Novel promoter

Abstract

The present disclosure relates to a novel promoter and a use thereof. More specifically, the present disclosure relates to a method for expressing a target gene operably linked to a novel promoter, and a vector comprising the novel promoter.

Description

New promoter

[0001] The present invention relates to a novel promoter and its use. The nucleic acid sequence of the novel promoter is characterized by being derived from the nucleic acid sequence of the MSTN promoter (myostatin promoter). The present invention also relates to a vector utilizing the novel promoter and its use.

[0002] Typically, genetic manipulation using viral vector systems has a limited capacity. This capacity may vary depending on the virus type. In particular, the most commonly used viral vector for genetic manipulation or therapy is the adeno-associated virus (AAV) vector, which has a capacity of approximately 4.7 kb. Therefore, to effectively utilize this limited capacity, it is necessary to use a small promoter.

[0003] To meet these needs, development of small-sized promoters has been carried out, and research has been continuously conducted to reduce the size by using the sequence of the wild-type promoter.

[0004] The present inventors sought to develop a novel promoter that is small in size but capable of expressing a target gene with high efficiency, using a wild-type MSTN promoter that is functionally linked to the MSTN gene (myostatin gene).

[0005] The present specification provides a sequence of a novel promoter, and further provides a nucleic acid comprising the same and a vector comprising the same.

[0006] To address the above technical challenges, the present disclosure provides a novel promoter; a nucleic acid comprising the novel promoter; and a vector comprising the novel promoter or a nucleic acid comprising the novel promoter. The inventors of the present disclosure have confirmed that a gene functionally linked to the novel promoter can be expressed.

[0007] Functionally linking a target gene to the novel promoter provided herein enables effective expression of the target gene. This is particularly useful for expressing the target gene using an expression vector.

[0008] Figure 1 is a schematic diagram of a nucleic acid sequence encoding GFP linked to the MSTN promoter (upstream 3 kb), a nucleic acid sequence encoding GFP linked to MP#1, a nucleic acid sequence encoding GFP linked to MP#2, and a nucleic acid sequence encoding GFP linked to MP#3.

[0009] Figure 2 is a graph comparing the mRNA expression levels of GFP expressed in cells after the nucleic acid sequences illustrated in Figure 1 were each inserted into the genome of the cells. At this time, All on the vertical axis means the result of linking the nucleic acid sequence encoding GFP to the MSTN promoter (upstream 3 kb). 1 on the vertical axis means linking the nucleic acid sequence encoding GFP to MP#1. 2 on the vertical axis means linking the nucleic acid sequence encoding GFP to MP#2. 3 on the vertical axis means linking the nucleic acid sequence encoding GFP to MP#3.

[0010] Figure 3 compares the fluorescence levels of GFP expressed in cells after each of the nucleic acid sequences illustrated in Figure 1 was inserted into the genome of the cells. a represents the result of linking a nucleic acid sequence encoding GFP to the MSTN promoter (upstream 3 kb). b represents the result of linking a nucleic acid sequence encoding GFP to MP#1. c represents the result of linking a nucleic acid sequence encoding GFP to MP#2. d represents the result of linking a nucleic acid sequence encoding GFP to MP#3.

[0011] Figure 4 is a schematic diagram of a nucleic acid sequence encoding GFP linked to the MSTN promoter (upstream 3 kb), a nucleic acid sequence encoding GFP linked to MP#32, a nucleic acid sequence encoding GFP linked to MP#31, and a nucleic acid sequence encoding GFP linked to MP#21.

[0012] Figure 5 is a graph comparing the mRNA expression levels of GFP expressed in cells after the nucleic acid sequences illustrated in Figure 4 were each inserted into the genome of the cells. At this time, All on the vertical axis means that the nucleic acid sequence encoding GFP was linked to the MSTN promoter (upstream 3 kb). -1 on the cell axis means that the nucleic acid sequence encoding GFP was linked to MP#32. -2 on the vertical axis means that the nucleic acid sequence encoding GFP was linked to MP#31. -3 on the vertical axis means that the nucleic acid sequence encoding GFP was linked to MP#21.

[0013] Figure 6 compares the fluorescence levels of GFP expressed in cells after each of the nucleic acid sequences illustrated in Figure 4 was inserted into the genome of the cells. Here, a refers to linking a nucleic acid sequence encoding GFP to the MSTN promoter (upstream 3 kb). b refers to linking a nucleic acid sequence encoding GFP to MP#32. c refers to linking a nucleic acid sequence encoding GFP to MP#31. d refers to linking a nucleic acid sequence encoding GFP to MP#21.

[0014] Figure 7 is a schematic diagram of a nucleic acid encoding GFP linked to the MSTN promoter (upstream 3 kb), a nucleic acid encoding GFP linked to the MSTN promoter (upstream 1.6 kb), a nucleic acid encoding GFP linked to MP#31, a nucleic acid encoding GFP linked to MP#21, and a nucleic acid encoding GFP linked to MP#1.

[0015] Figure 8 is a graph comparing the mRNA expression levels of GFP expressed in cells after the nucleic acid sequences illustrated in Figure 7 were each inserted into the genome of the cells. In this case, the graph regarding proliferating in Figure 8 is related to the expression level measured when mouse muscle cells were in the myoblast state. The graph regarding differentiated in Figure 8 is related to the expression level measured after mouse muscle cells differentiated into myocytes.

[0016] Figure 9 is a graph comparing the two graphs shown in Figure 8, in which the ratio of expression levels before and after differentiation into muscle cells is compared.

[0017] At this time, in FIGS. 8 and 9, 3 kb on the horizontal axis means that a nucleic acid encoding GFP is linked to the MSTN promoter (upstream 3 kb). 1.6 kb on the horizontal axis means that a nucleic acid encoding GFP is linked to the MSTN promoter (upstream 1.6 kb). -2 on the horizontal axis means that a nucleic acid encoding GFP is linked to MP#31. -3 on the horizontal axis means that a nucleic acid encoding GFP is linked to MP#21. 1 on the horizontal axis means that a nucleic acid encoding GFP is linked to MP#1.

[0018] Figure 10 is a comparison of the fluorescence levels of GFP expressed in cells after each nucleic acid sequence illustrated in Figure 7 was inserted into the genome of the cells. In this case, Figure 10 is a comparison of the fluorescence levels of GFP when mouse muscle cells are in the myoblast state.

[0019] Figure 11 compares the fluorescence levels of GFP expressed in cells after each nucleic acid sequence illustrated in Figure 7 was inserted into the genome of the cells. In this case, Figure 11 relates to the GFP fluorescence levels after mouse muscle cells differentiate into myocytes.

[0020] At this time, in FIGS. 10 and 11, a means that a nucleic acid encoding GFP is linked to MP#1. b means that a nucleic acid encoding GFP is linked to MP#21. c means that a nucleic acid encoding GFP is linked to MP#31. d means that a nucleic acid encoding GFP is linked to the MSTN promoter (upstream 1.6 kb). e means that a nucleic acid encoding GFP is linked to the MSTN promoter (upstream 3 kb).

[0021] Figure 12 shows the ratio of cells expressing GFP in each cell line after insertion of a nucleic acid encoding GFP linked to each of the CAG promoter, EF1a promoter, mPGK promoter, MP#1, MP#21, short MP#1, CMV enhancer MP#1, and MP#1 tail duplication into the genomes of various cell lines. Here, the horizontal axis represents the name of each cell line.

[0022] Figure 13 shows the GFP fluorescence levels in each cell line after insertion of nucleic acids encoding GFP linked to each of the CAG promoter, EF1a promoter, mPGK promoter, MP#1, MP#21, short MP#1, CMV enhancer MP#1, and MP#1 tail duplication into the genomes of various cell lines. Here, NC represents negative control. #1 represents MP#1. #2 + #1 represents MP#21. Short #1 represents short MP#1.

[0023] Figure 14 shows the GFP fluorescence level in each cell line compared to the negative control after insertion of a nucleic acid encoding GFP linked to each of the CAG promoter, EF1a promoter, mPGK promoter, MP#1, MP#21, short MP#1, CMV enhancer MP#1, and MP#1 tail duplication into the genome of various cell lines.

[0024] Figure 15 is a graph showing the values ​​shown in the graph of Figure 14 separated by each cell line.

[0025] Figure 16 is a graph comparing the values ​​shown in the graph of Figure 14 for each promoter (or new promoter candidate).

[0026] Figure 17 is a graph comparing the values ​​shown in the graph of Figure 14 for each promoter (or new promoter candidate).

[0027] Figure 18 shows the GFP fluorescence levels in the ISD0615 and C2C12 cell lines after insertion of nucleic acids encoding GFP linked to the CAG promoter, MP#1, M73, and M48, respectively, into the genomes of the cell lines. Here, NC represents a negative control. CAG represents a cell line into which the CAG promoter has been inserted. 243 represents a cell line into which MP#1 has been inserted. 73 represents a cell line into which M73 has been inserted. 48 represents a cell line into which M48 has been inserted.

[0028] Figure 19 shows the level of GFP fluorescence in a bovine primary fibroblast cell line after insertion of a nucleic acid encoding GFP linked to each of the CAG promoter, MP#1, M73, M50TSS, M100TSS, M100F, M150, M50F, and M50B into the genome of the cell line. Here, NC means negative control. CAG means a cell line into which the CAG promoter has been inserted. 243 means a cell line into which MP#1 has been inserted. 73 means a cell line into which M73 has been inserted. 50F means a cell line into which M50F has been inserted. 50B means a cell line into which M50B has been inserted. 50TSS means a cell line into which M50TSS has been inserted. 100F means a cell line into which M100F has been inserted. 100TSS means a cell line into which M100TSS has been inserted. 150 refers to the cell line into which M150 has been inserted.

[0029] Figure 20 is a graph showing the GFP fluorescence levels observed in cell lines derived from three bovine individuals after inserting nucleic acids linked to GFP encoding nucleic acids in MP#1, M73, M50TSS, M100TSS, M100F, M150, M50F, and M50B into the genomes of bovine primary fibroblast cell lines derived from each bovine individual, converted to values. Here, 243 refers to the cell line into which MP#1 has been inserted. 73 refers to the cell line into which M73 has been inserted. 50F refers to the cell line into which M50F has been inserted. 50B refers to the cell line into which M50B has been inserted. 50TSS refers to the cell line into which M50TSS has been inserted. 100F refers to the cell line into which M100F has been inserted. 100TSS refers to the cell line into which M100TSS has been inserted. 150 refers to the cell line into which M150 has been inserted.

[0030] Figure 21 shows the GFP fluorescence level in the cell line after insertion of nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the ISD0615 cell line.

[0031] Figure 22 shows the GFP fluorescence level in the cell line after insertion of nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the MDBK cell line.

[0032] Figure 23 shows the GFP fluorescence levels in cell lines after insertion of nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of a Bovine Myoblast cell line.

[0033] Figure 24 shows the GFP fluorescence level in a cell line after insertion of a nucleic acid encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of a 4T1 cell line.

[0034] Figure 25 shows the GFP fluorescence level in a cell line after insertion of a nucleic acid encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the C2C12 cell line.

[0035] Figure 26 shows the GFP fluorescence level in a cell line after insertion of a nucleic acid encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the 3T3-L1 cell line.

[0036] Figure 27 shows the GFP fluorescence level in the cell line after insertion of nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the HC11 cell line.

[0037] Figure 28 shows the GFP fluorescence levels in cell lines after insertion of nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the Hela cell line.

[0038] Figure 29 shows the GFP fluorescence level in a cell line after insertion of a nucleic acid encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of a human myoblast cell line.

[0039] Figure 30 shows the GFP fluorescence level in a cell line after insertion of a nucleic acid encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 into the genome of the HEK293T cell line.

[0040] In Figures 21 to 30, 243 means a cell line into which MP#1 is inserted, 150 means a cell line into which M150 is inserted, 100F means a cell line into which M100F is inserted, 100TSS means a cell line into which M100TSS is inserted, and 50TSS means a cell line into which M50TSS is inserted.

[0041] Figure 31 is a graph showing the ratio of cells expressing GFP in each cell line confirmed in Figures 21 to 30 converted into a value. At this time, the graph of Figure 31 is separated by the sequence inserted in each cell line (MP#1, M50TSS, M100TSS, M100F, and M150), and it is about comparing the ratio of cells expressing GFP in each cell line to the ratio of cells expressing GFP in the same type of cell line into which MP#1 has been inserted. For example, based on the ratio of cells expressing GFP in the 4T1 cell line into which MP#1 has been inserted, the ratio of cells expressing GFP in the 4T1 cell line into which M50TSS, M100TSS, M100F, and M150 have been inserted is calculated. That is, the ratio value of cells expressing GFP in the 4T1 cell line into which MP#1 is inserted (the second bar value from the left of the 243 indicator in Figure 31) is '1', and the ratio value of cells expressing GFP in the 4T1 cell line into which M150 is inserted (the second bar value from the left of the 150 indicator in Figure 31) is about '0.65'.

[0042] Figure 32 is a graph showing the GFP fluorescence levels confirmed in Figures 21 to 30 converted into values. At this time, the graph of Figure 31 is separated by the sequence inserted into each cell line (MP#1, M50TSS, M100TSS, M100F, and M150), and the GFP fluorescence level in each cell line is compared to the fluorescence level in the same type of cell line into which MP#1 was inserted. For example, the fluorescence values ​​in the 4T1 cell line into which M50TSS, M100TSS, M100F, and M150 were inserted are calculated based on the fluorescence value in the 4T1 cell line into which MP#1 was inserted. That is, the fluorescence value in the 4T1 cell line into which MP#1 was inserted (the second bar value from the left of the 243 indicator in Figure 32) is '1', and the fluorescence value in the 4T1 cell line into which M150 was inserted (the second bar value from the left of the 150 indicator in Figure 32) is about '0.5'.

[0043] In FIGS. 31 and 32, 243 refers to a cell line into which MP#1 is inserted, 150 refers to a cell line into which M150 is inserted, 100F refers to a cell line into which M100F is inserted, 100TSS refers to a cell line into which M100TSS is inserted, and 50TSS refers to a cell line into which M50TSS is inserted. Also, in FIGS. 31 and 32, 3t3 refers to the 3T3-L1 cell line, 4T1 refers to the 4T1 cell line, BoMyo refers to the bovine myoblast cell line, C2C12 refers to the C2C12 cell line, HC11 refers to the HC11 cell line, HEK293T refers to the HEK293T cell line, Hela refers to the Hela cell line, HuMyo refers to the human myoblast cell line, ISD0615 refers to the ISD0615 cell line, and MDBK refers to the MDBK cell line.

[0044] Figure 33 shows the GFP fluorescence levels in cell lines after insertion of nucleic acids encoding GFP linked to the EF1a promoter, Hu_M242, Hu_M198, Hu_M158, Hu_M108 and Hu_M73, into the genome of the C2C12 cell line.

