promoting muscle differentiation can be mixed at a specific mixing ratio.

[0398] The mixing ratio of the basic medium and the conditioned medium for promoting muscle differentiation can be 5% to 95%: 5% to 95%. For example, the mixing ratio of the basic medium and the conditioned medium can be selected from among 10%:90%, 20%:80%, 25%:75%, 30%:70%, 40%:60%, 50%:50%, 60%:40%, 70%:30%, 75%:25%, 80%:20%, and 90%:10%. Preferably, the basic medium and the conditioned medium can be mixed at a ratio selected from among 25%:75%, 50%:50%, 75%:25%, and 90%:10%. In one embodiment, the basic medium and the conditioned medium for promoting muscle differentiation can be mixed at a ratio of 90%:10%.

[0399]

[0400] The method for manufacturing the above-mentioned mixed medium for promoting muscle differentiation may optionally further include removing cell components, debris, etc. before or after mixing, if the above-mentioned conditioned medium contains cell components, etc.

[0401] This can be accomplished by appropriately utilizing known methods known in the art. For more information, please refer to the aforementioned <Conditioned Medium for Promoting Muscle Differentiation>.

[0402]

[0403] Composition comprising a culture medium for promoting muscle differentiation

[0404] As one aspect of the invention disclosed in the present application, a composition including the culture medium for promoting muscle differentiation is provided.

[0405] As another aspect, a composition is provided that includes an extract obtained by filtering impurities such as bacteria, bacteria, and debris from the culture medium through a TFF filter system.

[0406] In this specification, this is also referred to abbreviated as ‘composition for promoting muscle differentiation’.

[0407] The above composition may include the above-described conditioned medium for promoting muscle differentiation or the mixed medium for promoting muscle differentiation as an effective ingredient.

[0408] The above composition may include an extract obtained by filtering out impurities such as bacteria, bacteria, and debris from the above-mentioned conditioned medium or mixed medium as an effective ingredient.

[0409]

[0410] Additionally, the composition may further comprise optional components. These optional components may be appropriately selected based on the needs and objectives of those skilled in the art. For example, the composition may further comprise additional nutrients that are not contained in the basic medium used and are readily apparent to those skilled in the art.

[0411] The above composition for promoting muscle differentiation induces effective differentiation of target cells into muscle cells.

[0412]

[0413] 3. Methods for promoting muscle differentiation

[0414] Overview of methods to promote muscle differentiation

[0415] Another aspect of the present application relates to a method for promoting muscle differentiation.

[0416] The method for promoting muscle differentiation comprises contacting the genetically engineered cell described above with a target cell and culturing them together.

[0417] Alternatively, the method for promoting muscle differentiation comprises treating the target cell with the aforementioned composition for promoting muscle differentiation. This means treating the target cell with a secretion of a genetically engineered cell; a culture of a genetically engineered cell; an extract of a secretion of a genetically engineered cell; an extract of a culture of a genetically engineered cell; or a culture medium for promoting muscle differentiation comprising the same.

[0418]

[0419] target cell

[0420] In this application, “subject cell” means a cell to be differentiated into a muscle cell.

[0421] In one example, the target cell may be a muscle cell precursor cell, such as a myoblast or muscle satellite cell. In another example, the target cell may be a stem cell.

[0422]

[0423] Meanwhile, the target cells may be of mammalian origin.

[0424] The mammals include humans, cows, pigs, dogs, cats, mice, horses, goats, etc.

[0425] As an arbitrary specific example,

[0426] The above target cells may be bovine-derived myoblasts or muscle satellite cells.

[0427] The target cells may be human-derived myoblasts or muscle satellite cells.

[0428] The target cells may be canine-derived myoblasts or muscle satellite cells.

[0429] The above target cells may be cat-derived myoblasts or muscle satellite cells.

[0430] Alternatively, the target cell may be of mammalian origin other than human.

[0431]

[0432] contact

[0433] 'Contact' refers to a process for promoting (inducing) muscle differentiation of target cells using a muscle differentiation promoting substance (composition) disclosed by the present application.

[0434] The purpose of 'contact' is to provide an environment in which the aforementioned muscle differentiation promoting substance can interact with cell surface proteins (e.g., specific receptors, etc.) of the target cell.

[0435] In addition, the purpose of 'contact' is to provide an environment in which the aforementioned muscle differentiation promoting substance can be introduced into the interior of the target cell.

[0436]

[0437] Below, a specific example of a method for promoting muscle differentiation is described.

[0438]

[0439] Method Example 1:

[0440] The method for promoting muscle differentiation of the present application, in one embodiment,

[0441] (i) contacting a genetically engineered cell comprising a nucleic acid sequence of a mutated MSTN gene with a specific 12 bp sequence deleted with a target cell; and

[0442] (ii) Culturing the genetically engineered cells and target cells together in a basic medium

[0443] may include.

[0444] Contacting the genetically engineered cells with the target cells can be accomplished, for example, by placing the genetically engineered cells and the target cells together in the same culture vessel. Subsequently, the target cells and the genetically engineered cells are co-cultured. That is, the target cells and the genetically engineered cells are mixed and cultured.

[0445] At this time, the conditions and period required for cultivation can be arbitrarily adjusted by those skilled in the art as needed.

[0446] For example, genetically engineered fibroblasts containing a nucleic acid sequence of a mutated MSTN gene with a 12 bp sequence deletion can be cultured with wild-type myosatellite cells for about 3 to 7 days.

[0447] As another example, genetically engineered myoblasts containing a nucleic acid sequence of a mutated MSTN gene with a 12 bp sequence deletion can be cultured with wild-type myoblasts for about 3 to 10 days.

[0448] As another example, genetically engineered muscle satellite cells containing a nucleic acid sequence of a mutated MSTN gene with a 12 bp sequence deletion can be cultured with wild-type muscle satellite cells for about 2 to 10 days.

[0449]

[0450] In this process, the target cells are efficiently differentiated into muscle cells and muscle fibers by the paracrine effect of various secretory substances produced from the genetically engineered cells.

[0451] In this way, target cells, for example, myoblasts (target cells), differentiate well into myotube cells and further form muscle fibers by co-culturing or mixed-culturing with cells having a 12-bp deletion mutation in the MSTN gene.

[0452]

[0453] Method Example 2:

[0454] The method for promoting muscle differentiation of the present application, in one embodiment,

[0455] (i) culturing genetically engineered cells containing a nucleic acid sequence of a mutated MSTN gene with a specific 12 bp sequence deleted in a basal medium; and

[0456] (ii) treating target cells with a composition containing a secretion or culture of genetically engineered cells obtained by the above culture;

[0457] may include.

[0458]

[0459] The above 'treatment' provides an environment in which various substances included in the composition for promoting muscle differentiation can interact with cell surface proteins (e.g., specific receptors, etc.) of target cells, or an environment in which they can be introduced into the interior of target cells.

[0460] The process of (i) above is the same as that described in the culture medium for promoting muscle differentiation described above. That is, the method example 2 above relates to a method of obtaining / producing the culture medium for promoting muscle differentiation described above or a composition containing the same, and then treating the same to target cells according to process (ii).

[0461] At this time, the culture medium or composition containing cell secretions / culture products produced by culturing genetically engineered cells can be processed by adding them to the culture environment of the target cells simultaneously with or separately after production.

[0462] In one specific example, in the process (ii), before treating the target cells, a material is obtained by filtering the secretion or culture of the genetically engineered cells using centrifugation, chromatography, a 2-layer system, and / or a tangential flow filtration method, and a composition containing such a filtrate can be treated to the target cells.

[0463]

[0464] For example, by separately culturing genetically engineered cells to obtain a composition containing the secretions or cultures of the cells, and then adding this to a medium for culturing target cells and culturing the composition, the target cells can be differentiated into muscle cells or muscle fibers.

[0465] At this time, the secretion or culture can also be obtained by removing cellular components and debris from the medium in which the genetically engineered cells are cultured.

[0466]

[0467] As another example, genetically engineered cells can be cultured using a device that allows the secretions or cultures of the cells to be processed into the target cells at the same time as they are produced.

[0468] At this time, by using a 2-layer system to culture genetically engineered cells in the upper layer and target cells in the lower layer, the target cells can be differentiated into muscle cells or muscle fibers.

[0469] As a specific example, a method for promoting differentiation of wild-type myoblasts into myotube cells is provided.