[0045] Figure 34 shows the GFP fluorescence levels in cell lines after insertion of nucleic acids encoding GFP linked to the EF1a promoter, Hu_M242, Hu_M198, Hu_M158, Hu_M108 and Hu_M73, into the genome of the HepG2 cell line.

[0046] Figure 35 shows the GFP fluorescence levels in cell lines after insertion of nucleic acids encoding GFP linked to the EF1a promoter, Hu_M242, Hu_M198, Hu_M158, Hu_M108 and Hu_M73, into the genome of the PC3 cell line.

[0047] In FIGS. 33 to 35, Hu_M73 refers to a cell line into which Hu_M73 has been inserted, Hu_M108 refers to a cell line into which Hu_M108 has been inserted, Hu_M158 refers to a cell line into which Hu_M158 has been inserted, Hu_M198 refers to a cell line into which Hu_M198 has been inserted, Hu_M242 refers to a cell line into which Hu_M242 has been inserted, and EF1a refers to a cell line into which the EF1a promoter has been inserted.

[0048] Figure 36 shows the GFP fluorescence levels in cell lines after insertion of a nucleic acid encoding GFP linked to each of Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, Hu_M50TSS, M58TSS, and M50TSS into the genome of a HeLa cell line.

[0049] Figure 37 shows the expression level of SpCas9 protein in cell lines after nucleic acids encoding SpCas9 protein were inserted into the genome of HeLa cell lines, respectively, of Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, Hu_M50TSS, M58TSS, and M50TSS.

[0050] In FIGS. 36 and 37, Hu_M242 refers to a cell line into which Hu_M242 has been inserted, Hu_M158 refers to a cell line into which Hu_M158 has been inserted, Hu_M108 refers to a cell line into which Hu_M108 has been inserted, Hu_MM.192 refers to a cell line into which Hu_MM.192 has been inserted, Hu_MM.108 refers to a cell line into which Hu_MM.108 has been inserted, Hu_MM.58 refers to a cell line into which Hu_MM.58 has been inserted, Hu_50TSS refers to a cell line into which Hu_M50TSS has been inserted, 58TSS refers to a cell line into which M58TSS has been inserted, and M50TSS refers to a cell line into which 50TSS has been inserted.

[0051] Figure 38 shows the regions occupied by the trimmed sequences (MP#1, sM243, M73, M48, M50B, M50F, M50TSS, M100TSS, M100F, and M150) in the bovine wild-type MSTN promoter sequence in the experimental example.

[0052] Figure 39 shows a schematic diagram of sequences inserted into the genome of the cell line in Experimental Example 6.2.2. In this case, Hu_M198, Hu_M158, Hu_M108, and Hu_M73, in which 44 bp, 84 bp, 134 bp, or 169 bp in the 5' direction were removed from Hu_M242, show the corresponding positions in the sequence of Hu_M242.

[0053] Figure 40 shows a schematic diagram of the sequences inserted into the genome of the cell line in Experimental Example 6.2.4. Here, HHu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM58, and Hu_M50TSS trimmed from Hu_M242 show the corresponding positions in the sequence of Hu_M242.

[0054] 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.

[0055]

[0056] Some embodiments of the present application provide a nucleic acid having promoter activity.

[0057] At this time, the sequence of the nucleic acid having the promoter activity includes the nucleic acid sequence of SEQ ID NO: 19, and the length of the nucleic acid having the promoter activity is 50 bp to 243 bp.

[0058] In certain embodiments, the sequence of the nucleic acid having the promoter activity may be identical to a portion of the sequence of SEQ ID NO: 7.

[0059] In certain embodiments, the sequence of the nucleic acid having the promoter activity can be any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

[0060] In certain embodiments, the nucleic acid having the promoter activity can be characterized as functioning as a promoter for a desired nucleic acid encoding a desired protein.

[0061] In a specific embodiment, when the nucleic acid functioning as the promoter is used to express the desired protein from the desired nucleic acid, the 3' end of the nucleic acid functioning as the promoter may be operably linked to the 5' end of the desired nucleic acid.

[0062]

[0063] Some embodiments of the present application provide a nucleic acid for expressing a protein of interest comprising:

[0064] A first nucleic acid having promoter activity; and

[0065] A second nucleic acid encoding the target protein.

[0066] At this time, the sequence of the first nucleic acid having the promoter activity includes the nucleic acid sequence of SEQ ID NO: 19, and the length of the first nucleic acid having the promoter activity is 50 bp to 243 bp.

[0067] The second nucleic acid encoding the target protein is a nucleic acid encoding the target protein to be expressed in the cell,

[0068] The second nucleic acid is characterized in that it is operably linked to the 3' direction of the first nucleic acid.

[0069] In a particular embodiment, the sequence of the first nucleic acid having the promoter activity may be identical to a portion of the sequence of SEQ ID NO: 7.

[0070] In a specific embodiment, the sequence of the first nucleic acid having the promoter activity is any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

[0071]

[0072] Some embodiments of the present application provide methods for expressing a protein of interest in a cell.

[0073] At this time, the method includes a step of treating the vector to a target cell,

[0074] The above vector comprises a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein,

[0075] The sequence of the first nucleic acid having the above promoter activity includes the nucleic acid sequence of SEQ ID NO: 19, and the length of the first nucleic acid having the above promoter activity is 50 bp to 243 bp,

[0076] The second nucleic acid encoding the target protein is a nucleic acid encoding the target protein to be expressed in the cell,

[0077] The second nucleic acid is characterized in that it is operably linked to the 3' direction of the first nucleic acid.

[0078] In a particular embodiment, the sequence of the first nucleic acid having the promoter activity may be identical to a portion of the sequence of SEQ ID NO: 7.

[0079] In a particular embodiment, the sequence of the first nucleic acid having the promoter activity may be any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

[0080] In certain embodiments, the vector may be characterized as being a viral vector or a non-viral vector.

[0081] In a specific embodiment, the viral vector may be characterized as being one selected from the group consisting of a retroviral (retrovirus) vector, a lentiviral (lentivirus) vector, an adenoviral (adenovirus) vector, an adeno-associated viral (adeno-associated virus; AAV) vector, a vaccinia viral (vaccinia virus) vector, a poxviral (poxvirus) vector, and a herpes simplex viral (herpes simplex virus) vector.

[0082] In certain embodiments, the non-viral vector may be characterized as being a plasmid, phage, naked DNA, DNA complex, mRNA (transcript), or PCR amplicon.

[0083] In certain embodiments, the step of treating the target cell with the vector may be characterized as being performed in vitro, in vivo, or ex vivo.

[0084] 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.

[0085] Definition of terms

[0086] The definitions of terms used in this specification are as follows.

[0087] about

[0088] The term “about” as used herein means approximately to the extent of a quantity, and means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0089] nucleic acid

[0090] As used herein, the term "nucleic acid" refers to a region within a molecule or the entire molecule composed of DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA (double-stranded or single-stranded). Nucleic acid refers to a collection of nucleotides (a region within a molecule or the entire molecule). The terms nucleic acid or nucleic acid region may be used to refer to a region within a molecule. The terms nucleic acid or nucleic acid molecule may be used to refer to the entire molecule. The term "nucleic acid" should be appropriately interpreted according to the context, and the contents of each context including the description of the term "nucleic acid" will help one of ordinary skill in the art understand the meaning of the term nucleic acid.

[0091] upstream and downstream

[0092] The terms "upstream" and "downstream" as used herein are relative terms defining the linear positions of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) that are oriented in the 5' to 3' direction. The nucleic acid molecule may be DNA (double- or single-stranded), RNA (double- or single-stranded), or a hybrid of DNA and RNA.

[0093] For example, when it is described as "the first element is upstream of the second element in the nucleic acid molecule," it means that the first element is directly or indirectly connected to the 5' end of the second element. Alternatively, regardless of the two-dimensional or three-dimensional structure of the nucleic acid, it means that the first element is located closer to the 5' end of the second element than to the 3' end. For example, if a promoter is directly or indirectly connected to the 5' end of a gene of interest, the promoter can be described as being located upstream of the gene of interest.

[0094] As another example, when it is described as "the first element is downstream of the second element in the nucleic acid molecule," it means that the first element is directly or indirectly connected to the 3' end of the second element. Alternatively, regardless of the two-dimensional or three-dimensional structure of the nucleic acid, it means that the first element is located closer to the 3' end of the second element than the 5' end. For example, if a promoter is directly or indirectly connected to the 3' end of a gene of interest, the promoter can be described as being located downstream of the gene of interest.

[0095] operably linked

[0096] The term "operably linked" as used herein refers to any of a variety of possible arrangements of nucleic acid sequence elements such that each nucleic acid sequence element can perform its own function and each nucleic acid sequence element can interact with each other. Examples of nucleic acid sequence elements include, but are not limited to, a promoter, a polyadenine sequence, an intron, an exon, a gene of interest (a nucleic acid encoding a target gene or a protein of interest), and the like. The nucleic acid sequence elements are operably linked to control the presence or absence of expression or the level of expression of the gene of interest.

[0097] "connected" or "connected"

[0098] The terms "connected" or "linked" as used herein mean that two or more elements within a single conceptual structure are directly or indirectly connected (e.g., via another element such as a linker), and does not imply that no other additional elements may be present between the two or more elements. For example, a description such as "element B connected to element A" is intended to encompass, but is not limited to, both instances where one or more other elements are present between element A and element B (i.e., element A is connected to element B via one or more other elements) and instances where one or more other elements are not present between element A and element B (i.e., element A and element B are directly connected).

[0099] expression

[0100] As used herein, the term "expression" encompasses the production of a product (e.g., RNA, including pre-mRNA and mature-mRNA, peptides, or proteins) from genetic information (e.g., genomic DNA and a gene of interest, etc.). For example, as used herein, "expression of a gene of interest" may mean "transcription of the gene of interest into mRNA" and / or "translation of mRNA associated with the gene of interest." For example, when used herein as "regulating the expression of a gene of interest," the term "regulating the expression of a gene of interest" may be interpreted as "regulating the transcription of the gene of interest" and / or "producing a protein of interest from the gene of interest."

[0101] Sequence identity

[0102] The term "sequence identity" as used herein refers to the degree of similarity between two or more nucleotide sequences. For example, the term "sequence identity" is used with terms referring to a reference sequence and terms indicating a ratio (e.g., a percentage). For example, the term "sequence identity" can be used to describe a sequence that is similar or substantially identical to a reference nucleotide sequence. When described as "a sequence having at least 90% sequence identity to sequence A," the reference sequence is sequence A. For example, the percentage of sequence identity can be calculated by aligning a reference sequence with a sequence being the subject of the percent sequence identity measurement, and the percentage of sequence identity can be calculated including mismatches, deletions, and insertions for one or more nucleotides. The method for calculating and / or determining the percentage of sequence identity is not otherwise limited and may be calculated and / or determined through a reasonable method or algorithm available to a person skilled in the art.

[0103] Directionality of the sequence being initiated

[0104] Nucleotide sequences (e.g., DNA sequences, RNA sequences, DNA / RNA hybrid sequences) disclosed herein should be understood as being disclosed in the 5' to 3' direction, unless otherwise specified. Amino acid sequences disclosed herein should be understood as being disclosed in the N-terminal to C-terminal direction, unless otherwise specified. For sequences disclosed in an orientation other than the aforementioned orientation, the orientation for the other orientation is separately specified in the paragraph relating to the sequence.

[0105] vector

[0106] As used herein, unless otherwise specified, the term "vector" refers to any material capable of transporting genetic material into a cell. For example, a vector may be, but is not limited to, a DNA molecule containing the target genetic material, such as a nucleic acid encoding a protein of interest and / or a nucleic acid encoding an RNA of interest. The term encompasses all meanings that would be recognized by those skilled in the art and may be appropriately interpreted according to the context.

[0107] Promoter

[0108] In this application, the term "promoter" is broadly used to refer to a nucleic acid or region comprised of a set of nucleic acid sequences essential for transcription of a target gene, located near (e.g., upstream or 5' direction) the target gene, which controls the expression of the target gene (DNA sequence). A promoter may also refer to a nucleic acid or region to which a protein (e.g., transcription factor, RNA polymerase, etc.) binds to initiate transcription of a target gene near the promoter (e.g., downstream or 3' direction of the promoter). In this case, a nucleic acid that can become a promoter that controls the expression of the target gene when linked to the target gene is referred to as a nucleic acid having promoter activity.

[0109]

[0110] 1. New promoter of this disclosure

[0111] According to one aspect of the present disclosure, a novel promoter is disclosed. The novel promoter comprises a nucleic acid derived from the sequence of a known wild-type MSTN promoter. The novel promoter may be a nucleic acid that functions as a promoter for a gene (or nucleic acid) of interest. In this case, the gene of interest may refer to a gene encoding a protein of interest.

[0112] 1.1. The process of discovering new promoters

[0113] 1.1.1. Overview - The Process of Discovering New Promoters

[0114] The inventors of the present application conducted experiments to identify regions that play a key role in gene expression in the wild-type MSTN promoter to discover novel, short promoters. To identify this key region, the inventors analyzed the bovine wild-type MSTN promoter region and conducted research to shorten its length. As a result, a novel promoter containing a 50-bp core region was discovered. Considering the location of the core region in the bovine wild-type MSTN promoter, a region predicted to be the core region in wild-type MSTN promoters of other species was extracted and various experiments were conducted on the extracted region. The experimental results confirmed that the extracted region is the core region in wild-type MSTN promoters of other species, and thus, a novel promoter containing the extracted region was discovered.

[0115] 1.1.2. Wild-type MSTN promoter

[0116] The wild-type MSTN promoter is a promoter that is functionally linked to the MSTN gene (a gene encoding myostatin protein). The wild-type MSTN promoter is a promoter that has high activity specifically in muscle cells. Here, the wild-type MSTN promoter being a promoter that has high activity specifically in muscle cells means that a gene (e.g., the MSTN gene) that is functionally linked to the wild-type MSTN promoter is transcribed at a higher level in muscle cells (or tissues) than in other cells (or tissues).

[0117] In one example, the wild-type MSTN promoter may be derived from a bovine animal and is functionally linked to the MSTN gene in a sequence on the bovine genome. In one specific example, the sequence of the bovine wild-type MSTN promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

[0118] 1.1.3. Discovering a New Promoter (MP#1)

[0119] In one embodiment of the present disclosure, a novel promoter (MP#1) of 243 bp in length is disclosed.

[0120] The above 243 bp long novel promoter (MP#1) was derived from a sequence in the region adjacent to the coding sequence (CDS) encoding the MSTN protein among the sequences of the wild-type MSTN promoter in the bovine genome.

[0121] The present inventors divided the sequence from the start point (5'-AUG-3') of the coding sequence (CDS) encoding the MSTN protein in the bovine wild-type MSTN promoter sequence to a point approximately 1.6 kb upstream (in the 5' direction) into three regions. For convenience of explanation below, each of the three divided regions may be referred to as MP#1, MP#2, and MP#3 in descending order from the CDS. The nucleic acid sequence of MP#1 is SEQ ID NO: 7. The nucleic acid sequence of MP#2 is SEQ ID NO: 8. The nucleic acid sequence of MP#3 is SEQ ID NO: 9.

[0122] Experiments were conducted to determine the promoter activity of MP#1, MP#2, and MP#3, or the promoter activity when two of the three regions are linked. The inventors of the present application identified MP#1 as the core region based on the experimental results. The selection of MP#1 as the core region was derived from the following experimental results:

[0123] 1) Through Experimental Example 2, it was confirmed that the expression level of the gene linked to MP#1 was higher than the expression level of the gene linked to MP#2 or MP#3.

[0124] 2) Through Experimental Example 2, when comparing the cases where MP#1 and MP#2 were connected, the cases where MP#1 and MP#3 were connected, and the cases where MP#2 and MP#3 were connected, it was confirmed that the gene expression level was the lowest when MP#2 and MP#3 were connected. In other words, it was confirmed that the gene expression level was the lowest when MP#1 was excluded.

[0125] 3) Through Experimental Example 2, it was confirmed that the expression level of the gene linked to MP#1 was higher than the expression level of the gene linked to the wild-type MSTN promoter (a promoter of approximately 1.6 kb in length or a promoter of approximately 3 kb in length).

[0126] Through the above experimental results, the inventors of the present application deduced that MP#1 is a region that plays a key role in gene expression in the wild-type MSTN promoter, and completed a novel promoter (MP#1) of 243 bp in length.

[0127] 1.1.4. Further study on the MP#1 promoter

[0128] The inventors of the present application performed additional trimming of MP#1 to select a region that plays a key role in gene expression among the sequences of MP#1 in order to further develop a promoter of a shorter length. In particular, since a promoter with a length of approximately 170 bp or less is required to produce an adeno-associated viral (AAV) vector capable of expressing both a nucleic acid encoding the SpCas9 protein and a nucleic acid encoding a guide RNA, trimming was performed to exclude 73 bp in the 3' direction of MP#1. For convenience of explanation, the remaining 170 bp nucleic acid sequence after excluding 73 bp in the 3' direction of MP#1 may be referred to as 'sM243' (see sM243 in FIG. 38). The nucleic acid sequence of sM243 is SEQ ID NO: 15.

[0129] Experimental Example 3 shows that the expression level of the gene linked to sM243 is significantly lower than the expression level of the gene linked to MP#1. Based on the experimental results for sM243, the inventors of the present application determined that sM243 does not contain a region that plays a key role in gene expression among the sequences of MP#1.

[0130] The inventors of the present application confirmed that 170 bp in the 5' direction of MP#1 is not a core region, and further confirmed whether 73 bp in the 3' direction of MP#1 contains a core region. Trimming was performed to extract only 73 bp or 48 bp in the 3' direction from MP#1. For convenience of explanation, the 73 bp long nucleic acid sequence in the 5' direction of MP#1 may be referred to as 'M73' (see M73 in FIG. 38). For convenience of explanation, the 48 bp long nucleic acid sequence in the 5' direction of MP#1 may be referred to as 'M48' (see M48 in FIG. 38). The nucleic acid sequence of M73 is SEQ ID NO: 17. The nucleic acid sequence of M48 is SEQ ID NO: 18.

[0131] Experimental Example 4 shows that the expression levels of genes linked to a 73 bp nucleic acid sequence and a 48 bp nucleic acid sequence are significantly lower than the expression levels of genes linked to MP#1. The inventors of the present application determined through the experimental results that the 73 bp nucleic acid sequence and the 48 bp nucleic acid sequence do not include regions that play a key role in gene expression among the sequences of MP#1.

[0132] 1.1.5. Deriving the core region of the MP#1 promoter and discovering improved promoters.

[0133] Although MP#1 was divided into two regions centered on the region between the 170th and 171st nucleic acids from the 5' direction, it can be seen that neither of the two divided regions contained the core region.

[0134] Based on these results, the inventors of the present application predicted that the core region of MP#1 would include the boundary region of 170 bp and 73 bp in the nucleic acid sequence of MP#1, and therefore, trimmed the nucleic acid sequence of MP#1 to extract a 50 bp long nucleic acid sequence including the boundary of 170 bp and 73 bp. For convenience of explanation, the 50 bp long nucleic acid sequence including the boundary of 170 bp and 73 bp in the nucleic acid sequence of MP#1 may be referred to as 'M50TSS' (see M50TSS in FIG. 38). The nucleic acid sequence of the M50TSS is SEQ ID NO: 19.

[0135] Experimental Example 5 demonstrates that the expression level of the gene linked to the M50TSS nucleic acid sequence is similar to the expression level of the gene linked to MP#1. Therefore, the inventors of the present application have confirmed through the results of Experimental Example 5 that the M50TSS nucleic acid sequence includes a region that plays a key role in gene expression among the MP#1 sequences.

[0136] 1.1.6. Additional validation of the improved promoter.

[0137] To support the result that the nucleic acid sequence of M50TSS includes a region that plays a key role in gene expression among the sequences of MP#1, the inventors of the present application additionally conducted experiments to confirm the effects of two types of 100 bp long nucleic acid sequences including M50TSS; one type of 150 bp long nucleic acid sequence including M50TSS; and two types of 50 bp long nucleic acid sequences that do not include the 170 bp and 73 bp boundaries. For convenience of explanation, the two types of 100 bp long nucleic acid sequences including M50TSS may be referred to as 'M100TSS' and 'M100F', respectively (see M100TSS and M100F in FIG. 38). The nucleic acid sequence of the M100TSS is SEQ ID NO: 22. The nucleic acid sequence of the M100F is SEQ ID NO: 23. For convenience of explanation, a nucleic acid sequence having a length of 150 bp including M50TSS may be referred to as 'M150' (see M150 in FIG. 38). The nucleic acid sequence of M150 is SEQ ID NO: 24. For convenience of explanation, two types of nucleic acid sequences having a length of 50 bp that do not include the boundaries of 170 bp and 73 bp may be referred to as 'M50F' and 'M50B', respectively (see M50F and M50B in FIG. 38). The nucleic acid sequence of M50F is SEQ ID NO: 21. The nucleic acid sequence of M50B is SEQ ID NO: 20. The above contents are briefly summarized in Table 1 below as follows:

[0138] [Table 1]

[0139]

[0140]

[0141] Experimental Example 5 shows that the expression levels of genes linked to the nucleic acid sequences of M100TSS, M100F, and M150 are similar to the expression levels of genes linked to MP#1. On the other hand, Experimental Example 5 shows that the expression levels of genes linked to the nucleic acid sequences of M50F and M50B are significantly lower than the expression levels of genes linked to MP#1. In other words, it can be seen that the nucleic acid sequence including M50TSS has a level similar to that of MP#1, whereas the nucleic acid sequence not including M50TSS has very low promoter activity. Therefore, the inventors of the present application have further strengthened the result that the nucleic acid sequence of M50TSS includes a region that plays a key role in gene expression among the sequences of MP#1 through the results of Experimental Example 5. In addition, it was confirmed that the nucleic acid sequence including the nucleic acid sequence of M50TSS has promoter activity.

[0142] 1.1.7. Expansion of species

[0143] The nucleic acid sequences of the MSTN promoter show high sequence similarity between different species. In particular, the sequence corresponding to M50TSS, which includes the core region of the bovine wild-type MSTN promoter sequence, has been highly homologous and conserved in the wild-type MSTN promoter sequences of other evolutionary mammals. Therefore, the inventors of the present application extracted a sequence corresponding to a novel promoter discovered from the bovine wild-type MSTN promoter from the mammalian wild-type MSTN promoter sequences, and discovered a novel promoter derived from a mammal other than bovine. At this time, the nucleic acid sequences of the discovered novel promoters are nucleic acid sequences derived from the wild-type MSTN promoters of mammals such as bovine, human, canine, porcine, and equine. The sequence of the human wild-type MSTN promoter may be SEQ ID NO: 94. The sequence of the canine wild-type MSTN promoter may be SEQ ID NO: 95. The sequence of the porcine wild-type MSTN promoter may be SEQ ID NO: 96. The sequence of the wild-type MSTN promoter of the horse may be SEQ ID NO: 97.

[0144] A method for extracting a novel promoter from the wild-type MSTN promoter of mammals such as humans, canines, porcines, and equines is described in Experimental Example 6.

[0145] 1.2. New Promoter

[0146] In one aspect of the present disclosure, a promoter derived from the bovine wild-type MSTN promoter is disclosed. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 19. The promoter may be 50 bp (or 50 nt) or longer than 50 bp (or 50 nt). In some embodiments, the promoter may have a length of 50 nt to 1000 nt or 50 bp to 1000 bp. In some embodiments, the promoter may have a length of 50 nt to 500 nt or 50 bp to 500 bp. In some embodiments, the promoter may have a length of 50 nt to 300 nt or 50 bp to 300 bp. In some embodiments, the promoter may have a length of 50 nt to 243 nt or 50 bp to 243 bp. In some embodiments, the promoter may have a length of 50 nt to 242 nt or 50 bp to 242 bp. In some embodiments, the promoter may include a region of 50 bp or more including the transcription start site of the bovine wild-type MSTN promoter sequence. In this case, the transcription start site of the bovine wild-type MSTN promoter sequence means between the 169th and 170th nucleotides in the 5' direction of the nucleic acid sequence of MP#1.

[0147] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 22, 23, 24, and 25. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 22, 23, 24, and 25. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 22, 23, 24, and 25. In some embodiments, the nucleic acid sequence of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 22, 23, 24, and 25.

[0148] In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and can have a length of 50 bp to 243 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and can have a length of 50 bp to 242 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and can have a length of 50 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and can have a length of 58 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and can have a length of 100 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and may be 150 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and may be 243 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 19 and may be 50 to 243 bp in length and may have sequence identity with a portion of the nucleic acid sequence of SEQ ID NO: 7. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure is identical to a nucleic acid sequence of at least 50 bp in length contained in SEQ ID NO: 7 and may essentially comprise the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 7. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 7 or the nucleic acid sequence of SEQ ID NO: 19, wherein the nucleic acid sequence of the promoter may be identical to a portion of the sequence of SEQ ID NO: 7.

[0149] In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 7. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 19. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 22. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 23. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 24. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 25.

[0150] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90, 91, 92, and 93. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90, 91, 92, and 93. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90, 91, 92, and 93. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90, 91, 92, and 93.

[0151]

[0152] In one aspect of the present disclosure, a promoter derived from the human wild-type MSTN promoter is disclosed. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 32. The promoter may be 50 bp (or 50 nt) or longer than 50 bp (or 50 nt). In some embodiments, the promoter may have a length of 50 nt to 1000 nt or 50 bp to 1000 bp. In some embodiments, the promoter may have a length of 50 nt to 500 nt or 50 bp to 500 bp. In some embodiments, the promoter may have a length of 50 nt to 300 nt or 50 bp to 300 bp. In some embodiments, the promoter may have a length of 50 nt to 243 nt or 50 bp to 243 bp. In some embodiments, the promoter may have a length of 50 nt to 242 nt or 50 bp to 242 bp. In some embodiments, the promoter may include a region of 50 bp or more including the transcription start site of the human wild-type MSTN promoter sequence. In this case, the transcription start site of the human wild-type MSTN promoter sequence means between the 169th and 170th nucleotides in the 5' direction of the nucleic acid sequence of Hu_M242 (SEQ ID NO: 33).

[0153] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, and 40.

[0154] In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can have a length of 50 bp to 243 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can have a length of 50 bp to 242 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can have a length of 50 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can have a length of 58 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can have a length of 100 bp. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and may be 150 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and may be 242 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and may be 50 to 242 bp in length and may have sequence identity with a portion of the nucleic acid sequence of SEQ ID NO: 33. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure is identical to a nucleic acid sequence of at least 50 bp in length contained in SEQ ID NO: 33 and may essentially comprise the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 33. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 33 or the nucleic acid sequence of SEQ ID NO: 32, wherein the nucleic acid sequence of the promoter may be identical to a portion of the sequence of SEQ ID NO: 33.

[0155] In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 32. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 33. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 34. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 35. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 36. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 37. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 38. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 39. In a specific embodiment, the nucleic acid sequence of the promoter of the present disclosure may be the nucleic acid sequence of SEQ ID NO: 40.

[0156] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and 41. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and 41. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and 41. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and 41.

[0157]

[0158] In one aspect of the present disclosure, a promoter derived from a canine wild-type MSTN promoter is disclosed. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 48. The promoter may be 50 bp (or 50 nt) or longer than 50 bp (or 50 nt). In some embodiments, the promoter may have a length of 50 nt to 1000 nt or 50 bp to 1000 bp. In some embodiments, the promoter may have a length of 50 nt to 500 nt or 50 bp to 500 bp. In some embodiments, the promoter may have a length of 50 nt to 300 nt or 50 bp to 300 bp. In some embodiments, the promoter may have a length of 50 nt to 243 nt or 50 bp to 243 bp. In some embodiments, the promoter may have a length of 50 nt to 242 nt or 50 bp to 242 bp. In some embodiments, the promoter may include a region of 50 bp or more including a transcription start site of the wild-type MSTN promoter sequence of the dog. In this case, the transcription start site of the wild-type MSTN promoter sequence of the dog means between the 169th and 170th nucleotides in the 5' direction of the nucleic acid sequence of Ca_M243 (SEQ ID NO: 42).

[0159] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, and 56. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, and 56. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, and 56. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, and 56.

[0160] In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and can be from 50 bp to 243 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and can be from 50 bp to 242 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and can be from 50 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and can be from 58 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can be from 100 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and may be 150 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and may be 242 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 48 and may be 50 to 242 bp in length and may have sequence identity with a portion of the nucleic acid sequence of SEQ ID NO: 42. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure is identical to a nucleic acid sequence of at least 50 bp in length contained in SEQ ID NO: 42 and may essentially comprise the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 42. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 42 or the nucleic acid sequence of SEQ ID NO: 48, wherein the nucleic acid sequence of the promoter may be identical to a portion of the sequence of SEQ ID NO: 42.