[0470] Genetically engineered fibroblasts containing a nucleic acid sequence of a mutated MSTN gene with a 12BP sequence deleted are cultured for about 5 to 10 days to obtain a conditioned medium for the fibroblasts;

[0471] The method may include culturing wild-type muscle satellite cells in a mixed medium in which the conditioned medium of the above-mentioned fibroblasts is mixed with a basic medium. In this case, the nucleic acid sequence of the mutated MSTN gene with a deleted 12BP sequence may be composed of the nucleotides of SEQ ID NO: 12.

[0472] In another specific example, a method for promoting differentiation of wild-type myoblasts into myotube cells is provided.

[0473] Genetically engineered myoblasts containing a nucleic acid sequence of a mutated MSTN gene with a 12BP sequence deleted are cultured for about 5 to 7 days to obtain a conditioned medium for the fibroblasts;

[0474] The method may include culturing wild-type muscle satellite cells in a mixed medium in which the conditioned medium of the above-mentioned fibroblasts is mixed with a basic medium. In this case, the nucleic acid sequence of the mutated MSTN gene with a deleted 12BP sequence may be composed of the nucleotides of SEQ ID NO: 12.

[0475]

[0476] As another example, by treating the filtered extract with a device capable of filtering the secretions or cultures of genetically engineered cells and then culturing the target cells, the target cells can be differentiated into muscle cells or muscle fibers.

[0477] At this time, the secretions or cultures of genetically engineered cells can be filtered using a tangential flow filtration method. Specifically, the secretions or cultures of genetically engineered cells can be filtered using a TFF pump system (Cytiva, Minimate EVO system).

[0478] As a specific example, a method for promoting differentiation of wild-type myoblasts into myotube cells is provided.

[0479] Genetically engineered CHO cells containing a nucleic acid sequence of a mutated MSTN gene with a 12BP sequence deleted are cultured for about 1 to 10 days to obtain a conditioned medium;

[0480] The method may include culturing wild-type muscle satellite cells in a mixed medium in which the above-mentioned conditioned medium is mixed with a basic medium. In this case, the nucleic acid sequence of the mutated MSTN gene with a deleted 12BP sequence may be a sequence encoding any one of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 29.

[0481]

[0482] Differentiation of target cells into muscle cells

[0483] In the method of the present invention, when target cells treated with secretions or cultures of genetically engineered cells are cultured, the target cells are differentiated into muscle cells by the paracrine effect of various cell secretions.

[0484] That is, when the secretion or culture of cells having a 12bp deletion mutant MSTN gene is treated and cultured in target cells (myoblasts, muscle satellite cells, etc.), the target cells differentiate well into myotube cells and further form muscle fibers well.

[0485] Culturing target cells for differentiation into muscle cells can be determined depending on the type, condition, proliferation rate, etc. of the target cells.

[0486] At this time, the culture conditions for culturing the target cells can take into consideration the culture period, culture temperature, number of cells, whether a support is used, number of cells, confluence, etc.

[0487] The target cells can be cultured using a support, optionally during culture.

[0488] For example, the support may be Matrigel, a scaffold, a microcarrier, etc.

[0489] The target cells can be cultured for a certain period of time to achieve a confluency of approximately 50% to 100%. For example, the cells can exhibit a confluency of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0490] In any example, the myoblasts may be cultured until they reach a confluency of at least about 70%. In any other example, the myoblasts may be cultured until they reach a confluency of at least about 70%.

[0491] At this time, subculture can be performed at an appropriate confluency. Cell subculture can be performed approximately 1 to 50 times.

[0492]

[0493] The number of target cells can be about 1x10^3 / well (or 1 plate) to 1x10^10 / well (or 1 plate), but is not limited thereto. As an example, myoblasts can be seeded at a number of 3x10^4 to 4x10^4 / well of 6 wells. As another example, muscle satellite cells can be seeded at a number of 3x10^4 to 4x10^4 / well of 6 wells.

[0494] The culture temperature of the target cells may be approximately 25 to 40°C. This temperature may be the incubation temperature of the device used to culture the cells. Additionally, the culture pH may be approximately 6 to 8. This pH may be controlled using carbon dioxide. For example, a cell incubator with a temperature of approximately 37°C and a carbon dioxide content of approximately 5% may be used.

[0495] Various culture conditions for culturing target cells can be changed and used depending on the purpose of the experiment.

[0496]

[0497] In this way, by using the culture medium for promoting muscle differentiation disclosed in the present application or a composition including the same, target cells can be effectively differentiated into muscle cells, for example, myotube cells, and further, muscle fibers can be effectively formed.

[0498]

[0499] 4. Use of the composition for promoting muscle differentiation

[0500] As described above, the composition for promoting muscle differentiation of the present application has the function of effectively inducing or promoting differentiation into muscle cells.

[0501] Therefore, the present invention solves the problems of the past by significantly improving the low efficiency of the conventional artificial differentiation method into muscle cells.

[0502] Accordingly, the present application can provide various industrial applications that utilize these effects.

[0503]

[0504] 4-1) Cosmetic composition

[0505] The present application may provide, as another specific example, a cosmetic composition comprising the culture medium for promoting muscle differentiation.

[0506]

[0507] Effective ingredient of the cosmetic composition of the present application

[0508] The above-described cosmetic composition for promoting muscle differentiation is characterized in that it contains, as an active ingredient, a secretion or culture of genetically engineered cells containing the nucleic acid sequence of the 12 bp deleted MSTN gene described above; or a conditioned medium containing the same.

[0509]

[0510] The above-mentioned cosmetic composition for promoting muscle differentiation may contain, based on the total weight of the cosmetic composition, a secretory substance of genetically engineered cells containing a nucleic acid sequence of a 12-bp deleted MSTN gene or a conditioned medium containing the same, in an amount of about 1 to 99 wt%. For example, the composition may contain about 10 to 80 wt%. Preferably, the composition may contain about 20 to 70 wt%.

[0511]

[0512] Additional components of a cosmetic composition for promoting muscle differentiation

[0513] In addition to the above-mentioned effective ingredient, the above-mentioned cosmetic composition may further include additional components necessary for manufacturing cosmetics.

[0514] The above additional components may be physiologically acceptable, contributing to the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient. Furthermore, the term "additional component" is used to refer to materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems, within the scope of reasonable judgment and commensurate with a reasonable benefit / risk ratio.

[0515] The above additional components may be, but are not limited to, carriers, excipients, diluents, preservatives, etc.

[0516] The above additional components may include, but are not limited to, water-based components, oil-based components, surfactants, moisturizers, thickeners, pigments, sunscreens, preservatives, fragrances, alkaline agents, and physiologically active ingredients.

[0517] The aqueous component may be, for example, purified water, ethanol, saline solution, etc.

[0518] The oily components can be, for example, vegetable oils, animal oils, synthetic oils, natural oils, waxes, fatty acids, etc.

[0519] The surfactant can be, for example, a cationic surfactant, an anionic surfactant, a nonionic surfactant, or an amphoteric surfactant.

[0520] Humectants can be, for example, glycerin, sorbitol, hyaluronate, chondroitin sulfate, etc.

[0521] The thickener may be, for example, carboxyvinyl polymer, carboxymethylcellulose, aluminum silicate, etc.

[0522] The pigment may be, for example, a lake, a tar pigment, an inorganic pigment, an organic pigment, etc.

[0523] Sunscreens can be, for example, benzophenone derivatives, cinnamic acid derivatives, para-aminobenzoic acid derivatives, etc.

[0524] Antioxidants can be, for example, vitamin E, AHA (Alpha Hydroxy Acid), BHA (Beta Hydroxy Acid), PHA (Poly Hydroxy Acid), PHA (Poly Hydroxy Acid), BHT (Butylated Hydroxy Toluene), etc.

[0525] Preservatives can be, for example, methylparaben, propylparaben, etc.

[0526] Fragrances can be, for example, vegetable fragrances, animal fragrances, synthetic fragrances, etc.

[0527] The alkaline agent may be, for example, sodium hydroxide, potassium hydroxide, triethanolamine, etc.

[0528] Physiologically active ingredients may include, for example, sulfur-free agents, collagen, placental extract, propolis, royal jelly, lanolin, retinol, vitamin B, ascorbic acid, and arbutin.

[0529]

[0530] Formulation of a cosmetic composition for promoting muscle differentiation

[0531] The above cosmetic composition can be formulated in various forms.

[0532] For example, the cosmetic composition can be formulated as a solution (lotion-type composition), a concentrated solution, a gel, an ointment, an emulsion (cream, milk), a vesicle dispersion, a powder, a dense powder, a paste, a solid, etc.