[0161]

[0162] In one aspect of the present disclosure, a promoter derived from the wild-type MSTN promoter of porcine is disclosed. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 63. The promoter may be 50 bp (or 50 nt) or longer than 50 bp (or 50 nt). In some embodiments, the promoter may have a length of 50 nt to 1000 nt or 50 bp to 1000 bp. In some embodiments, the promoter may have a length of 50 nt to 500 nt or 50 bp to 500 bp. In some embodiments, the promoter may have a length of 50 nt to 300 nt or 50 bp to 300 bp. In some embodiments, the promoter may have a length of 50 nt to 243 nt or 50 bp to 243 bp. In some embodiments, the promoter may have a length of 50 nt to 271 nt or 50 bp to 271 bp. In some embodiments, the promoter may include a region of 50 bp or more including a transcription start site of a wild-type MSTN promoter sequence of a pig. In this case, the transcription start site of the wild-type MSTN promoter sequence of the pig means between the 169th and 170th nucleotides in the 5' direction of the nucleic acid sequence of Po_M241 (SEQ ID NO: 57).

[0163] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, and 71. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, and 71. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, and 71. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, and 71.

[0164] In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and can be from 50 bp to 243 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and can be from 50 bp to 242 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and can be from 50 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and can be from 58 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 32 and can be from 100 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and may be 150 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and may be 241 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 63 and may be 50 to 241 bp in length and may have sequence identity with a portion of the nucleic acid sequence of SEQ ID NO: 57. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure is identical to a nucleic acid sequence of at least 50 bp in length contained in SEQ ID NO: 57 and may essentially comprise the nucleic acid sequence from position 143 to position 192 of the nucleic acid sequence of SEQ ID NO: 57. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence from position 143 to position 192 of the nucleic acid sequence of SEQ ID NO: 57 or the nucleic acid sequence of SEQ ID NO: 63, wherein the nucleic acid sequence of the promoter may be identical to a portion of the sequence of SEQ ID NO: 57.

[0165]

[0166] In one aspect of the present disclosure, a promoter derived from the wild-type MSTN promoter of an equine is disclosed. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 78. The promoter may be 50 bp (or 50 nt) or longer than 50 bp (or 50 nt). In some embodiments, the promoter may have a length of 50 nt to 1000 nt or 50 bp to 1000 bp. In some embodiments, the promoter may have a length of 50 nt to 500 nt or 50 bp to 500 bp. In some embodiments, the promoter may have a length of 50 nt to 300 nt or 50 bp to 300 bp. In some embodiments, the promoter may have a length of 50 nt to 243 nt or 50 bp to 243 bp. In some embodiments, the promoter may have a length of 50 nt to 271 nt or 50 bp to 271 bp. In some embodiments, the promoter may include a region of 50 bp or more including the transcription start site of the wild-type MSTN promoter sequence of the horse. In this case, the transcription start site of the wild-type MSTN promoter sequence of the horse means between the 169th and 170th nucleotides in the 5' direction of the nucleic acid sequence of Eq_M243 (SEQ ID NO: 72).

[0167] In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, and 86. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure may comprise a sequence complementary to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, and 86. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, and 86. In some embodiments, the nucleic acid sequence of the promoter of the present disclosure can comprise a sequence complementary to a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, and 86.

[0168] In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and can be from 50 bp to 243 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and can be from 50 bp to 242 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and can be from 50 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and can be from 58 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and can be from 100 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and may be 150 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and may be 243 bp in length. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 78 and may be 50 to 241 bp in length and may have sequence identity with a portion of the nucleic acid sequence of SEQ ID NO: 72. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure is identical to a nucleic acid sequence of at least 50 bp in length contained in SEQ ID NO: 72 and may essentially comprise the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 72. In some specific embodiments, the nucleic acid sequence of the promoter of the present disclosure comprises the nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 72 or the nucleic acid sequence of SEQ ID NO: 78, wherein the nucleic acid sequence of the promoter may be identical to a portion of the sequence of SEQ ID NO: 72.

[0169] In some embodiments, the promoter may be double-stranded. In some embodiments, the promoter may be composed of DNA.

[0170] 1.3. Novel promoters containing enhancers

[0171] According to one aspect of the present disclosure, a novel promoter is disclosed that further comprises a known enhancer from the promoter disclosed in the Table of Contents <1.2. Novel Promoter> of the present application. Alternatively, according to one aspect of the present disclosure, a novel promoter is disclosed in which a known enhancer is covalently linked to the 5' end of the novel promoter disclosed in the Table of Contents <1.2. Novel Promoter> of the present application.

[0172] The enhancer of the above notice may be a CMV enhancer.

[0173] The nucleic acid sequence of the enhancer of the above-mentioned notice may include any one sequence selected from the sequence of SEQ ID NO: 4 and its complementary sequence, and a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% or more sequence identity with the sequence of SEQ ID NO: 4 and its complementary sequence.

[0174] The nucleic acid sequence of the novel promoter further comprising the known enhancer may comprise any one sequence selected from a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% sequence identity to the sequence of SEQ ID NO: 17 and a complementary sequence thereof. In a specific embodiment, the nucleic acid sequence of the novel promoter further comprising the known enhancer may be the sequence of SEQ ID NO: 17 or a complementary sequence thereof.

[0175] In some embodiments, the promoter may be double-stranded. In some embodiments, the promoter may be composed of DNA.

[0176] 1.4. Promoter Effect

[0177] The novel promoter of the present disclosure may be operably linked (or covalently linked) to a gene of interest. Alternatively, the gene of interest may be operably linked to the novel promoter of the present disclosure.

[0178] The novel promoter may be operably linked upstream or downstream of a gene of interest. In some specific embodiments, the novel promoter may be operably linked upstream of the gene of interest. In some specific embodiments, the gene of interest may be operably linked downstream of the novel promoter. The novel promoter may be operably linked to the 5' or 3' end of the gene of interest. In some specific embodiments, the novel promoter may be operably linked to the 5' end of the gene of interest. In some specific embodiments, the gene of interest may be operably linked to the 3' end of the novel promoter.

[0179] When the nucleic acid having the above promoter activity is used to express a desired protein from a desired gene (or nucleic acid), the 3' end of the nucleic acid functioning as the promoter can be operably linked to the 5' end of the desired nucleic acid.

[0180] The gene of interest refers to a gene whose transcription and / or expression is desired within a cell. In some embodiments, the gene of interest may be exogenous DNA derived from outside the wild-type target cell (e.g., an unmanipulated target cell). In other embodiments, the gene of interest may be endogenous DNA located in the genome of the wild-type target cell (e.g., an unmanipulated target cell).

[0181] The target gene is a gene encoding a target protein. In some embodiments, the target protein may be a protein not produced in a wild-type target cell (e.g., an unmanipulated target cell). In other embodiments, the target protein may be a protein produced in a wild-type target cell (e.g., an unmanipulated target cell).

[0182] A gene of interest operably linked to the novel promoter can be transcribed and / or expressed. The novel promoter can initiate or induce transcription and / or expression of the gene of interest operably linked to the novel promoter.

[0183] The novel promoter may contain a nucleic acid capable of being bound to or recognized by a transcription factor. The novel promoter may be bound to or recognized by a transcription factor.

[0184] The novel promoter may contain a nucleic acid to which RNA polymerase can bind or recognize. The novel promoter may bind to or be recognized by RNA polymerase.

[0185] 1.5. Features of New Promoters

[0186] In a cell or tissue, the gene of interest may be transcribed at a higher level (or in a greater amount of mRNA) when the gene of interest is operably linked to the novel promoter than when the gene of interest is operably linked to the wild-type MSTN promoter. In certain embodiments, in a muscle cell or muscle tissue, the gene of interest may be transcribed at a higher level (or in a greater amount of mRNA) when the gene of interest is operably linked to the novel promoter than when the gene of interest is operably linked to the wild-type MSTN promoter.

[0187] Unlike the wild-type MSTN promoter, the novel promoter described above is not a promoter with high activity specifically in muscle cells compared to other cells. That is, the transcription level (or expression level) of a target gene operably linked to the novel promoter may be higher in other types of cells than in muscle cells.

[0188] Examples of other types of cells, not just muscle cells, include fat cells, epithelial cells, etc. That is, the novel promoter does not have a high level of promoter activity specifically for muscle cells, but can have an equivalent / similar level of promoter activity in most cells and tissues.

[0189] The results of Experimental Examples 2 and 5 show that the novel promoter of the present disclosure has promoter activity in cells of various species and cells of various tissues.

[0190]

[0191] 2. Nucleic acid containing a novel promoter

[0192] 2.1. Nucleic acid containing a promoter

[0193] According to one aspect of the present disclosure, a nucleic acid having promoter activity is disclosed. The nucleic acid having promoter activity can function as a promoter for a target gene to which it is operably linked (or functionally linked). The nucleic acid may be a nucleic acid that functions as a promoter for the target gene (or nucleic acid). In this case, the target gene may be a nucleic acid encoding a target protein.

[0194] The sequence of the nucleic acid having the promoter activity of the present disclosure may be the same nucleic acid sequence as the novel promoter disclosed in the table of contents of the present application <1.2. Novel promoter> and <1.3. Novel promoter including enhancer>.

[0195] A nucleic acid having the promoter activity of the present disclosure may be operably linked (or covalently linked) to a gene of interest. Alternatively, the gene of interest may be operably linked to a nucleic acid having the promoter activity of the present disclosure.

[0196] The nucleic acid having the promoter activity can be operably linked upstream or downstream of the gene of interest. In some specific embodiments, the nucleic acid having the promoter activity can be operably linked upstream of the gene of interest. In some specific embodiments, the gene of interest can be operably linked downstream of the nucleic acid having the promoter activity. The nucleic acid having the promoter activity can be operably linked to the 5' or 3' end of the gene of interest. In some specific embodiments, the nucleic acid having the promoter activity can be operably linked to the 5' end of the gene of interest. In some specific embodiments, the gene of interest can be operably linked to the 3' end of the nucleic acid having the promoter activity.

[0197] When the nucleic acid having the above promoter activity is used to express a desired protein from a desired gene (or nucleic acid), the 3' end of the nucleic acid functioning as the promoter can be operably linked to the 5' end of the desired nucleic acid.

[0198] The gene of interest refers to a gene whose transcription and / or expression is desired within a cell. In some embodiments, the gene of interest may be exogenous DNA derived from outside of a wild-type target cell (e.g., an unmanipulated target cell). In other embodiments, the gene of interest may be a sequence encoding endogenous DNA located in the genome of a wild-type target cell (e.g., an unmanipulated target cell).

[0199] The target gene is a gene encoding a target protein. In some embodiments, the target protein may be a protein not produced in a wild-type target cell (e.g., an unmanipulated target cell). In other embodiments, the target protein may be a protein produced in a wild-type target cell (e.g., an unmanipulated target cell).

[0200] The nucleic acid having the above promoter activity may contain a nucleic acid to which a transcription factor can bind or recognize. The nucleic acid having the above promoter activity may bind to or be recognized by a transcription factor.

[0201] The nucleic acid having the above promoter activity may include a nucleic acid to which RNA polymerase can bind or recognize. The nucleic acid having the above promoter activity can bind to or be recognized by RNA polymerase.

[0202] 2.2. Nucleic acid containing a promoter and a gene of interest

[0203] In one aspect of the present disclosure, a nucleic acid is disclosed, comprising a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein. In this case, the nucleic acid may be for expressing the target protein.

[0204] At this time, the first nucleic acid may be operably linked to the second nucleic acid. Alternatively, the second nucleic acid may be operably linked to the first nucleic acid. The first nucleic acid may be operably linked upstream or downstream of the second nucleic acid. In some specific embodiments, the first nucleic acid may be operably linked upstream of the second nucleic acid. The second nucleic acid may be operably linked upstream or downstream of the first nucleic acid. In some specific embodiments, the second nucleic acid may be operably linked downstream of the first nucleic acid.

[0205] The first nucleic acid comprises a novel promoter described in the table of contents <1. Novel Promoter of the Present Disclosure> of the present application or a nucleic acid having promoter activity described in the table of contents <2.1. Nucleic Acid Comprising Promoter> of the present application. In some specific embodiments, the first nucleic acid may be a novel promoter described in the table of contents <1. Novel Promoter of the Present Disclosure> of the present application or a nucleic acid having promoter activity described in the table of contents <2.1. Nucleic Acid Comprising Promoter> of the present application.

[0206] The second nucleic acid refers to a nucleic acid that is intended to be transcribed and / or expressed within a cell. In some embodiments, the second nucleic acid may be exogenous DNA derived from outside the wild-type subject cell (e.g., an unmanipulated subject cell). In other embodiments, the second nucleic acid may be endogenous DNA located in the genome of the wild-type subject cell (e.g., an unmanipulated subject cell). In some embodiments, the target protein may be a protein that is not produced in the wild-type subject cell (e.g., an unmanipulated subject cell). In other embodiments, the target protein may be a protein that is produced in the wild-type subject cell (e.g., an unmanipulated subject cell).

[0207] The first nucleic acid may contain a nucleic acid to which RNA polymerase can bind or recognize. The first nucleic acid may bind to or be recognized by RNA polymerase.

[0208] In muscle cells, the second nucleic acid may be transcribed at a higher level (or in a greater amount of mRNA) when it is operably linked to the first nucleic acid than when it is operably linked to the wild-type MSTN promoter.

[0209] The second nucleic acid operably linked to the first nucleic acid can be transcribed and / or expressed in cells other than muscle cells. Examples of cells other than muscle cells include adipocytes and epithelial cells. In other words, the first nucleic acid does not have promoter activity specifically in muscle cells, but can also have promoter activity in most cells and tissues.

[0210]

[0211] 3. Vector containing a novel promoter

[0212] 3.1. Construction of a vector containing a novel promoter

[0213] In one aspect of the present disclosure, a vector is disclosed that includes a novel promoter described in <1. Novel Promoter of the Present Disclosure> of the Table of Contents of the present application or a nucleic acid having promoter activity described in <2. Nucleic Acid Comprising Novel Promoter> of the Table of Contents of the present application. In this case, the novel promoter or the nucleic acid having promoter activity is referred to as a first nucleic acid (or first region) having promoter activity. The vector may further include a second nucleic acid (or second region) encoding a target protein.

[0214] 3.2. Structure of nucleic acids included in vectors

[0215] In some embodiments, the vector may comprise a nucleic acid comprising a first nucleic acid (or first region) having promoter activity and a second nucleic acid (or second region) encoding a target protein. In this case, the first nucleic acid may be operably linked to the second nucleic acid. In this case, the first nucleic acid and the second nucleic acid may be linked in a 5' to 3' direction or a 3' to 5' direction in that order. Alternatively, the first nucleic acid and the second nucleic acid may be linked in an upstream to downstream direction or a downstream to upstream direction in that order.

[0216] In certain embodiments, the nucleic acid contained in the vector may be double-stranded. In certain embodiments, the nucleic acid contained in the vector may be single-stranded. In certain embodiments, the nucleic acid contained in the vector may be composed of DNA.

[0217] 3.3. Additional components of nucleic acids included in the vector

[0218] The vector disclosed in one aspect of the present disclosure may optionally further comprise a regulatory / control component, wherein the regulatory / control component may be operably linked to a first nucleic acid or a second nucleic acid included in the vector.

[0219] The above regulatory / control elements may be, but are not limited to, enhancers, introns, termination signals, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), WPREs, splice acceptors, 2A sequences, and / or replication origins.

[0220] The above-mentioned control / regulation components can be arranged in an appropriate order within the vector as needed by a person skilled in the art.

[0221] 3.4. Types of vectors

[0222] The vector disclosed in one aspect of the present disclosure may be a viral vector or a non-viral vector. At this time, the viral vector may be one or more viral vectors selected from the group consisting of a retroviral vector, a lentiviral vector, an adenovirus vector, an adeno-associated viral (adeno-associated virus; AAV) vector, a vaccinia viral (vaccinia virus) vector, a poxviral (poxvirus) vector, and a herpes simplex viral (herpes simplex virus) vector. At this time, the non-viral vector may be, but is not limited to, a plasmid, a phage, naked DNA, a DNA complex, an mRNA (transcript), or a PCR amplicon. For example, the plasmid may be a pcDNA series, pSC101, pGV1106, pACYC177, ColE1, It may be selected from the group consisting of pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19.

[0223] 3.5. Embodiments in which vectors are used: Expression vectors

[0224] The vector of the present disclosure can be used as an expression vector.

[0225] The expression vector described above refers to a vector capable of expressing a desired gene within a target cell into which the vector has been treated or introduced. In this case, the desired gene is contained in the nucleic acid of the expression vector and is operably linked to a novel promoter or a nucleic acid having promoter activity of the present disclosure. In certain specific embodiments, the expression vector may be a vector comprising a nucleic acid comprising a first nucleic acid and a second nucleic acid among the vectors disclosed in the table of contents <3. Vector Comprising a Novel Promoter> of the present application.

[0226] 3.6. Embodiment in which the vector is used: Delivery of the CRISPR / Cas gene editing system

[0227] The vector of the present disclosure can be used to deliver a CRISPR / Cas gene editing system. For example, the vector can be used to deliver a nucleic acid encoding a Cas protein. In another example, the vector can be used to deliver a nucleic acid encoding a Cas protein and a nucleic acid encoding a guide RNA.

[0228] In some embodiments, the vector may comprise a nucleic acid comprising a first nucleic acid (or first region) having promoter activity and a second nucleic acid (or second region) encoding a Cas protein. The first nucleic acid may be operably linked to the second nucleic acid. The first nucleic acid and the second nucleic acid may be linked in a 5' to 3' direction or a 3' to 5' direction, or the first nucleic acid and the second nucleic acid may be linked in an upstream to downstream direction or a downstream to upstream direction.

[0229] In some embodiments, the vector may comprise a nucleic acid comprising a first nucleic acid (or first region) having promoter activity, a second nucleic acid (or second region) encoding a Cas protein, and a third nucleic acid (or third region) encoding a guide RNA. In this case, the first nucleic acid may be operably linked to the second nucleic acid, the first nucleic acid may be operably linked to the third nucleic acid, or the first nucleic acid may be operably linked to the second nucleic acid and the third nucleic acid. In this case, the first nucleic acid, the second nucleic acid, and the third nucleic acid may be linked in the order of 5' to 3' or 3' to 5'. Alternatively, the first nucleic acid, the third nucleic acid, and the second nucleic acid may be linked in the order of 5' to 3' or 3' to 5'. Alternatively, the third nucleic acid, the first nucleic acid, and the second nucleic acid may be linked in the order of 5' to 3' or 3' to 5'.

[0230] In some embodiments, the vector is an adeno-associated viral (adeno-associated virus; AAV) vector, and the length of the nucleic acid that can be delivered via the adeno-associated virus vector is about 4.7 kb.

[0231] In some embodiments, the Cas protein is selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Campylobacter jejuni, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus caldus), Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira Arthrospira platensis, Arthrospira sp., Lyngbya sp.), Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus or Acaryochloris marina. In some specific examples, the Cas protein may be SpCas9 protein (Cas9 from Streptococcus pyogenes). The sequence of the nucleic acid encoding the SpCas9 protein may be SEQ ID NO: 98.

[0232]

[0233] 4. Cells containing a novel promoter

[0234] The vector disclosed in the table of contents of this application, <3. Vectors Containing Novel Promoters>, can be used to produce artificially engineered cells. In this case, the term "artificially engineered cell" refers to a cell containing the novel promoter of the present disclosure or a nucleic acid having promoter activity. Various examples of such artificially engineered cells are disclosed below.

[0235] 4.1. Cells containing expression vectors

[0236] In one aspect of the present disclosure, an artificially engineered cell comprising an expression vector is disclosed. The expression vector refers to a vector comprising a nucleic acid comprising a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein, among the vectors described in the table of contents of the present application, <3. Vectors Comprising Novel Promoters>. The first nucleic acid contained in the expression vector can function as a promoter within the artificially engineered cell.

[0237] The above expression vector can transcribe and / or express a second nucleic acid encoding a target protein or express the target protein. Specifically, the expression vector can transcribe and / or express a second nucleic acid encoding a target protein or express the target protein in an artificially engineered cell containing the expression vector. That is, the artificially engineered cell can transcribe and / or express a second nucleic acid encoding a target protein or express the target protein through the expression vector it contains.

[0238]

[0239] 5. Method of expressing the desired gene

[0240] 5.1. Overview of Target Gene Expression Methods

[0241] In one aspect of the present disclosure, a method for expressing a target gene (a nucleic acid encoding a target protein) in a target cell is disclosed. The method may include treating or introducing a vector into the target cell. Here, the vector refers to an expression vector and includes a novel promoter or a nucleic acid having promoter activity of the present disclosure.

[0242] As a result of treating or introducing the vector into the target cell, the target protein may be expressed in the target cell. In this case, the target cell may exhibit a significant increase in expression of the target protein compared to before treating the vector.

[0243] The target cell may be a human cell or a non-human animal cell.

[0244] The target cells may be cells obtained from a human or non-human animal.

[0245] The above target cell may be a cell existing within a human or non-human animal body.

[0246] The above processing or introduction method is not particularly limited as long as it can deliver the vector into the target cell.

[0247] The treatment or introduction of the vector into the target cell may be by electroporation, a gene gun, sonication, magnetofection, a nanoparticle method, and / or a temporary cell compression or squeezing method. Alternatively, the treatment or introduction of the vector into the target cell may be by cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation, lipofection, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection, and / or nanoparticle-mediated nucleic acid delivery (see Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023), but is not limited thereto.

[0248] Treating or introducing the vector into the target cells can be performed in vitro, ex vivo, or in vivo.

[0249] 5.2. How to use expression vectors

[0250] The method for expressing the target gene of the present disclosure may be a method for expressing the target gene (an exogenous gene or a second nucleic acid of the present disclosure) within a target cell. The method may include treating or introducing an expression vector into the target cell.

[0251] At this time, the expression vector refers to a vector including a nucleic acid including a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein among the vectors described in the table of contents <3. Vector including a novel promoter> of the present application. The first nucleic acid included in the expression vector can function as a promoter in a target cell. The expression vector can transcribe and / or express an exogenous gene (the second nucleic acid of the present disclosure) or express a target protein. The expression vector can transcribe and / or express an exogenous gene (the second nucleic acid of the present disclosure) or express a target protein in a target cell.

[0252] As a result of treating or introducing the expression vector into the target cell, the target protein can be expressed in the target cell. Specifically, the target protein can be expressed as the second nucleic acid is transcribed and / or expressed from the expression vector introduced into the target cell. In this case, the target cell can exhibit a significant increase in expression of the target protein compared to before treatment with the expression vector.

[0253]

[0254] 6. Purpose of new promoters

[0255] Gene expression regulation plays a crucial role in the life sciences. Representative examples of fields requiring gene expression regulation include bioreactors and therapeutics. Various factors are involved in gene expression regulation, and among them, the promoter sequence is a crucial regulatory element that determines the initiation of transcription of a target gene. Therefore, the novel promoter or nucleic acid with promoter activity of the present disclosure can be used in bioreactors or therapeutics.

[0256] The above bioreactor is intended to have the effect of mass-producing proteins or substances required in the medical and industrial fields. Typically, the bioreactor may comprise cells capable of producing a target protein. In this case, the cells used as the bioreactor may comprise the novel promoter of the present disclosure and a nucleic acid encoding the target protein, and the nucleic acid encoding the target protein may be operably linked to the novel promoter.

[0257] The therapeutic agent may have the effect of expressing a target protein necessary for treating a specific disease in the body. In this case, the therapeutic agent may include a vector capable of expressing the target protein. Specifically, the vector disclosed in the table of contents of the present application, <3. Vector Comprising a Novel Promoter>, may be used in the therapeutic agent. Typically, the therapeutic agent may include an AAV vector comprising the novel promoter of the present disclosure and a nucleic acid encoding the target protein, wherein the nucleic acid encoding the target protein may be operably linked to the novel promoter.

[0258]

[0259] Possible embodiments of the invention

[0260] Below, we list possible embodiments of the invention provided in this specification. The following embodiments provided in this paragraph are merely illustrative of the invention. Therefore, the invention provided in this specification should not be construed as limited to these embodiments. Furthermore, the brief descriptions provided with the embodiment numbers are merely for convenience in distinguishing between the embodiments and should not be construed as limitations on the invention disclosed in this specification.

[0261] Promoter

[0262] Example 1, promoter

[0263] Promoter.

[0264] Example 2, sequences included

[0265] In Example 1, the promoter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one nucleic acid sequence selected from the group consisting of:

[0266] Sequence numbers 7, 19, 22, 23, 24, 25, 26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, and 93.

[0267] Example 3, same sequence

[0268] In any one of Examples 1 to 2, the promoter comprises any one nucleic acid sequence selected from the group consisting of: or consists of any one nucleic acid sequence selected from the group consisting of:

[0269] Sequence numbers 7, 19, 22, 23, 24, 25, 26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, and 93.

[0270] Example 4, length of promoter

[0271] In any one of Examples 1 to 3, the length of the promoter is 50bp, 51bp, 52bp, 53bp, 54bp, 55bp, 56bp, 57bp, 58bp, 59bp, 60bp, 61bp, 62bp, 63bp, 64bp, 65bp, 66bp, 67bp, 68bp, 69bp, 70bp, 71bp, 72bp, 73bp, 74bp, 75bp, 76bp, 77bp, 78bp, 79bp, 80bp, 81bp, 82bp, 83bp, 84bp, 85bp, 86bp, 87bp, 88bp, 89bp, 90bp, 91bp, 92bp, 93bp, 94bp, 95bp, 96bp, 97bp, 98bp, 99bp, 100bp, 101bp, 102bp, 103bp, 104bp, 105bp, 106bp, 107bp, 108bp, 109bp, 110bp, 111bp, 112bp, 113bp, 114bp, 115bp, 116bp, 117bp, 118bp, 119bp, 120bp, 121bp, 122bp, 123bp, 124bp, 125bp, 126bp, 127bp, 128bp, 129bp, 130bp, 131bp, 132bp, 133bp, 134bp, 135bp, 136bp, 137bp, 138bp, 139bp, 140bp, 141bp, 142bp, 143bp, 144bp, 145bp, 146bp, 147bp, 148bp, 149bp, 150bp, 151bp, 152bp, 153bp, 154bp, 155bp, 156bp, 157bp, 158bp, 159bp, 160bp, 161bp, 162bp, 163bp, 164bp, 165bp, 166bp, 167bp, 168bp, 169bp, 170bp, 171bp, 172bp, 173bp, 174bp, 175bp, 176bp, 177bp, 178bp, 179bp, 180bp, 181bp, 182bp, 183bp, 184bp, 185bp, 186bp, 187bp, 188bp, 189bp, 190bp, 191bp, 192bp, 193bp,194bp, 195bp, 196bp, 197bp, 198bp, 199bp, 200bp, 201bp, 202bp, 203bp, 204bp, 205bp, 206bp, 207bp, 208bp, 209bp, 210bp, 211bp, 212bp, 213bp, 214bp, 215bp, 216bp, 217bp, 218bp, 219bp, 220bp, 221bp, 222bp, 223bp, 224bp, 225bp, 226bp, 227bp, Any one selected from among 228bp, 229bp, 230bp, 231bp, 232bp, 233bp, 234bp, 235bp, 236bp, 237bp, 238bp, 239bp, 240bp, 241bp, 242bp, and 243bp, or within a range of two selected numbers from the aforementioned numbers.

[0272] Example 5, range of promoter lengths

[0273] In any one of Examples 1 to 4, the length of the promoter is 50 bp to 243 bp or 50 bp to 242 bp.

[0274] Example 6, wild-type MSTN promoter origin of cattle

[0275] In any one of Examples 1 to 5, the promoter is derived from a wild-type MSTN promoter sequence of bovine,

[0276] The above promoter comprises a nucleic acid sequence of SEQ ID NO: 19; or comprises a nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 7,

[0277] The sequence of the above promoter is identical to a portion of the sequence of SEQ ID NO: 7.

[0278] Example 7, derived from the human wild-type MSTN promoter

[0279] In any one of Examples 1 to 5, the promoter is derived from a human wild-type MSTN promoter sequence,

[0280] The above promoter comprises a nucleic acid sequence of SEQ ID NO: 32; or comprises a nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 33,

[0281] The sequence of the above promoter is identical to a portion of the sequence of SEQ ID NO: 33.

[0282] Example 8, derived from the wild-type MSTN promoter of dogs

[0283] In any one of Examples 1 to 5, the promoter is derived from a wild-type MSTN promoter sequence of a canine,

[0284] It is identical to the nucleic acid sequence of 50 bp or more in length included in sequence number 42,

[0285] The above promoter comprises a nucleic acid sequence of SEQ ID NO: 48; or comprises a nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 42,

[0286] The sequence of the above promoter is identical to a portion of the sequence of SEQ ID NO: 42.

[0287] Example 9, derived from the wild-type MSTN promoter of pigs

[0288] In any one of Examples 1 to 5, the promoter is derived from a wild-type MSTN promoter sequence of porcine,

[0289] It is identical to the nucleic acid sequence of 50 bp or more in length included in sequence number 57,

[0290] The above promoter comprises a nucleic acid sequence of SEQ ID NO: 63; or comprises a nucleic acid sequence from position 143 to position 192 of the nucleic acid sequence of SEQ ID NO: 57,

[0291] The sequence of the above promoter is identical to a portion of the sequence of SEQ ID NO: 57.

[0292] Example 10, derived from the wild-type MSTN promoter of a horse

[0293] In any one of Examples 1 to 5, the promoter is derived from the wild-type MSTN promoter sequence of an equine,

[0294] It is identical to the nucleic acid sequence of 50 bp or more in length included in sequence number 72,

[0295] The above promoter comprises a nucleic acid sequence of SEQ ID NO: 78; or comprises a nucleic acid sequence from position 145 to position 194 of the nucleic acid sequence of SEQ ID NO: 72.