[0533] As a specific example, the cosmetic composition of the present application may be formulated in various forms, such as a facial cream, hand cream, moisturizing cream, sunscreen cream, cream powder, lotion, microemulsion, ointment, etc. In addition, the cosmetic composition of the present application may be formulated as a pressurized pack containing a propellant that can be applied in the form of a foam or spray.

[0534]

[0535] 4-2) Feed composition

[0536] The present application, as another specific example, can provide a feed composition including the culture medium for promoting muscle differentiation.

[0537] The feed composition of the present application contains essential nutrients that must be supplied for the maintenance and growth of the subject. These essential nutrients may be provided as a conventionally known feed composition or may be mixed and provided by the provider.

[0538] The essential nutrients may be carbohydrates, proteins, fats, minerals, vitamins, water, etc. Carbohydrates may be, for example, corn, wheat, oats, barley, etc. Proteins may be, for example, crude protein, chicken meal, pork meal, ostrich meat meal, soybean meal, rapeseed meal, wheat bran, rice bran, barley bran, cottonseed meal, sesame cake, brewer's grains, etc. Fats may be, for example, vegetable oil, corn DDGS, choice white grease, etc.

[0539] The feed composition of the present application is characterized in that, in addition to the known components, it further comprises a secretion or culture of genetically engineered cells containing the nucleic acid sequence of the MSTN gene with the 12 bp deletion described above; or a conditioned medium containing the same.

[0540] The feed composition of the present application acts to increase muscle mass in a subject by promoting (or inducing) muscle differentiation in muscle satellite cells and myoblasts of the subject ingesting it. In other words, it can be provided as a functional feed that helps in muscle production and / or enhancement in the subject. In this case, the subject may be, but is not limited to, cows, chickens, pigs, goats, sheep, rabbits, dogs, cats, mice, rats, etc.

[0541] The above feed composition may optionally further comprise the following additional components.

[0542] The above additional components may be physiologically acceptable, contributing to the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient or / and essential nutrients. Furthermore, the term "additional ingredients" is used to refer to materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems, within the scope of reasonable judgment and commensurate with a reasonable benefit / risk ratio.

[0543] Additional components may be, for example, antifungal agents, antioxidants, anticoagulants, emulsifiers, binders, etc.

[0544] The above feed composition can be formulated into various forms depending on the extrusion molding method, moisture content, etc. For example, it can be formulated into dry, semi-wet, wet, and powdered forms.

[0545] The feed composition according to the present invention has an excellent muscle-enhancing effect by strengthening the muscles of the subject. Therefore, even with the same feed intake per subject's body weight, it has a high muscle-enhancing effect.

[0546]

[0547] 4-3) Composition for producing muscle tissue of cultured meat

[0548] Cultured meat is a meat-like food product created by culturing animal cells. Various cells are isolated from muscle and used in cultured meat production. The most commonly used cells are muscle satellite cells, myoblasts, or fibroblasts. However, as mentioned above, the efficiency of differentiating these muscle satellite cells, myoblasts, or fibroblasts into muscle remains significantly low.

[0549] The composition for promoting muscle differentiation of the present invention overcomes these conventional problems and has the effect of very effectively differentiating muscle satellite cells, myoblasts, or fibroblasts into muscle. Therefore, the present invention can be very usefully utilized in the cultured meat industry.

[0550] In the process of muscle differentiation of conventional muscle satellite cells, it will be possible to produce muscle tissue of excellent quality by adding the composition for promoting muscle differentiation of the present invention.

[0551]

[0552] Possible implementation examples of this application

[0553] Below, examples of implementations provided in this application are listed as examples.

[0554] Therefore, the invention provided in this application should not be construed as limited to the following implementation examples. Furthermore, the brief descriptions provided with the implementation example numbers are merely for convenience in distinguishing between the embodiments and should not be construed as limitations on the invention disclosed in this application.

[0555]

[0556] Implementation Example 1: Genetically Modified Cells (1)

[0557] A genetically engineered cell containing a mutated MSTN gene sequence.

[0558]

[0559] Example 2: Genetically engineered cells (2)

[0560] In embodiment 1, the genetically engineered cell wherein the mutated MSTN gene sequence is any one of the following nucleic acid sequences:

[0561] SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0562]

[0563] Example 3: Genetically engineered cells (3)

[0564] In any one of embodiments 1 to 2, the genetically engineered cell wherein the mutated MSTN gene sequence is a nucleic acid sequence encoding any one of the following amino acid sequences:

[0565] Sequence number 13, Sequence number 19, Sequence number 24, and Sequence number 29.

[0566]

[0567] Example 4: Genetically engineered cells (4)

[0568] A genetically engineered cell according to any one of embodiments 1 to 3, wherein the genetically engineered cell comprises the mutated MSTN gene sequence in its genome.

[0569]

[0570] Example 5: Genetically engineered cells (5)

[0571] In any one of embodiments 1 to 4, the genetically engineered cell expresses a mutated MSTN protein, wherein the amino acid sequence of the mutated MSTN protein is any one of the following amino acid sequences:

[0572] Sequence number 13, Sequence number 19, Sequence number 24, and Sequence number 29.

[0573]

[0574] Example 6: Genetically engineered cells (6)

[0575] In any one of embodiments 1 to 5, the genetically engineered cell has an endogenous MSTN gene that is knocked out or knocked down.

[0576]

[0577] Example 7: Genetically engineered cells (7)

[0578] A genetically engineered cell according to any one of embodiments 1 to 6, wherein the genetically engineered cell is a mammalian cell.

[0579]

[0580] Example 8: Genetically engineered cells (8)

[0581] In any one of embodiments 1 to 7, the mammal is a genetically engineered cell selected from the group consisting of a human, a cow, a pig, a dog, a cat, a mouse, a horse, a goat, and a hamster.

[0582]

[0583] Example 9: Genetically engineered cells (9)

[0584] In any one of embodiments 1 to 8, the genetically engineered cell is any one selected from the following:

[0585] Somatic cells, stem cells, progenitor cells, muscle cells, fibroblasts, keratinocytes, mucosal cells, adipocytes, epithelial cells, vascular endothelial cells, bone marrow cells, neurons, myoblasts, and muscle satellite cells.

[0586]

[0587] Example 10: Genetically engineered cells (10)

[0588] In any one of embodiments 1 to 9, the genetically engineered cell is a small cell in which an endogenous MSTN gene in the genome has been deleted, and the endogenous MSTN gene in which the deletion has occurred comprises a nucleic acid sequence of SEQ ID NO: 4.

[0589]

[0590] Example 11: Genetically engineered cells (11)

[0591] In any one of embodiments 1 to 9, the genetically engineered cell is a small cell in which an endogenous MSTN gene in the genome has been deleted, and the deleted endogenous MSTN gene comprises a nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0592] The genetically engineered cell is a CHO cell comprising the mutated MSTN gene sequence in its genome.

[0593]

[0594] Embodiment 12: Composition for promoting muscle differentiation

[0595] A composition for promoting muscle differentiation, comprising a culture medium of any one of the genetically engineered cells of embodiments 1 to 11; a secretion medium of the genetically engineered cells; an extract of the culture medium of the genetically engineered cells; or an extract of the secretion medium of the genetically engineered cells.

[0596]

[0597] Implementation Example 13: Method for Promoting Muscle Differentiation (1)

[0598] Methods for promoting muscle differentiation, including:

[0599] Contacting the genetically engineered cell of any one of embodiments 1 to 11 with the target cell and culturing them together.

[0600]

[0601] Implementation Example 14: Method for Promoting Muscle Differentiation (2)

[0602] Methods for promoting muscle differentiation, including:

[0603] Contacting a target cell with a culture medium of any one of embodiments 1 to 11 of genetically engineered cells; a secretion medium of the genetically engineered cells; an extract of the culture medium of the genetically engineered cells; an extract of the secretion medium of the genetically engineered cells; or a composition for promoting muscle differentiation of embodiment 12.

[0604]

[0605] Implementation Example 15: Method for Promoting Muscle Differentiation (3)

[0606] Methods for promoting muscle differentiation, including:

[0607] Filtering the culture or secretion of any one of the genetically engineered cells of embodiments 1 to 11 using centrifugation, chromatography, a 2-layer system, and / or a tangential flow filtration method; and

[0608] The filtered extract is brought into contact with the target cells.

[0609]

[0610] Implementation Example 16: Method for Promoting Muscle Differentiation (4)

[0611] A method for promoting muscle differentiation, wherein the target cell is differentiated into a muscle cell in any one of embodiments 13 to 15.

[0612]

[0613] Implementation Example 17: Method for Promoting Muscle Differentiation (5)

[0614] A method for promoting muscle differentiation according to any one of embodiments 13 to 15, wherein the target cell is a mammalian cell.

[0615]

[0616] Implementation Example 18: Method for Promoting Muscle Differentiation (6)

[0617] A method for promoting muscle differentiation in embodiment 17, wherein the mammal is any one of a human, a cow, a pig, a dog, a cat, a mouse, a horse, a goat, and a hamster.

[0618]

[0619] Implementation Example 19: Method for Promoting Muscle Differentiation (7)

[0620] A method for promoting muscle differentiation according to any one of embodiments 17 to 18, wherein the target cell is a muscle cell precursor cell, a myoblast, a muscle satellite cell, or a stem cell.

[0621]

[0622] Embodiment 20: Cosmetic composition

[0623] A functional cosmetic composition comprising a culture medium of any one of the genetically engineered cells of Embodiments 1 to 11; a secretion medium of the genetically engineered cells; an extract of the culture medium of the genetically engineered cells; an extract of the secretion medium of the genetically engineered cells; or a composition for promoting muscle differentiation of Embodiment 12.

[0624]

[0625] Embodiment 21: Composition for functional feed

[0626] A culture medium of any one of the genetically engineered cells of embodiments 1 to 11; a secretion medium of the genetically engineered cells; an extract of the culture medium of the genetically engineered cells; an extract of the secretion medium of the genetically engineered cells; or a composition for promoting muscle differentiation of embodiment 12, and

[0627] A composition for functional feed containing essential nutrients.

[0628]

[0629] Implementation Example 22: Use for generating muscle tissue from cultured meat

[0630] Use of the composition for promoting muscle differentiation of embodiment 12 for generating muscle tissue of cultured meat.

[0631]

[0632]

[0633] Form for implementing the application

[0634] Hereinafter, the present application will be described in more detail through examples.

[0635] These examples are only intended to more specifically explain the present application, and it will be apparent to a person skilled in the art to which the present application pertains that the scope of the present application is not limited by these examples.

[0636]

[0637] ingredient

[0638] ● Culture vessels and materials

[0639] The following culture dishes and flasks were used to conduct these experiments:

[0640] 6 well cell culture plate (SPL, 30006).

[0641] A dish with an added transwell insert having 0.4 μm pores (Corning, Cat.# 3412).

[0642] 175 cm 2 A cell culture flask having an area of ​​(SPL, Cat.#71175).

[0643] 100mm cell culture dish (SPL, 20100).

[0644] 75T plate (SPL, 70075).

[0645] Matrigel (Corning, Cat. #356234).

[0646] Phosphate-buffered saline (PBS, Gibco, Cat. #20012050)

[0647] Penicillin / streptomycin (Gibco, Cat. #15140122).

[0648] Dulbecco's modified Eagle's medium (DMEM, Gibco, Cat. #11995065).

[0649] Fetal Bovine Serum (FBS, Gibco, Cat. #26140079).

[0650] Horse serum (HS, Gibco, Cat. #16050122).

[0651]

[0652] ● Cells

[0653] Genetically engineered muscle satellite cells and fibroblasts were obtained from the muscle tissue of a transgenic cow produced by the applicant.

[0654] Specifically, the following were collected and used from the transgenic cattle disclosed in WO 2023-0130639 (2023.09.12).

[0655] i) Muscle satellite cells obtained from bovine muscle tissue containing the nucleic acid sequence of SEQ ID NO: 12. Hereinafter referred to as muscle satellite cells containing the 12BP deleted MSTN gene (-12bp MSTN comprised myosatellite cells);

[0656] ii) Fibroblasts obtained from bovine ear tissue containing the nucleic acid sequence of SEQ ID NO: 12. Hereinafter referred to as fibroblasts containing the 12-bp deleted MSTN gene (-12bp MSTN comprised fibroblasts).

[0657] As a control, wild-type muscle satellite cells (hereinafter referred to as muscle satellite cells comprising the wild-type MSTN gene (WT-MSTN comprised myosatellite cells) or wild-type muscle satellite cells) obtained from the muscle tissue of wild-type cattle that had not been genetically modified were collected and used.

[0658]

[0659] ● Basic badge

[0660] DMEM supplemented with 100 U / ml penicillin / streptomycin and 20% fetal bovine serum (FBS).

[0661]

[0662] Manufacturing Example 1: Obtaining and Maintaining Muscle Satellite Cells Containing 12BP Deleted MSTN Gene and Wild-Type Muscle Satellite Cells

[0663] (1) Tissue sampling

[0664] Muscle satellite cells containing the 12bp deleted MSTN gene and wild-type muscle satellite cells were sampled from muscle tissues of cows with the 12bp deleted MSTN gene and wild-type cows, respectively.

[0665] Specifically, muscle tissue was sampled from the longissimus dorsi muscle or biceps femoris muscle of each cow.

[0666] Each sampled tissue was washed at least three times in PBS containing 10% povidone once and 10% penicillin / streptomycin.

[0667]

[0668] (2) Mechanical dissociation

[0669] (1) 1 cm of tissue obtained from 3 Samples were allocated to 100 mm dishes. Tissues were minced using a sterile blade (Ailee surgical blade No. 10) and transferred to 15 ml tubes (SPL, Cat. #50015). After resuspension in phosphate-buffered saline (PBS) containing 10% penicillin / streptomycin, centrifugation (1600 rpm, 3 min) and washing to remove the supernatant were repeated three times.

[0670]

[0671] (3) Enzymatic dissociation

[0672] Collagenase I (Gibco TM , Cat. #17100-017) at a concentration of 500 U / ml in HBSS (Hanks' Balanced Salt Solution; Gibco TM , Cat. #14025092) was diluted in a solution. The sample of (2) was resuspended in the diluted enzyme solution and reacted in a 37°C incubator for 1 hour, and vortexed once every 5 minutes during the reaction. Through this process, dissociated muscle tissue could be obtained.

[0673]

[0674] (4) Matrigel coating

[0675] To perform primary cell culture using the dissociated muscle tissue obtained through the processes (1) to (3), matrigel was coated on a cell culture plate as follows.

[0676] Matrigel about 8.7μg / cm 3 The solution was diluted in PBS to a concentration of 100% and spread on a cell culture plate (100 mm cell culture dish). It was placed in a 37°C incubator for 1 hour. The PBS was removed immediately before cell culture and dried for 10 minutes before use.

[0677]

[0678] (5) Tissue seeding, acquisition and maintenance of muscle satellite cells

[0679] To obtain muscle satellite cells from the obtained dissociated muscle tissue for tissue seeding, the following pre-plating and muscle satellite cell identification processes were additionally performed.

[0680] Specifically, pre-plating was performed to filter out cells other than muscle satellite cells, and the differentiation process of cells obtained from pre-plating into myotube cells was confirmed to confirm whether the cells obtained from pre-plating were muscle satellite cells.

[0681]

[0682] i) Pre-plating

[0683] After adding an enzyme solution and an equal amount of basic medium to the dissociated muscle tissue obtained in the above process (3), centrifugation (1600 rpm, 3 min) was performed and the supernatant was removed to prepare muscle tissue for seeding. The muscle tissue was resuspended in the basic medium and then spread on a Matrigel-coated plate prepared in (4).

[0684] Afterwards, when the cells proliferated from the seeded muscle tissue reached approximately 70-80% confluence in the culture dish, the cells were detached by treatment with trypsin-EDTA. The detached cells were spread on a 100 mm cell culture plate without Matrigel coating along with the basic medium and cultured in a 37°C incubator for 1 hour.

[0685] After culture, the supernatant, which was expected to contain muscle satellite cells, was cultured together with the basic medium on a Matrigel-coated plate.

[0686]

[0687] ii) Identification of muscle satellite cells

[0688] To confirm whether the cells obtained through pre-plating were actual muscle satellite cells, some cells were collected and tested to see whether differentiation into myotube cells occurred.

[0689] To this end, the obtained cells were cultured in a DMEM solution containing 2% horse serum and 100 U / ml of penicillin / streptomycin, with the medium replaced approximately every 2 to 3 days. This was observed for 4 to 10 days to confirm whether multinucleated cells, a characteristic of muscle cells, were formed well, confirming that the obtained cells were muscle satellite cells.