[0296] The sequence of the above promoter is identical to a portion of the sequence of SEQ ID NO: 72.

[0297] Example 11, a promoter containing an enhancer

[0298] A promoter comprising a nucleic acid sequence of any one of Examples 1 to 10; and a nucleic acid sequence of a known enhancer.

[0299] Example 12, Linkage with enhancers

[0300] In Example 11, the nucleic acid sequence of the known enhancer is linked in the 5' direction to the nucleic acid sequence of the promoter of any one of Examples 1 to 10.

[0301] Example 13, Types of Enhancers

[0302] In any one of Examples 11 to 12, the known enhancer is a CMV enhancer, and the nucleic acid sequence of the CMV enhancer comprises any one sequence selected from the sequence of SEQ ID NO: 4 and a complementary sequence thereof, and a sequence having a sequence identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% or more with the sequence of SEQ ID NO: 4 and a complementary sequence thereof.

[0303] Example 14, Effect of Promoter

[0304] In any one of Examples 1 to 13, the promoter may be operably linked (or covalently linked) to a gene of interest,

[0305] The above promoter can initiate or induce transcription and / or expression of the desired gene.

[0306] Example 15, Type of target gene

[0307] In any one of Examples 1 to 14, the gene of interest has one or more of the following characteristics:

[0308] The above target gene is a gene whose transcription and / or expression is desired within a cell;

[0309] The gene of interest is exogenous DNA derived from outside the wild-type target cell (e.g., an unmanipulated target cell);

[0310] The above target gene is an endogenous DNA located in the genome of a wild-type target cell (e.g., an unmanipulated target cell); and

[0311] The above target gene is a gene encoding a target protein.

[0312] Nucleic acid containing a promoter

[0313] Example 16, Nucleic acid containing a promoter

[0314] A nucleic acid comprising the promoter of any one of Examples 1 to 15.

[0315] Example 17, Nucleic acid having promoter activity

[0316] A nucleic acid having promoter activity, comprising a nucleic acid sequence of any one of the promoters of Examples 1 to 15; or consisting of a nucleic acid sequence of any one of the promoters of Examples 1 to 15.

[0317] Example 18, nucleic acid comprising a first nucleic acid and a second nucleic acid

[0318] A nucleic acid comprising a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein.

[0319] Example 19, first nucleic acid

[0320] In Example 18, the first nucleic acid is a nucleic acid having the promoter activity of any one of Examples 1 to 15; a nucleic acid of Example 16; or a nucleic acid having the promoter activity of Example 17.

[0321] Example 20, second nucleic acid

[0322] In any one of Examples 18 to 19, the second nucleic acid is a nucleic acid for transcription and / or expression in a cell,

[0323] The second nucleic acid is exogenous DNA derived from outside the wild-type target cell (e.g., unmanipulated target cell); or endogenous DNA located in the genome of the wild-type target cell (e.g., unmanipulated target cell).

[0324] Example 21, target protein

[0325] In any one of Examples 18 to 20, the target protein is a protein that is not produced in a wild-type target cell (e.g., an unmanipulated target cell); or a protein that is produced in a wild-type target cell (e.g., an unmanipulated target cell).

[0326] Example 22, Connection Relationship

[0327] In any one of Examples 18 to 21, the first nucleic acid is operably linked upstream (or 5' direction) of the second nucleic acid,

[0328] The above first nucleic acid can initiate or induce transcription and / or expression of the above second nucleic acid.

[0329] vector containing a promoter

[0330] Example 23, vector containing a promoter

[0331] A vector comprising a promoter of any one of Examples 1 to 15 or a nucleic acid of any one of Examples 16 to 22.

[0332] Example 24, Additional Components

[0333] In Example 23, the vector further comprises a regulatory / control element, wherein the regulatory / control element is an enhancer, an intron, a termination signal, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a WPRE, a splice acceptor, a 2A sequence, and / or an origin of replication.

[0334] Example 25, Types of Vectors

[0335] In any one of Examples 22 to 24, the vector is a viral vector or a non-viral vector.

[0336] Example 26, Types of Viral Vector

[0337] In Example 25, the viral vector is at least one viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenovirus vector, an adeno-associated viral (adeno-associated virus; AAV) vector, a vaccinia viral (vaccinia virus) vector, a poxviral (poxvirus) vector, and a herpes simplex viral (herpes simplex virus) vector.

[0338] Example 27, Types of nonviral vectors

[0339] In Example 25, the non-viral vector is a plasmid, phage, naked DNA, DNA complex, mRNA (transcript), or PCR amplicon.

[0340] The above plasmids are selected from the group consisting of pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19.

[0341] Example 28, expression vector

[0342] In any one of Examples 23 to 27, the vector is a vector for expressing a desired gene.

[0343] Example 29, Delivery of the CRISPR / Cas gene editing system

[0344] In any one of Examples 23 to 27, the vector comprises a nucleic acid encoding a Cas protein and / or a nucleic acid encoding a guide RNA,

[0345] The nucleic acid encoding the Cas protein and / or the nucleic acid encoding the guide RNA is operably linked to the promoter of any one of Examples 1 to 15 or the nucleic acid of any one of Examples 16 to 22.

[0346] Cells containing promoters

[0347] Example 30, Cells Containing a Promoter

[0348] A cell comprising a promoter of any one of Examples 1 to 15; a nucleic acid of any one of Examples 16 to 22; or a vector of any one of Examples 23 to 29.

[0349] Example 31, Cellular Effects

[0350] In Example 31, the cell can express a desired gene and / or a desired protein.

[0351] Method of expressing the desired gene

[0352] Example 32, Method for Expression of a Target Gene

[0353] A method for expressing a nucleic acid encoding a target gene or target protein within a target cell,

[0354] The method comprises treating or introducing into the target cell a vector of any one of Examples 23 to 29.

[0355] Example 33, Type of target cell

[0356] In Example 32, the target cell is a human cell, a non-human animal cell, a cell obtained from a human, a cell obtained from a non-human animal, a cell existing in a human body; or a cell existing in a non-human animal body.

[0357] Example 34, Vector Introduction Method 1

[0358] In any one of Examples 32 to 33, treating or introducing the vector into the target cell is performed in vitro, ex vivo, or in vivo.

[0359] Example 35, Vector Introduction Method 2

[0360] In any one of Examples 32 to 34, treating or introducing the vector into the target cell is performed using electroporation, a gene gun, sonication, magnetofection, a nanoparticle method, a transient cell compression or squeezing method, a cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation, lipofection, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection, and / or nanoparticle-mediated nucleic acid delivery.

[0361]

[0362]

[0363] Below, the invention provided by this specification is described in more detail through experimental examples. These experimental examples are intended solely to illustrate the subject matter disclosed by this specification, and it will be apparent to those skilled in the art that the scope of the subject matter disclosed by this specification is not limited by these experimental examples.

[0364] Experimental Example 1. Experimental Method

[0365] Experimental Example 1.1. Design of a Novel Promoter Candidate

[0366] The inventors of this application designed candidate sequences for novel promoters by trimming the sequence of the wild-type MSTN promoter. They conducted experiments to confirm the effectiveness of the designed sequences, and the results identified regions within the wild-type MSTN promoter that play a key role in gene expression. Accordingly, they designed novel promoters containing these key regions.

[0367] Information about the wild-type MSTN promoter, trimmed sequences of the wild-type MSTN promoter, known promoters, and known enhancers used in the experiment is described in Table 2 below.

[0368] [Table 2]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381] Experimental Example 1.2. Vector cloning

[0382] A nucleic acid sequence encoding green fluorescent protein (GFP), a fluorescent protein, was linked to a known promoter sequence or a portion of the MSTN promoter sequence and cloned into a transposon vector. Here, when the promoter sequence and the nucleic acid sequence encoding GFP were linked, the promoter sequence disclosed in this experimental example and the nucleic acid sequence encoding GFP were linked in the 5' to 3' direction. At this time, the promoter sequence and the nucleic acid sequence encoding GFP were directly linked without any other nucleic acid sequence between them.

[0383] The known promoters used in the experiment include the CAG promoter, the EF1a promoter, and the mPGK promoter.

[0384] Experimental Example 1.3. Cells and Cell Culture Methods Used in the Experiment

[0385] The types of cells used in the experiments are shown in Table 3. C2C12, 3T3-L1, C127:LT, MCF7, HeLa, A549, PC-3, human myoblast, and MDBK cells were purchased from ATCC. CAL-62 cells were purchased from the German Collection of Microorganisms and Cell Cultures. ISD0615 cells were produced directly by the inventors and are cells for which immortalization has been confirmed. Specific information about ISD0615 cells can be found in the paper "WooJae Choi, Eunji Kim, Soo-Young Yum, ChoongIl Lee, JiHyun Lee, JoonHo Moon, Sisitha Ramachandra, Buddika Oshadi Malaweera, JongKi Cho, Jin-Soo Kim, SeokJoong Kim & Goo Jang (2015) Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells, Prion, 9:4, 278-291, DOI: 10.1080 / 19336896.2015.1071459".

[0386] The cells used in the experiment were cultured in a 20% fetal bovine serum (FBS, gibco) mixed Dulbecco's Modified Eagle's Medium high glucose (DMEM, gibco) medium at 37°C (38°C for MDBK and SD0615) in a 5% CO2 environment.

[0387] Except for C2C12 cells, passage was performed when the culture area was 80-90% full, and C2C12 cells were passaged earlier when 60-70% full to prevent spontaneous differentiation during culture.

[0388] For differentiation of C2C12 cells, C2C12 cells were cultured under general culture conditions until 80-90% confluent, and then 2% horse serum was added and cultured for 7 days.

[0389] Bovine primary fibroblasts were isolated and cultured from bovine ear tissue. Bovine ear tissue was sampled using a biopsy punch and minced into pieces less than 1 mm in size using a sterilized blade. The minced tissue was placed in HBSS (Hank's balanced salt solution) containing 0.1% collagenase and incubated for 16–24 hours at 38°C in a 5% CO2 atmosphere. Upon completion of the incubation, an equal volume of cell culture medium was added, centrifuged at 1500–2000 rpm for 3 minutes, and the supernatant was removed. The pellet collected at the bottom was resuspended in cell culture medium and cultured in cell culture dishes (SPL, 20060) until cell growth began.

[0390] Bovine myoblasts were isolated and cultured from the Longissimus dorsi m. or Biceps femoris m. After isolation, the culture method was as follows: Povidone 10% once, penicillin & streptomycin 10% PBS (Gibco™, Cat. #20012050) washes were performed three or more times. 1 cm 3Samples were allocated to a 100 mm dish size. They were chopped using a sterile blade (Ailee surgical blade No. 10) and transferred to a 15 ml tube (SPL, Cat. #50015). After resuspension in 10% penicillin / streptomycin in PBS, the cells were centrifuged at 1600 rpm for 3 minutes and washed three times. Matrigel (Cornin, Cat. #356234) was diluted in PBS to a concentration of approximately 8.7 μg / cm3 and spread on a cell culture plate. The cells were incubated in a 37°C incubator for 1 hour. The PBS was removed immediately before incubation and dried for 10 minutes before use. Collagenase I (Gibco™, Cat. #17100-017) was diluted in HBSS solution (Gibco™, Cat. #14025092) to a concentration of 500 U / ml. Samples were resuspended with diluted enzyme solution and incubated at 37℃ for 1 hour. The mixture was vortexed every 5 minutes during the reaction. After adding an equal volume of culture media to the enzyme solution, the sample was centrifuged at 1600 rpm for 3 minutes, and the supernatant was removed. The sample was resuspended in DMEM, 20% FBS, and spread on a prepared Matrigel-coated plate. When the cells grew to 70–80% confluent in the culture dish, the cells were detached using trypsin-EDTA treatment. The detached cells were spread on a Matrigel-free plate with culture media, and incubated in a 37℃ incubator for 1 hour. The supernatant was spread on a new Matrigel-coated plate with culture media. The medium was changed every 2–3 days in DMEM, 2% Horse serum (Gibco™, Cat. #16050122). The cells were observed for 4–10 days to determine whether multinucleated cells had formed.

[0391] In all cell experiments, the medium was changed once every 2–3 days.

[0392] [Table 3]

[0393]

[0394]

[0395] Experimental Example 1.4. qRT-PCR

[0396] RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Cat. no. 74106), and 1 μg of the extracted RNA was used in EcoDry Premix (Takara, Cat. no. 639543) to synthesize cDNA.

[0397] The amount of GFP gene (GFP mRNA) was measured using SYBR Green in QuantStudio 1 (Applied Biosystems, Model no. A42870).

[0398] Relative cycle threshold (CT) values ​​were analyzed by normalizing them to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with three replicates per sample.

[0399] Experimental Example 1.5. Transfection

[0400] After mixing 750 ng of the experimental vector (vector of Experimental Example 1.2) and 750 ng of the transposase-encoding vector, a reaction solution was prepared by diluting the volume to 15 μl using R buffer. After that, cells were mixed into the reaction solution as a single cell suspension and transfection was performed. At this time, the NEON transfection system was used, and electroporation was performed under different conditions (cell number, voltage, etc.) for each cell type.

[0401] The conditions for each type of cell used in the experiment (number of cells, electroporation conditions, etc.) are as shown in Table 4 below.

[0402] [Table 4]

[0403]

[0404]

[0405] Experimental Example 1.6. FACS Analysis

[0406] The GFP fluorescence intensity expressed in cells was measured using a flow cytometer (BD FACSAria-II). Specifically, cells to be analyzed were mixed in PBS (gibco) as a single cell suspension and loaded. After irradiating single cells with a 488-nm coherent sapphire laser, the emitted GFP signal was analyzed using flowjo software.

[0407] Experimental Example 1.7. Western Blot

[0408] Cells were lysed using RIPA buffer and protease inhibitor, centrifuged, and the supernatant was quantified for protein concentration using the Bradford assay. The quantified proteins were mixed with sample buffer, electrophoresed on a 10% acrylamide gel, and transferred to a PVDF membrane. The membrane was blocked with skim milk powder for 1 hour and then incubated with primary antibody (anti-Cas9, 7A9-3A3, Cat# ab191468, Abcam) diluted 1:1,000 at 4°C. After three washes, the membrane was incubated with HRP-conjugated secondary antibody diluted 1:5,000 for 1 hour at room temperature, and then washed three times with TBS-T. Finally, ECL reagent was applied, and images were acquired using iBright1500.

[0409]

[0410] Experimental Example 2. Discovery of a Novel Promoter (MP#1)

[0411] Experimental Example 2.1. Trimming Process for Wild-Type Bovine MSTN

[0412] The assumed MSTN gene promoter sequence was isolated from wild-type bovine cells by PCR. The isolated sequence is approximately 3 kb upstream (in the 5' direction) from the start point (5'-AUG-3') of the coding sequence (CDS) encoding the myostatin (MSTN) protein. For convenience of explanation, the sequence up to approximately 3 kb is referred to as the wild-type MSTN promoter of bovine (upstream 3 kb).