[0690]

[0691] iii) Maintenance of muscle satellite cells

[0692] In order to confirm the effect of the composition for promoting muscle differentiation of the present invention on target cells, the inventors of the present invention proliferated muscle satellite cells and wild-type muscle satellite cells containing the 12BP deleted MSTN gene obtained from the above process ii), but maintained them so that differentiation did not proceed, and used them in subsequent experiments.

[0693] The above-mentioned muscle satellite cells, by nature, exist in a dormant state. When cultured or otherwise stimulated to proliferate, they transform into myoblasts, which then differentiate into myotubes. Therefore, to prevent muscle satellite cells from differentiating into myotubes, confluency was prevented above 70% during culture. This prevented spontaneous differentiation into myotubes, thereby maintaining muscle satellite cells.

[0694] That is, the cells obtained from process ii) were cultured in each culture dish using basic medium, and the basic medium was replaced every 2 to 3 days, and subculture was performed to control the culture so as not to reach 70% confluency.

[0695] In the following examples, the muscle satellite cells obtained through the above process were appropriately seeded and used. Since the muscle satellite cells become myoblasts through culture / proliferation, the following examples describe muscle differentiation of myoblasts.

[0696]

[0697] Manufacturing Example 2: Obtaining and Maintaining Fibroblasts Containing 12BP Deleted MSTN Gene

[0698] (1) Tissue sampling

[0699] Fibroblasts containing the 12bp deleted MSTN gene were sampled from the ear tissue of a cow with a 12bp deleted MSTN gene.

[0700] The sampled tissue was washed three or more times in PBS containing 10% povidone once and 10% penicillin / streptomycin.

[0701]

[0702] (2) Mechanical dissociation

[0703] (1) 1 cm of tissue obtained from 3Samples were allocated to 100 mm dishes. Tissues were minced using a sterile blade (Ailee surgical blade No. 10) and transferred to 15 ml tubes (SPL, Cat. #50015). After resuspension in phosphate-buffered saline (PBS) containing 10% penicillin / streptomycin, centrifugation (1600 rpm, 3 min) and washing to remove the supernatant were repeated three times.

[0704]

[0705] (3) Enzymatic dissociation

[0706] Collagenase I (Gibco TM , Cat. #17100-017) at a concentration of 500 U / ml in HBSS (Hanks' Balanced Salt Solution; Gibco TM , Cat. #14025092) was diluted in the solution. The sample of (2) was resuspended in the diluted enzyme solution. The reaction was performed in a 37℃ incubator for 9 to 24 hours without vortexing. Through this process, dissociated ear tissue could be obtained.

[0707]

[0708] (4) Matrigel coating

[0709] In order to perform primary cell culture using the dissociated ear tissue obtained through the processes (1) to (3), matrigel was coated on a cell culture plate as follows.

[0710] Matrigel about 8.7μg / cm 3The solution was diluted in PBS to a concentration of 100% and spread on a cell culture plate (100 mm cell culture dish). It was placed in a 37°C incubator for 1 hour. The PBS was removed immediately before cell culture and dried for 10 minutes before use.

[0711]

[0712] (5) Tissue seeding, acquisition and culture of fibroblasts

[0713] The following process was performed to obtain fibroblasts by tissue seeding using the obtained dissociated ear tissue.

[0714] After adding an enzyme solution and an equal amount of basic medium to the dissociated ear tissue obtained in the process (3) above, centrifugation (1600 rpm, 3 min) was performed and the supernatant was removed to prepare ear tissue for seeding. The ear tissue for seeding was resuspended in the basic medium and then spread on the Matrigel-coated plate prepared in (4).

[0715] Afterwards, when the cells proliferated from the seeded ear tissue reached approximately 70-80% confluency, fibroblasts were isolated through trypsin-EDTA treatment and centrifugation. The isolated fibroblasts were cultured in basal medium on 100 mm cell culture plates without Matrigel coating, maintained through subculture, and used for experiments at appropriate times.

[0716]

[0717] Manufacturing Example 3: Production of CHO cells with MSTN gene knockout (KO)

[0718] (1) sgRNA design targeting the MSTN gene

[0719] To design sgRNA targeting the MSTN gene in CHO cells, the hamster MSTN gene sequence was obtained from the NCBI database.

[0720] The acquired sequences were inserted into the RGEN system's Cas-Designer, and the identified candidates were filtered by appropriate GC ratio, out-of-frame score, and mismatch (to reduce off-targets) to select four candidate sgRNA sequences. The sequence information of the selected sgRNAs, including their sequences, is shown in Table 2. The four selected sgRNAs were synthesized using the IVT gRNA synthesis kit and used in the experiments.

[0721] [Table 2]

[0722]

[0723]

[0724] (2) Introduction of sgRNA and Cas9 protein into CHO cell lines

[0725] RNPs with a ratio of 2:1 of Cas9 protein and selected sgRNA were prepared using a NEON device in CHO cells (Chinese Hamster Ovary; CCL-6) TM ) were injected into the cells and cultured for 2 days, and then T7E1 assay was performed. Mutation efficiency in cells into which each of the four types of sgRNAs was inserted was observed at a similar level (Fig. 6).

[0726]

[0727] (3) Selection and genetic analysis of single cell lines with KO of the MSTN gene

[0728] Single-cell culture was performed using CHO cells introduced with sgRNAs (2 or 3 in Table 2). Ten single cells that had been stably cultured in a 35 mm dish were selected and subjected to genomic DNA PCR (Fig. 7). In single cell #5 of Fig. 7, a faint band (the upper box in the box located at #5 in Fig. 7) and a dark band (the lower box in the box located at #5 in Fig. 7) were observed.

[0729] As a result of sequencing for each band, the dark band (the lower box in the box located at #5 in Figure 7) was confirmed to have a 277 bp deletion centered on the position targeted by sgRNA #2 in Table 2. The sequence of the deleted 277 bp is shown in SEQ ID NO: 36. The faint band (the upper box in the box located at #5 in Figure 7) was confirmed to have a 31 bp deletion and a 2 bp insertion of 5'-CC-3'. The sequence of the deleted 31 bp is shown in SEQ ID NO: 37.

[0730] #5 Single Cell was identified as a bi-allelic heterozygous KO cell for the MSTN gene, as two bands of 277 bp and 31 bp were confirmed to be deleted, respectively. #5 Single Cell has a mutated MSTN gene sequence of SEQ ID NO: 45 and a mutated MSTN gene sequence of SEQ ID NO: 46 in its genome.

[0731] As a result of performing western blot on the culture solution of the wild-type CHO cell line and the culture solution of the #5 single cell line, which were each concentrated (200X), it was confirmed that a band due to the anti-MSTN antibody was observed in the culture solution of the wild-type CHO cell line, but not in the culture solution of the #5 single cell line (Fig. 8).

[0732]

[0733] Manufacturing Example 4: Production of CHO cells containing the MSTN gene with 12BP deletion

[0734] (1) Vector construction for introduction of the 12BP deleted MSTN gene

[0735] The inventors of the present invention constructed vectors for the introduction of a 12BP deleted MSTN gene using three transposon systems (Piggybac: PB, Sleeping Beauty: SB, and Tol2). Each transposon vector (PB, SB, and Tol2) was cloned with the 12BP deleted MSTN gene linked to one of three promoters (CAG, EF1a, ceM243) and an antibiotic resistance gene (Neomycin: Neo, Blasticidin: Bla, Neomycin: Neo), and sequenced to construct vectors. Figure 9 shows the sequences contained in the three constructed vectors.

[0736] The sequence of the CAG promoter used in Manufacturing Example 4 is SEQ ID NO: 38.

[0737] The sequence of the EF1a promoter used in Manufacturing Example 4 is SEQ ID NO: 39.

[0738] The sequence of the ceM243 promoter used in Manufacturing Example 4 is SEQ ID NO: 40.

[0739] The sequence of the 12BP deleted MSTN gene used in Manufacturing Example 4 is SEQ ID NO: 17. SEQ ID NO: 17 is a nucleic acid sequence encoding the -4aa MSTN protein of bovine (SEQ ID NO: 13) condon-optimized for canine.

[0740]

[0741] (2) Vector insertion and verification

[0742] After the manufactured transposon vectors (PB, SB, Tol2) and transposase were introduced into the #5 single cell line manufactured in Manufacturing Example 3 using a NEON device, the cells in which the vectors were selected were sequentially selected using the characteristics of the antibiotic resistance genes (Puromycin, Blasticidin, Neomycin) included in the vectors (1st: PB-Puromycin, 2nd: SB-Blasticidine, 3rd: Tol2-Neomycin).

[0743] After selection, genomic DNA was extracted from the cells and genomic DNA PCR experiments were performed using primers that could identify each of the three vectors (Fig. 10A). As a result, it was confirmed that all three vectors were inserted into the cells (Fig. 10B).

[0744]

[0745] Example 1: Comparison of muscle differentiation results of myoblasts

[0746] Cells for performing Example 1 were cultured as follows.

[0747] Myoblasts and wild-type myoblasts containing the 12BP deleted MSTN gene obtained through Manufacturing Example 1 were seeded at a cell culture area of ​​1 square centimeter in a 6-well cell culture plate at a cell number of 35,000 and cultured in a basic medium.

[0748] In this experiment, the inventors observed whether myoblasts containing the mutated MSTN gene and wild-type myoblasts differentiated into myotube cells by culturing them to a confluency of 70% or more.

[0749]

[0750] The differentiation of myoblasts containing the 12BP deleted MSTN gene and wild-type myoblasts into myotubes was confirmed and is shown in Figure 3.

[0751] As a result, in wild-type myoblasts (described as wild type in Fig. 3), no myotubes or myofibers were found even after 7 days of culture, whereas in myoblasts containing the 12BP deleted MSTN gene (described as MSTN KO in Fig. 3), myotubes or myofibers were found from 4 days of culture.

[0752] That is, myoblasts containing the MSTN gene with 12BP deletion were found to have myotubes or muscle fibers from about 3 to 4 days after culture, confirming that myoblasts containing the MSTN gene with 12BP deletion undergo muscle differentiation into myotubes or muscle fibers more quickly than wild-type myoblasts.

[0753]

[0754] After confirming these results, the inventors sought to confirm the paracrine effect of muscle satellite cells containing the MSTN gene with a 12BP deletion.

[0755]

[0756] Example 2: Confirmation of paracrine effects in 12BP-deleted myoblasts.

[0757] In order to confirm whether there was a paracrine effect on the cultured product (secretion) of myoblasts containing the MSTN gene with 12BP deletion in Example 1, an experimental design was conducted as shown in Fig. 4(a).

[0758] Wild-type myoblasts were cultured on the bottom of the transwell in all groups: the control group (indicated as Blank in Fig. 4(a)), the WT co-culture group, and the MSTN KO co-culture group.

[0759] In the upper layer of the transwell of the control group (indicated as Blank in Fig. 4(a)), only the basic medium was added without culturing cells, in the upper layer of the transwell of the WT co-culture group, wild-type myoblasts were cultured, and in the upper layer of the transwell of the MSTN KO co-culture group, myoblasts containing the MSTN gene with 12BP deletion were cultured.

[0760]

[0761] To be more specific, the cells for performing Example 2 were cultured as follows.

[0762] To design as in Fig. 4(a), the control group was cultured in basal medium by seeding 40,000 cells / well of wild-type myoblasts obtained through Manufacturing Example 1 only in a 6-well plate (bottom), and 2 ml of basal medium without cells was added to the transwell insert.

[0763] The WT co-culture group was cultured with 2 ml of basal medium by seeding wild-type myoblasts obtained through Manufacturing Example 1 at a density of 40,000 cells / well in both the transwell insert (top) and the 6-well plate (bottom).

[0764] The MSTN KO co-culture group was cultured with 2 ml of basic medium by seeding 40,000 cells / well of myoblasts containing the 12BP deleted MSTN gene obtained through Manufacturing Example 1 in a transwell insert (top), and wild-type myoblasts obtained through Manufacturing Example 1 were seeded in a 6-well plate (bottom) at 40,000 cells / well and cultured with 2 ml of basic medium.

[0765]

[0766] The above-mentioned cells were cultured, and the results of observing wild-type myoblasts in the lower layer of the transwell of each group are shown in Figure 4(b).

[0767] In the control group (indicated as Blank in Fig. 4(b)), only wild-type myoblasts were observed until about day 6. That is, when no material was supplied from the upper layer of the transwell, wild-type myoblasts cultured on the lower layer of the transwell did not differentiate into myotubes.

[0768] In the WT co-culture group (indicated as +WT in Fig. 4(b)), only wild-type myoblasts were found until day 5, and some myotubes or myofibers were not found until about day 6. In other words, when secretions of wild-type myoblasts were supplied from the upper layer of the transwell, wild-type myoblasts cultured in the lower layer of the transwell began to differentiate into myotubes around day 6.

[0769] In contrast, in the MSTN KO co-culture group (indicated as +MSTN KO in Fig. 4(b)), myotubes or myofibers were found from about day 3. That is, when secretions of myoblasts containing the MSTN gene with 12BP deletion were supplied from the upper layer of the transwell, wild-type myoblasts cultured in the lower layer of the transwell began to differentiate into myotubes from about day 3, and it was confirmed that differentiation into myotubes had progressed significantly by about day 5 to 6.

[0770] Based on these results, it can be seen that various secretory substances secreted by myoblasts containing the 12BP deleted MSTN gene cultured in the upper layer of the transwell affect the microenvironment of wild-type myoblasts in the lower layer of the transwell, promoting muscle differentiation into myotubes or myofibers. In particular, because the transwell insert prevents cells from passing through the lower layer of the transwell, the lower layer of the transwell contains various secretory substances secreted by myoblasts containing the 12BP deleted MSTN gene.

[0771] That is, it can be seen that the secreted substances secreted by myoblasts containing the MSTN gene with 12BP deletion when cultured have a paracrine effect that promotes or induces muscle differentiation.

[0772]

[0773] Example 3: Confirmation of muscle differentiation promotion ability using fibroblasts containing the MSTN gene with 12BP deletion.

[0774] The present inventors sought to determine whether similar effects were observed not only in myoblasts but also in fibroblasts, another type of somatic cell, as cells having the 12BP deleted MSTN gene.

[0775] To this end, the differentiation of wild-type myoblasts was observed using a mixed medium containing the conditioned medium of fibroblasts containing the 12BP deleted MSTN gene and the basic medium.

[0776] As the basic medium used, DMEM solution containing 2% horse serum and 100 U / ml of penicillin / streptomycin was used.

[0777] And, the conditioned medium of fibroblasts containing the 12BP deleted MSTN gene was obtained through the following process.

[0778] Fibroblasts containing the 12BP deleted MSTN gene obtained through Manufacturing Example 2 were seeded at 2x10^6 cells each on three 75T plates containing 12 ml of DMEM medium containing 10% FBS and 100 U / ml of penicillin / streptomycin, and cultured. After culturing, when the cells reached 90% confluency, they were washed twice with PBS and replaced with 20 ml of DMEM medium containing 100 U / ml of penicillin / streptomycin and FBS-free, and cultured for 5 more days. When the cell confluency reached 100%, 60 ml of the culture was obtained as a conditioned medium.

[0779] And, the obtained conditioned medium was used in this experiment after removing cells, debris, etc. through the following process.

[0780] A Tangential Flow Filtration (TFF) pump system (Cytiva, Minimate EVO system) was used to remove cells, debris, etc. from the obtained conditioned medium.

[0781] A TFF pump system was installed and a 100 kDa filter (Cytiva, 100K Minimate capsule with Omega membrane, cat. no. OA100C12) was attached. The filter was washed by flowing 100 ml of DW through the pump system at a speed of 120 RPM.

[0782] The obtained conditioned medium (60 ml) and PBS (40 ml) were mixed to prepare a conditioned medium + PBS solution (100 ml). The conditioned medium + PBS solution was loaded into the sample tank of the TFF pump system. Filtering was performed at 60 RPM until it was concentrated 25-fold to 4 ml. The concentrated 4 ml was obtained in its entirety, dispensed, stored at -80°C, and used in the experiment. Afterwards, to analyze the exosomes contained in the concentrated 4 ml, the number of particles with a size of 50 nm to 200 nm was measured using a nanoparticle analyzer. The particle number measurement results showed that 200 μl contained 3.56 x 10^8 particles.

[0783] A mixed medium was prepared by mixing the conditioned medium without cell components obtained by the above method and the basic medium.

[0784] At this time, DMEM solution containing 2% horse serum and 100 U / ml of penicillin / streptomycin was used as the basic medium.

[0785] The mixed medium of the present invention was prepared by mixing 1800 ul of the above basic medium and 200 ul of a conditioned medium of fibroblasts containing the 12BP deleted MSTN gene that performed the TFF pump system.