[0413] The present inventors extracted a sequence from the MSTN protein CDS starting point to about 1.6 kb upstream (in the 5' direction) among the sequences of the wild type MSTN promoter of bovine (upstream 3 kb), and for convenience of explanation, the sequence up to about 1.6 kb is referred to as the wild type MSTN promoter of bovine (upstream 1.6 kb).

[0414] The present inventors extracted a portion of the sequence included in the wild type MSTN promoter of bovine (upstream 1.6 kb) into three fragments. The three fragments were classified as MP#1, MP#2, and MP#3 in order of proximity to the CDS of the MSTN protein (from the 3' direction). MP#1 is identical to the sequence from position 1396 to position 1638 of the wild type MSTN promoter of bovine (upstream 1.6 kb) sequence, MP#2 is identical to the sequence from position 878 to position 1415 of the wild type MSTN promoter of bovine (upstream 1.6 kb) sequence, and MP#3 is identical to the sequence from position 1 to position 571 of the wild type MSTN promoter of bovine (upstream 1.6 kb) sequence. Each fragment was divided based on the enhancer box (E box) located within the sequence. At this time, the sequence of the E-box is 5'-CANNTG-3' (N means any one of A, T, C, and G). Specifically, the criteria for dividing each fragment are explained. In MP#1, E1 (the E box closest to the CDS of the MSTN protein) and E2 (the E box second closest to the CDS of the MSTN protein) are located. In MP#2, E3 (the E box third closest to the CDS of the MSTN protein), E4 (the E box fourth closest to the CDS of the MSTN protein), E5 (the E box fifth closest to the CDS of the MSTN protein), and E6 (the E box sixth closest to the CDS of the MSTN protein) are located.MP#3 contains E7 (the seventh closest E box to the CDS of the MSTN protein), E8 (the eighth closest E box to the CDS of the MSTN protein), E9 (the ninth closest E box to the CDS of the MSTN protein), and E10 (the tenth closest E box to the CDS of the MSTN protein).

[0415] For further comparison of MP#1, MP#2, and MP#3, the inventors designed MP#21 connecting MP#1 and MP#2, MP#31 connecting MP#1 and MP#3, and MP#32 connecting MP#2 and MP#3.

[0416] The sequences of wild type MSTN promoter of bovine (upstream 3 kb), wild type MSTN promoter of bovine (upstream 1.6 kb), MP#1, MP#2, MP#3, MP#21, MP#31, and MP#32 are disclosed in Table 2. The MP#1 may be referred to as M243 in the experimental examples or drawings of the present application.

[0417] Experimental Example 2.1. Identification of a fragment containing the core region of the wild-type MSTN promoter.

[0418] The present inventors sought to determine whether the wild-type MSTN promoter of bovine (upstream 3 kb), MP#1, MP#2, and MP#3 each functioned as a promoter. In addition, the present inventors sought to compare the promoter activity levels of the wild-type MSTN promoter of bovine (upstream 3 kb), MP#1, MP#2, and MP#3. To this end, nucleic acids encoding GFP linked to the wild-type MSTN promoter of bovine (upstream 3 kb), MP#1, MP#2, and MP#3 were inserted into the genome of mouse muscle cells (C2C12 cell line). The method used for insertion into the genome was the transfection method disclosed in Experimental Example 1.5.

[0419] A schematic diagram of the sequence of the nucleic acids used as the experimental subjects in Experimental Example 2.1 is shown in Figure 1. The mRNA levels of GFP expressed in cells into which each nucleic acid was inserted are measured according to the method of Experimental Example 1.4, and the results are shown in Figure 2. The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Figure 3.

[0420] The first schematic diagram in Fig. 1 (a schematic diagram connected in the order of 3-2-1-GFP) represents the connection of a nucleic acid sequence encoding GFP to the wild-type MSTN promoter of bovine (upstream 3 kb). The experimental results for the first schematic diagram are shown in All on the vertical axis of the graph in Fig. 2 and in a in Fig. 3.

[0421] The second schematic diagram of Fig. 1 (a schematic diagram connected in the 1-GFP order) indicates that a nucleic acid sequence encoding GFP is connected to MP#1. The experimental results for the second schematic diagram are shown in 1 on the vertical axis of the graph of Fig. 2 and b of Fig. 3.

[0422] The third schematic diagram of Fig. 1 (a schematic diagram connected in the 2-GFP sequence) indicates that a nucleic acid sequence encoding GFP is connected to MP#2. The experimental results for the third schematic diagram are shown in 2 on the vertical axis of the graph of Fig. 2 and c in Fig. 3.

[0423] The fourth schematic diagram in Fig. 1 (a schematic diagram connected in the 3-GFP sequence) indicates that a nucleic acid sequence encoding GFP is connected to MP#3. The experimental results for the second schematic diagram are shown in 3 on the vertical axis of the graph in Fig. 2 and c in Fig. 3.

[0424] The experimental results of Experimental Example 2.1 demonstrate that MP#1, MP#2, and MP#3 have promoter activity. In particular, MP#1 was found to have higher promoter activity than not only MP#2 and MP#3, but also the wild type MSTN promoter of bovine (upstream 3 kb). Based on the results of Experimental Example 2.1, the present inventors determined that the region that plays a key role in gene expression in the sequence of the wild type MSTN promoter of bovine (upstream 3 kb) is included in MP#1.

[0425] Experimental Example 2.2. Verification for MP#1

[0426] To support the finding that the core region of the wild-type MSTN promoter of bovine (upstream 3 kb) is included in MP#1, the present inventors conducted additional experiments. Specifically, nucleic acids encoding GFP linked to the wild-type MSTN promoter of bovine (upstream 3 kb), MP#32, MP#31, and MP#21 were inserted into the genome of mouse muscle cells (C2C12 cell line). The method used for insertion into the genome was the transfection method disclosed in Experimental Example 1.5.

[0427] A schematic diagram of the sequence of the nucleic acids used as the experimental subject in Experimental Example 2.2 is shown in Fig. 4. The mRNA levels of GFP expressed in cells into which each nucleic acid was inserted are measured according to the method of Experimental Example 1.4 and the results are shown in Fig. 5. The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Fig. 6.

[0428] The first schematic diagram of Fig. 4 (a schematic diagram connected in the order of 3-2-1-GFP) represents the connection of a nucleic acid sequence encoding GFP to the wild-type MSTN promoter of bovine (upstream 3 kb). The experimental results for the first schematic diagram are shown in All on the vertical axis of the graph of Fig. 5 and in a of Fig. 6.

[0429] The second schematic diagram of Fig. 4 (a schematic diagram connected in the 3-2-GFP sequence) indicates that a nucleic acid sequence encoding GFP is connected to MP#32. The experimental results for the second schematic diagram are shown in -1 of the vertical axis of the graph of Fig. 5 and b of Fig. 6.

[0430] The third schematic diagram of Fig. 4 (a schematic diagram connected in the order of 3-1-GFP) indicates that a nucleic acid sequence encoding GFP is connected to MP#31. The experimental results for the third schematic diagram are shown at -2 on the vertical axis of the graph of Fig. 5 and c in Fig. 6.

[0431] The fourth schematic diagram of Fig. 4 (a schematic diagram connected in the order of 2-1-GFP) indicates that a nucleic acid sequence encoding GFP is connected to MP#21. The experimental results for the fourth schematic diagram are shown at -3 on the vertical axis of the graph of Fig. 5 and d in Fig. 6.

[0432] The experimental results of Experimental Example 2.2 show that the construct containing MP#1 exhibits higher promoter activity than the construct without MP#1. In other words, these experimental results support the notion that MP#1 contains a region that plays a critical role in promoter activity in the wild-type MSTN promoter.

[0433] Experimental Example 2.3. Additional Verification for MP#1

[0434] To further verify the results of Experimental Example 2.2, the inventors inserted nucleic acids encoding GFP linked to the wild type MSTN promoter of bovine (upstream 3 kb), the wild type MSTN promoter of bovine (upstream 1.6 kb), MP#31, MP#21, and MP#1, respectively, into the genome of mouse muscle cells (C2C12 cell line). The method used for insertion into the genome was the transfection method disclosed in Experimental Example 1.5.

[0435] A schematic diagram of the sequence of the nucleic acid that was the subject of the experiment in Experimental Example 2.3 is shown in Figure 7.

[0436] The mRNA levels of GFP expressed in cells into which each nucleic acid was inserted are measured according to the method of Experimental Example 1.4, and the results are shown in Figs. 8 and 9. Here, the result value shown in Fig. 8 for proliferating is the value measured when the mouse muscle cells are in the myoblast state. The result value shown in Fig. 8 for differentiated is the value measured after the mouse muscle cells have differentiated into myocytes. The graph in Fig. 9 shows the value obtained by dividing (the value after differentiation into myocytes) by (the value in the myoblast state). That is, through the graph in Fig. 9, the level of change in the level of promoter activity before and after differentiation can be compared.

[0437] The GFP fluorescence levels observed in cells into which each nucleic acid was inserted are shown in Figures 10 and 11. Here, Figure 10 shows the GFP fluorescence levels observed when mouse muscle cells were in the myoblast state. Figure 11 shows the GFP fluorescence levels observed after mouse muscle cells differentiated into myocytes.

[0438] The inventors of the present application have once again verified through the experimental results of Experimental Example 2.3 that a region that plays an important role in promoter activity in the wild-type MSTN promoter is included in MP#1.

[0439]

[0440] Experimental Example 3. Additional Study on the MP#1 Promoter 1

[0441] Experimental Example 3.1. Additional Trimming Process for the MP#1 Promoter

[0442] In Experimental Example 2, MP#1, which was confirmed to include the core region of the MSTN promoter, is 243 bp in length. To further reduce the length of the promoter, the inventors of the present application performed trimming to exclude 73 bp in the 3' direction of MP#1. For convenience of explanation, the remaining 170 bp of nucleic acid sequence after excluding 73 bp in the 3' direction of MP#1 may be referred to as 'short MP#1' or 'sM243'. The sequence of short MP#1 (or sM243) is described in Table 2.

[0443] Experimental Example 3.2. Proportion of cells expressing GFP in each cell line

[0444] The present inventors conducted experiments in Experimental Examples 3.2 and 3.3 to confirm the effects of short MP#1 (or sM243) and the effects of MP#1 on various types of cells.

[0445] In Experimental Examples 3.2 and 3.3, nucleic acids encoding GFP were linked to each of the CAG promoter, the EF1a promoter, the mPGK promoter, MP#1, MP#21, short MP#1 (or sM243), the CMV enhancer MP#1, and the MP#1 tail duplication, and were inserted into the genomes of various cell lines. At this time, the method used for insertion into the genome is the transfection method disclosed in Experimental Example 1.5. Here, the types of cell lines used are C2C12, 3T3-L1, C127:LT, HeLa, ISD 1st, A549, Cal-62, ISD 2nd, MDBK, PC-3, and MCF7. The CMV enhancer MP#1 refers to a nucleic acid sequence in which the CMV enhancer of SEQ ID NO: 4 is linked to the 5' of MP#1. The above MP#1 tail duplication refers to a nucleic acid sequence in which short MP#1 is connected to the 3' of MP#1.

[0446] To minimize the impact of transfection efficiency for each promoter and cell type on the GFP expression level in each experiment, the inventors sought to determine the proportion of cells expressing GFP in each experiment. Accordingly, the proportion of cells expressing GFP in each case was measured using the analysis method of Experimental Example 1.6, and the results are shown in Figure 12.

[0447] Experimental Example 3.3. Intensity of GFP expression in each cell line

[0448] Using the method of Experimental Example 1.6, the GFP fluorescence level in each experiment of Experimental Example 3.2 was also measured. At this time, the inventors divided the measured GFP fluorescence level in each experiment by the proportion of cells expressing GFP. The results are shown in Figures 13 to 17.

[0449] The results of Experimental Example 3 show that linking a known enhancer to MP#1 increases its activity toward the promoter.

[0450] The results of Experimental Example 3 demonstrate that MP#1 does not exhibit promoter activity specifically in muscle cells, but rather exhibits promoter activity in other cell types as well. Furthermore, the results of Experimental Example 3 demonstrate that MP#1 exhibits promoter activity not only in bovine cells but also in cells of other species, including mice and humans. In other words, those skilled in the art will recognize, based on the experimental results of the present disclosure, that MP#1 exhibits the potential to be a ubiquitous promoter.

[0451] The results of Experimental Example 3 show that among the sequences of MP#1, the region that plays a key role in gene expression is not included in short MP#1 (or sM243).

[0452]

[0453] Experimental Example 4. Additional Study on the MP#1 Promoter 2

[0454] Experimental Example 4.1. Additional Trimming Process for the MP#1 Promoter

[0455] In Experimental Example 2, MP#1, which was confirmed to include the core region of the MSTN promoter, has a length of 243 bp. The inventors of the present application confirmed through Experimental Example 3 that sM243, which is 170 bp long in the 5' direction of MP#1, does not include the core region. Based on these results, the inventors of the present application predicted that the core region would be included in 73 bp in the 3' direction of MP#1, and therefore trimmed MP#1 to extract only 73 bp or 48 bp in the 3' direction. For convenience of explanation, the sequence obtained by extracting only 73 bp in the 3' direction of MP#1 may be referred to as 'M73'. For convenience of explanation, the sequence obtained by extracting only 48 bp in the 3' direction of MP#1 may be referred to as 'M48'. The sequences of M73 and M48 are described in Table 2.

[0456] Experimental Example 4.2. Confirmation of the effects of M73 and M48

[0457] To confirm the effects of M73 and M48, the inventors conducted the experiment of Experimental Example 4.2.

[0458] Nucleic acids encoding GFP linked to the CAG promoter, MP#1, M73, and M48 were inserted into the genomes of various cell lines. The method used for insertion into the genome was the transfection method disclosed in Experimental Example 1.5. The cell lines used here were ISD0615 and C2C12. In Experimental Example 4.2, Kozak consensus sequences (5'-GCCACC-3') were added between the promoter sequence and the nucleic acid encoding GFP. That is, 5'-GCCACC-3' was added between the CAG promoter, MP#1, M73, and M48 and the nucleic acid encoding GFP.

[0459] The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Fig. 18. When observing the fluorescence in Fig. 18, the fluorescence in cells into which M73 and M48 were inserted was confirmed to be very dark, at a level not different from that of sM243 in Experimental Example 3, and the proportion of cells expressing fluorescence also decreased. That is, Fig. 18 shows that the expression levels of genes linked to M73 and M48 are significantly lower than the expression levels of genes linked to MP#1.