[0786] As a control for the mixed medium of the present invention, a control mixed medium was also prepared by mixing 1800 ul of DMEM solution containing 2% horse serum and 100 U / ml of penicillin / streptomycin and 200 ul of PBS.

[0787]

[0788] Meanwhile, as target cells, wild-type myoblasts obtained from Manufacturing Example 1 were seeded at 1x10^5 cells each in two 35 mm dishes and cultured in basic medium. Afterwards, when the cell confluency reached 90-95%, they were washed with PBS and prepared.

[0789] After treating the prepared wild-type myoblasts with a control group cultured in a mixed medium (indicated as PBS in Figure 5) and a group cultured in a mixed medium of the present invention (indicated as MSTN KO in Figure 5), differentiation of the myoblasts was observed on the 1st, 3rd, 5th, and 8th days, respectively.

[0790] The results are shown in Figure 5.

[0791] When wild-type myoblasts were cultured in a control mixed medium, no myotubes or myofibers were observed in the wild-type myoblasts for up to 8 days (indicated by PBS in Figure 5).

[0792] In contrast, when wild-type myoblasts were cultured in the mixed medium of the present invention, myotubes or myofibers were observed in the wild-type myoblasts on the 8th day (indicated as MSTN KO in Fig. 5).

[0793] That is, it was confirmed that the mixed medium containing the conditioned medium of fibroblasts containing the 12BP deleted MSTN gene rapidly induced differentiation of wild-type myoblasts into myotubes or myofibers.

[0794]

[0795] Through the above examples 2 and 3, both muscle satellite cells containing the 12BP deleted MSTN gene and fibroblasts containing the 12BP deleted MSTN gene clearly confirmed the paracrine effect on the muscle differentiation promotion effect.

[0796] That is, the secreted substance secreted by cells containing the MSTN gene with 12BP deletion has an excellent effect of effectively promoting muscle differentiation, and thus can be utilized in various fields.

[0797]

[0798] Example 4: Confirmation of muscle differentiation promotion ability using CHO cells containing the MSTN gene with 12BP deletion 1

[0799] (1) Cultivation of cell lines

[0800] Genomic DNA PCR and sequencing, and RT-PCR were performed on the CHO cell line containing the 12BP deleted MSTN gene produced in Manufacturing Example 4 to confirm whether the 12BP deleted MSTN gene was introduced. As a result, it was confirmed that i) a cell line into which only the PB vector was inserted; ii) a cell line into which both the PB vector and the Tol2 vector were inserted; and iii) a cell line into which all of the PB vector, Tol2 vector, and SB vector were inserted were produced. Figures 11 and 12 show the results of confirming whether vectors were introduced into i) a cell line into which only the PB vector was inserted.

[0801] A wild-type CHO cell line or a CHO cell line containing the 12BP deleted MSTN gene produced in Manufacturing Example 4 was cultured through the following process.

[0802] In a 175T dish, 16 ml of F12 medium (hereinafter referred to as F12 medium) without phenol red (hereinafter referred to as F12 medium) containing 4 ml of FBS (20% FBS) was added, and the cell line was added at 4 x 10^6 / 1 well and cultured. After 48 hours of culture, the supernatant was discarded, washed once with PBS, and the medium was replaced with 80 ml of F12 medium (FBS X). After 5 days, 40 ml of the supernatant was harvested in two 50 ml tubes each and centrifuged (2000 rpm, 5 min, RT) to remove impurities.

[0803]

[0804] (2) Isolation of cell line culture extract

[0805] Extracts from the culture medium of a wild-type CHO cell line or a CHO cell line containing the 12BP deleted MSTN gene produced in Preparation Example 4 were separated using a TFF device (Minimate). Figure 13 shows the TFF device settings.

[0806]

[0807] The cell culture pretreatment method is as follows.

[0808] To separate floating cells, centrifugation was performed at 1650 rpm for 5 minutes.

[0809] The supernatant was separated using a 0.2 um filter. At this time, since the size of bacteria and fungi is larger than about 0.5 um, most microorganisms, including bacteria, were filtered using a 0.2 um filter to sterilize and remove fine particles.

[0810] After separation, it was extracted immediately using TFF or stored frozen at -80℃ until use. When using, it was dissolved in a 4℃ refrigerator.

[0811]

[0812] The method for separating cell culture extract using a TFF device is as follows.

[0813] The dedicated filter and TFF device were set up as shown in Fig. 13.

[0814] When installing the filter, connect the hose coming from the pump to the area marked Retention and the hose going into the sample container.

[0815] A hose was connected to a 500ml flask for waste at the location marked Vent.

[0816] When connecting the rubber tube to the pump, make sure to place it in the center and make sure there are no leaks.

[0817] 200 ml of 3rd DW was added to the sample container, and the pump rpm was increased from 50 to 100 rpm while maintaining the appropriate pressure (1 to 1.5) for washing.

[0818] When approximately 5 ml of tertiary DW remained in the sample container, the pump was stopped and the tertiary DW in the sample container was removed.

[0819] The pretreated sample was placed in a sample container with a minimum volume of 100 ml and a maximum volume of 500 ml.

[0820] Starting with the pump rpm at 30, the rpm was gradually increased or decreased while maintaining the appropriate pressure (1 to 1.5) and checking the amount of sample in the sample container.

[0821] When the desired volume was reached (usually 5 ml), the pump was stopped and PBS equivalent to four times the amount of sample added in step 7 was added to the sample container.

[0822] The pump rpm was started at 30 and gradually increased or decreased while maintaining the appropriate pressure (1 to 1.5).

[0823] When the desired volume was reached (usually 5 ml), the pump was stopped, the upper valve was closed, and the sample remaining in the tube was collected by regenerating for 10 seconds. The remaining extract in the sample container was then collected in a 15 ml tube.

[0824] 0.5N NaOH was added to the sample container in an amount equal to the amount of sample added in step 7.

[0825] Starting with 50 rpm of the pump, the RPM was gradually increased or decreased while maintaining the appropriate pressure (1.2 to 1.7).

[0826] When there was approximately 5 ml of 0.5N NaOH left in the sample container, the pump was stopped and the 0.5N NaOH in the sample container was discarded.

[0827] After washing the 500 ml Erlenmeyer flask and sample container with water, they were dried and the filter holder was cleaned.

[0828] The recovered cell culture extract was stored in a deep freezer.

[0829]

[0830] (3) Confirmation of the muscle differentiation promotion ability of the culture medium 1

[0831] Extracts of the culture medium of a wild-type CHO cell line (WT) or a CHO cell line containing the 12BP deleted MSTN gene prepared in Preparation Example 4 were treated with bovine, rat, and mouse myoblast cell lines using a TFF device.

[0832] As a result, in the sample treated with the extract of the culture medium of the wild-type CHO cell line (WT), differentiation of all three types of myoblasts into myotubes was inhibited (WT in Figure 14). On the other hand, in the sample treated with the extract of the culture medium of the CHO cell line containing the 12BP deleted MSTN gene produced in Preparation Example 4, differentiation of all three types of myoblasts into myotubes was activated (-12BP MSTN in Figure 14).

[0833]

[0834] (4) Confirmation of the muscle differentiation promotion ability of the culture medium 2

[0835] 1) Culture medium of wild-type CHO cell line (WT) (WT in Media of Fig. 15); 2) Culture medium of CHO cell line containing 12BP deleted MSTN gene prepared in Preparation Example 4 (-12BP MSTN in Media of Fig. 15); 3) Sample separated by TFF device from culture medium of wild-type CHO cell line (WT in TFF of Fig. 15); and 4) Sample separated by TFF device from culture medium of CHO cell line containing 12BP deleted MSTN gene prepared in Preparation Example 4 (-12BP MSTN in TFF of Fig. 15) were treated to C2C12 (mouse myoblast cell line) cell line. Thereafter, muscle differentiation efficacy was confirmed through ICC analysis using muscle differentiation marker (Desmin, muscle-specific intermediate filament protein, a marker for confirming differentiation and structural stability of myotubes) and nuclear marker (DAPI). The results are shown in Fig. 15. Through Fig. 15, it was confirmed that the number of myotube cells stained with desmin increased in 2) compared to 1). It was confirmed that the number of myotube cells stained with desmin increased in 4) compared to 3). It was confirmed that the number of myotube cells stained with desmin increased in 4) compared to 2). The result of analyzing the fusion index to quantify the results of Fig. 15 is the graph in Fig. 16.

[0836]

[0837] Proteins from C2C12 cell lines treated with TFF separation samples were extracted and western blot experiments were performed using muscle differentiation markers, desmin and Myh1 / 2 antibodies. As a result, it was confirmed that the expression level of muscle differentiation markers increased in the CHO cell treatment group containing -12BP MSTN (Fig. 17).

[0838]

[0839] (5) Confirmation of the muscle differentiation promotion ability of the culture medium 3

[0840] i) Samples separated from the culture medium of the wild-type CHO cell line (WT) using a TFF device (WT in Figs. 18 and 19); ii) extracts from the culture medium of the CHO cell line into which the PB vector and the Tol2 vector were inserted (CAG+EF1a in Figs. 18 and 19); and iii) extracts from the culture medium of the CHO cell line into which the PB vector, the Tol2 vector, and the SB vector were inserted (CAG+EF1a+ceM243 in Figs. 18 and 19) were treated to the C2C12 (mouse myoblast) cell line. Afterwards, the muscle differentiation efficacy was confirmed through ICC analysis using a muscle differentiation marker (Desmin, a muscle-specific intermediate filament protein, a marker that confirms the differentiation and structural stability of myotubes) and a nuclear marker (DAPI). The results are shown in Fig. 18. Through Fig. 18, it can be confirmed that the number of myotube cells stained with desmin is greater in ii) and iii) than in i). Additionally, it can be confirmed that the number of myotube cells stained with desmin increases in iii) compared to ii). The result of analyzing the fusion index to quantify the results of Fig. 18 is the graph in Fig. 19.

[0841]

[0842] Example 5: Confirmation of muscle differentiation promotion response using CHO cells containing the MSTN gene with 12BP deletion 2

[0843] A CHO cell containing a 12BP deleted MSTN gene is produced. The production method is mostly the same as the method for producing a CHO cell containing a 12BP deleted MSTN gene in Manufacturing Example 4. At this time, the difference between the production method of Example 5 and the method of Manufacturing Example 4 is that the nucleic acid sequence of the 12BP deleted MSTN gene inserted into the CHO cell is any one of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33. At this time, the CHO cell used may be one in which the endogenous MSTN gene has not been artificially manipulated, and thus the MSTN gene may not be knocked out or knocked down.

[0844] CHO cells containing the 12BP deleted MSTN gene produced in Example 5 are cultured in the same manner as in Example 4, and the obtained culture solution is extracted in the same manner as in Example 4.

[0845] Extracts of the culture medium of a wild-type CHO cell line (WT) or a CHO cell line containing the 12BP deleted MSTN gene prepared in Example 5 are treated with bovine, canine, rat, and mouse myoblast cell lines using a TFF device.

[0846] As a result, in the sample treated with the extract of the culture medium of the wild-type CHO cell line (WT), differentiation of all three types of myoblasts into myotube cells was inhibited. On the other hand, in the sample treated with the extract of the culture medium of the CHO cell line containing the 12BP deleted MSTN gene prepared in Example 5, differentiation of all four types of myoblasts into myotube cells was activated.

[0847]

[0848] Example 6: Preparation of cosmetics for promoting muscle differentiation

[0849] A functional cosmetic is manufactured using the conditioned medium of the fibroblasts described in Example 3 or the CHO cells described in Examples 4 to 5.

[0850] The above conditioned medium is one in which debris, etc. have been removed using the TFF pump system of Example 3 described above. The conditioned medium prepared in Example 3 contains secreted substances of 12BP-deleted fibroblasts, such as various growth factors, cytokines, exosomes, etc.

[0851]

[0852] In order to manufacture a functional cosmetic having a muscle differentiation promoting effect, in addition to the above-mentioned conditioned medium, an aqueous component, an oily component, a surfactant, a moisturizer, a thickener, a pigment, a UV blocker, a preservative, a fragrance, an alkaline agent, a physiologically active ingredient, etc. are added as needed.

[0853] During manufacturing, a cosmetic manufacturing method known in the art, such as a solubilization process, an emulsification process, a dispersion process, or an aerosol process, is performed.

[0854] The solubilization process is a process that dissolves substances that are not soluble in water in a transparent state by forming micelles, which are small aggregates of surfactants (solubilizers), and giving them the property of dissolving them.

[0855] Emulsification is a process in which water and oil, which do not mix, are artificially mixed using an emulsifying device and an emulsifier.

[0856] The dispersion process is a process of uniformly mixing solid particles such as pigments into a liquid.

[0857] The aerosol process is a process for making products by discharging liquid from a pressure vessel using the pressure of a gas.

[0858] After going through the solubilization process, it is manufactured into toner, essence, etc.

[0859] After going through the emulsification process, it is manufactured into creams, lotions, etc.

[0860] After going through the dispersion process, it is manufactured into foundation, lipstick, powder, etc.

[0861] After going through the aerosol process, it is manufactured into a spray, etc.

Claims

1. A method for inducing muscle differentiation (myogenesis), wherein the method is as follows: Genetically engineered cells are cultured in basal medium; and Treating the target cells to be differentiated into muscle cells with the conditioned medium obtained by culturing the genetically engineered cells above. Includes, At this time, the genetically engineered cell contains a mutated MSTN gene in its genome, and the mutated MSTN gene is a nucleic acid encoding an MSTN protein mutant in which four specific amino acids are deleted from the wild-type MSTN protein. A method wherein the MSTN protein variant is any one sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO:

29.

2. In paragraph 1, The above method, A method characterized in that it further comprises removing cellular components from a conditioned medium obtained by culturing the cells before treating the target cells.

3. In paragraph 2, A method further comprising preparing a mixed medium by mixing the basic medium after removing the above cell components.

4. In paragraph 1, A method wherein the sequence of the above-mentioned mutated MSTN gene is any one sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO:

33.

5. In paragraph 1, A method characterized in that the cell containing the above-mentioned mutated MSTN gene in its genome is a somatic cell, a progenitor cell, or a stem cell.

6. In paragraph 1, A method characterized in that the cell containing the above-mentioned mutated MSTN gene in its genome is selected from among muscle satellite cells (myosatellite cells), myoblasts, fibroblasts, and muscle cells.

7. In paragraph 1, A method characterized in that the cell containing the above-mentioned mutated MSTN gene in its genome is of bovine animal origin.

8. In paragraph 1, A method characterized in that the cell containing the above-mentioned mutated MSTN gene in its genome is a CHO cell.

9. In paragraph 1 or 2, A method characterized in that the target cell is a muscle satellite cell (myosatellite cell) or a myoblast.

10. In paragraph 1 or 2, A method characterized in that the target cell is of mammalian origin.

11. In paragraph 10, A method characterized in that the mammal is selected from among humans, cows, pigs, dogs, and cats.

12. In paragraph 1 or paragraph 3, The above basic medium is characterized in that it is selected from among DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimal Essential Medium), RPMI-1640 (Roswell Park Memorial Institute 1640), Ham's F-12 (Ham's Nutrients Mixture F12), and Opti-MEM.

13. In paragraph 12, The above basic medium further contains serum or antibiotics, The above serum is selected from among fetal bovine serum (FBS), bovine calf serum (BCS), horse serum (HS), human serum, and newborn calf serum (NCS). A method characterized in that the above antibiotic is selected from penicillin, streptomycin, and vancomycin.

14. In paragraph 1, A method characterized in that the above culture is cultured for 3 to 15 days.

15. In paragraph 1, A method wherein the above conditioned medium contains exosomes, cytokines, hormones or neurotransmitters secreted from cells.

16. In paragraph 2, A method characterized in that the removal of the above cellular components is performed by a method selected from among centrifugation, filtration, precipitation, and column use.

17. A composition for inducing muscle differentiation comprising a conditioned medium obtained by the method of paragraph 1 or 2, The composition is characterized in that it induces muscle differentiation of target cells into myotube cells and muscle fibers.

18. In paragraph 17, The above composition for inducing muscle differentiation further comprises a basic medium, At this time, the composition is characterized in that the conditional medium and the basic medium are included in a ratio of 10%:90%, 20%:80%, 25%:75%, 30%:70%, 40%:60%, 50%:50%, 60%:40%, 70%:30%, 75%:25%, 80%:20% or 90%:10%.

19. In paragraph 17, A composition characterized in that the target cell is a muscle satellite cell (myosatellite cell) or a myoblast.

20. A functional cosmetic composition comprising a composition selected from any one of claims 17 to 19.

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