[0460] The results of Experimental Example 4 show that among the sequences of MP#1, the regions that play a key role in gene expression are not included in M73 and M48.

[0461]

[0462] Experimental Example 5. Derivation of the core region of the MP#1 promoter and discovery of an improved promoter.

[0463] Experimental Example 5.1. Deriving the Core Region of the MP#1 Promoter

[0464] The length of MP#1, which was confirmed to include the core region of the MSTN promoter in Experimental Example 2, is 243 bp. The inventors of the present application confirmed through Experimental Example 3 that sM243, which is 170 bp long in the 5' direction of MP#1, does not include the core region. In addition, the inventors of the present application confirmed through Experimental Example 4 that M73, which is 73 bp long in the 3' direction of MP#1, does not include the core region. Since sM243 and M73 do not overlap, the nucleic acid sequence of MP#1 is completed by connecting 170 bp and 73 bp. In other words, even though MP#1 was divided into two regions centered on the area between the 170th and 171st nucleic acids in the 5' direction, neither of the two separated regions contained the core region.

[0465] Based on these results, the inventors of the present application predicted that the core region of MP#1 would include the boundaries of 170 bp and 73 bp in the nucleic acid sequence of MP#1, and therefore, trimmed the nucleic acid sequence to extract a 50 bp long nucleic acid sequence including the boundaries of 170 bp and 73 bp in the nucleic acid sequence of MP#1. For convenience of explanation, the 50 bp long nucleic acid sequence including the boundaries of 170 bp and 73 bp in the nucleic acid sequence of MP#1 may be referred to as 'M50TSS'. In order to further verify the effect of M50TSS, the inventors of the present application additionally extracted a sequence including M50TSS and a sequence not including M50TSS. Specifically, from the sequence of MP#1, two types of nucleic acid sequences each having a length of 100 bp including M50TSS; one type of nucleic acid sequence having a length of 150 bp including M50TSS; An additional experiment was conducted to confirm the effects of two types of 50-bp long nucleic acid sequences that do not include the boundaries of 170 bp and 73 bp. For convenience of explanation, the two types of 100-bp long nucleic acid sequences that include M50TSS may be referred to as 'M100TSS' and 'M100F', respectively. For convenience of explanation; the 150-bp long nucleic acid sequence that includes M50TSS may be referred to as 'M150'. For convenience of explanation, the two types of 50-bp long nucleic acid sequences that do not include the boundaries of 170 bp and 73 bp may be referred to as 'M50F' and 'M50B', respectively. The sequences of M50TSS, M100TSS, M100F, M150, M50F, and M50B are described in Table 2. Figure 38 shows the regions occupied by MP#1 (or M243), sM243, M73, M48, M50B, M50F, M50TSS, M100TSS, M100F, and M150, respectively, in the bovine wild-type MSTN promoter sequence. From Figure 38, it can be easily seen that M243, M100TSS, M100F, and M150, which showed promoter activity, all include the core region, M50TSS.

[0466] Experimental Example 5.2. Confirming the Effectiveness of M50TSS: Key Areas

[0467] The inventors of the present invention conducted the experiment of Experimental Example 5.2 to confirm the effects of M50TSS, M100TSS, M100F, M150, M50F, and M50B.

[0468] Nucleic acids encoding GFP were linked to the CAG promoter, MP#1, M73, M50TSS, M100TSS, M100F, M150, M50F, and M50B, respectively, and were inserted into the genomes of various cell lines. At this time, the method used for insertion into the genome is the transfection method disclosed in Experimental Example 1.5. Here, the types of cell lines used were bovine primary fibroblasts from three different individuals. In Experimental Example 5, Kozak consensus sequences (5'-GCCACC-3') were added between the promoter sequence and the nucleic acid encoding GFP. That is, 5'-GCCACC-3' was added between the CAG promoter, MP#1, M73, M50TSS, M100TSS, M100F, M150, M50F, and M50B and the nucleic acid encoding GFP.

[0469] The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Figures 19 and 20. The results in Figures 19 and 20 show that the cell lines into which M50TSS, M100TSS, M100F, and M150 were inserted showed fluorescence brightness with no statistically significant difference compared to the cell line into which MP#1 was inserted. Therefore, the results of Experimental Example 5.2 demonstrate that M50TSS contains the core region of MP#1.

[0470] Experimental Example 5.3. Confirming the Effect of M50TSS: Ubiquitous Promoter

[0471] The present inventors conducted the experiment of Experimental Example 5.3 to confirm whether M50TSS and sequences containing M50TSS are promoters showing the possibility of being ubiquitous promoters.

[0472] Nucleic acids encoding GFP linked to each of MP#1, M50TSS, M100TSS, M100F, and M150 were inserted into the genomes of various cell lines. At this time, the method used for insertion into the genome is the transfection method disclosed in Experimental Example 1.5. Here, the types of cell lines used are three types of bovine cells (fibroblasts, myoblasts, MDBK cell line), three types of human cells (myoblasts, HeLa cell line, HEK293T cell line), and four types of mouse cells (4T1 cell line, 3T3-L1 cell line, C2C12 cell line, HC11 cell line). In Experimental Example 5, Kozak consensus sequences (5'-GCCACC-3') were added between the promoter sequence and the nucleic acid encoding GFP. That is, 5'-GCCACC-3' was added between the CAG promoter, MP#1, M50TSS, M100TSS, M100F, and M150 and the nucleic acid encoding GFP.

[0473] The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Figures 21 to 32. The results in Figures 21 to 32 show that sequences containing M50TSS and M50TSS can induce GFP gene expression in all cell lines tested.

[0474] The results of Experimental Example 5.3 show that the sequences containing M50TSS and M50TSS do not have promoter activity specific to muscle cells, but also have promoter activity in other types of cells. Furthermore, the results of Experimental Example 5.3 show that the sequences containing M50TSS and M50TSS have promoter activity not only in bovine cells but also in cells of other species, such as mice and humans. In other words, those skilled in the art can recognize, through the experimental results of the present specification, that the sequences containing M50TSS and M50TSS are promoters that exhibit the potential of being ubiquitous promoters.

[0475]

[0476] Experimental Example 6. Trimming of MSTN promoters of various species.

[0477] Experimental Example 6.1. Trimming of MSTN Promoters of Various Species

[0478] The inventors of the present application, through the processes of Experimental Examples 2 to 5, trimmed the bovine wild-type MSTN promoter sequence to discover a novel promoter that is shorter than the wild-type sequence but exhibits promoter activity. At this time, the core region of the bovine wild-type MSTN promoter sequence identified in Experimental Examples 2 to 5 was highly homologous and conserved in the wild-type MSTN promoter sequences of evolutionarily other mammals. Based on the fact that the core region of the MSTN promoter among mammals is highly homologous and conserved, it was speculated that novel promoters could be discovered from the wild-type MSTN promoter sequences of mammals other than bovines.

[0479] Accordingly, the inventors of the present application extracted sequences corresponding to the novel promoters discovered in Experimental Examples 2 to 5 from the wild-type MSTN promoter sequences of mammals other than cattle, thereby discovering novel promoter sequences. The specific trimming methods and the results confirming the effectiveness of the trimmed sequences are described in Experimental Examples 6.2 to 6.5.

[0480] Experimental Example 6.2. Trimming the Human Wild-Type MSTN Promoter

[0481] Experimental Example 6.2.1. Trimming Process

[0482] The sequence homologous to MP#1 in the bovine wild-type MSTN promoter sequence was extracted from the human wild-type MSTN promoter sequence. The sequences at the homologous positions were selected based on the following criteria: 1) transcription start site (TSS); 2) E box; and 3) distance from CDS of the MSTN protein. The length of the sequence selected from the human wild-type MSTN promoter sequence is 242 bp, and for convenience of explanation, the 242-bp sequence can be referred to as Hu_M242. The sequence of Hu_M242 is a nucleic acid sequence of SEQ ID NO: 33, and when the sequence of Hu_M242 is compared with the sequence of MP#1, it shows approximately 95% homology.

[0483] The inventors of the present application further trimmed Hu_M242 in a direction that removes a portion of the 5' direction of Hu_M242. For example, 44 bp, 84 bp, 134 bp, or 169 bp of the 5' direction of Hu_M242 were removed. For convenience of explanation, the sequence from which 44 bp was removed may be referred to as Hu_M198, and the sequence of Hu_M198 is the nucleic acid sequence of SEQ ID NO: 34. For convenience of explanation, the sequence from which 84 bp was removed may be referred to as Hu_M158, and the sequence of Hu_M158 is the nucleic acid sequence of SEQ ID NO: 35. For convenience of explanation, the sequence from which 134 bp was removed may be referred to as Hu_M108, and the sequence of Hu_M108 is the nucleic acid sequence of SEQ ID NO: 36. For convenience of explanation, the sequence with 169 bp removed may be referred to as Hu_M73, and the sequence of Hu_M73 is the nucleic acid sequence of sequence number 37.

[0484] The inventors of the present application further trimmed the sequences of Hu_M242, Hu_M158, and Hu_M108 in the 3' direction by removing 50 bp. For convenience of explanation, the sequence from which 50 bp is removed in the 3' direction from the Hu_M242 sequence is referred to as Hu_MM.192, and the sequence of Hu_MM.192 is SEQ ID NO: 38. For convenience of explanation, the sequence from which 50 bp is removed in the 3' direction from the Hu_M158 sequence is referred to as Hu_MM.108, and the sequence of Hu_MM.108 is SEQ ID NO: 39. For convenience of explanation, the sequence from which 50 bp is removed in the 3' direction from the Hu_M108 sequence is referred to as Hu_MM.58, and the sequence of Hu_MM.58 is SEQ ID NO: 40.

[0485] The inventors of the present application performed trimming to extract sequences corresponding to M50TSS, M243, M150, M100F, M100TSS, and M58TSS included in MP#1 from the sequence of the human wild-type MSTN promoter. For convenience of explanation, the sequence corresponding to M50TSS in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M50TSS, and the sequence of Hu_M50TSS is the nucleic acid sequence of SEQ ID NO: 32. When the sequence of Hu_M50TSS is compared with the sequence of M50TSS, it shows 100% homology. For convenience of explanation, the sequence corresponding to M243 in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M243, and the sequence of Hu_M243 is the nucleic acid sequence of SEQ ID NO: 26. For convenience of explanation, the sequence corresponding to M150 in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M150, and the sequence of Hu_M150 is the nucleic acid sequence of SEQ ID NO: 27. For convenience of explanation, the sequence corresponding to M100F in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M100F, and the sequence of Hu_M100F is the nucleic acid sequence of SEQ ID NO: 28. For convenience of explanation, the sequence corresponding to M100TSS in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M100TSS, and the sequence of Hu_M100TSS is the nucleic acid sequence of SEQ ID NO: 29. For convenience of explanation, the sequence corresponding to M150 in the sequence of the human wild-type MSTN promoter may be referred to as Hu_M150, and the sequence of Hu_M150 is the nucleic acid sequence of SEQ ID NO: 27. The sequence corresponding to M58TSS in the promoter sequence may be referred to as Hu_M58TSS, and the sequence of Hu_M58TSS is the nucleic acid sequence of SEQ ID NO: 41.

[0486] Experimental Example 6.2.2. Confirming the Effect of Trimmed Sequences 1

[0487] The present inventors conducted the experiment of Experimental Example 6.2.2 to confirm the effects of Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73.

[0488] Nucleic acids encoding GFP linked to the EF1a promoter, Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73, respectively, were inserted into the genomes of various cell lines. At this time, the method used for insertion into the genome is the transfection method disclosed in Experimental Example 1.5. Here, the types of cell lines used are C2C12, HepG2, and PC3 cells. At this time, Kozak consensus sequences (5'-GCCACC-3') were added between the promoter sequence and the nucleic acid encoding GFP. That is, 5'-GCCACC-3' was added between the EF1a promoter, Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73 and the nucleic acid encoding GFP. Figure 39 is a schematic diagram of the sequences inserted into the genomes of the cell lines. Through Figure 39, it is easy to see the regions occupied by Hu_M198, Hu_M158, Hu_M108, and Hu_M73 in the sequence of Hu_M242, respectively.

[0489] The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Figures 33 to 35. The results in Figures 33 to 35 confirmed that all cell lines into which Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73 were inserted expressed fluorescence. In addition, among Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73, Hu_M158 showed the highest fluorescence expression.

[0490] Therefore, the results of Experimental Example 6.2.2 show that Hu_M242, Hu_M198, Hu_M158, Hu_M108, and Hu_M73 all have promoter activity. In addition, the results of Experimental Example 6.2.2 show that the core region in the human wild-type MSTN promoter is included in Hu_M158. In addition, a person skilled in the art can know from the results of Experimental Example 6.2.2 that the core sequences of the MSTN promoters identified in Experimental Examples 2 to 5 are highly homologous and conserved among mammals, and therefore, a novel promoter can be discovered by extracting a sequence corresponding to the novel promoter discovered in Experimental Examples 2 to 5 from the wild-type MSTN promoter sequences of mammals other than cows.

[0491] Experimental Example 6.2.3. Confirming the Effect of Trimmed Sequences 2

[0492] The present inventors conducted the experiment of Experimental Example 6.2.3 to confirm the effects of Hu_M243, Hu_M150, Hu_M100F, Hu_M100TSS, and Hu_M58TSS.

[0493] Nucleic acids encoding GFP linked to each of Hu_M243, Hu_M150, Hu_M100F, Hu_M100TSS, and Hu_M58TSS are inserted into the genomes of various cell lines. At this time, the transfection method disclosed in Experimental Example 1.5 is used as the genome insertion method. The target cell line for insertion is one or more of the cell lines listed in Table 3.

[0494] The fluorescence level of GFP observed in each nucleic acid-inserted cell is observed. As a result of the observation,

[0495] Fluorescence expression was confirmed in cell lines inserted with Hu_M243, Hu_M150, Hu_M100F, Hu_M100TSS, and Hu_M58TSS.

[0496] Experimental Example 6.2.4. Determining Cas9 Protein Expression Levels

[0497] The present inventors conducted the experiment of Experimental Example 6.2.4 to confirm the effects of Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, and Hu_M50TSS.

[0498] A nucleic acid encoding SpCas9 protein (SEQ ID NO: 98) and a nucleic acid encoding GFP were linked to each of Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, Hu_M50TSS, M58TSS, and M50TSS, and the nucleic acid was inserted into the genome of various cell lines. At this time, the method used for insertion into the genome is the transfection method disclosed in Experimental Example 1.5. Here, the type of cell line used is HeLa cells. At this time, the nucleic acid encoding Cas9 protein and the nucleic acid encoding GFP were linked with a 2A linker sequence. The M58TSS refers to a 58 bp sequence that includes one more TATA box in the 5' direction of M50TSS. The sequence of the M58TSS is the nucleic acid sequence of SEQ ID NO: 25. Figure 40 is a schematic diagram of the sequence inserted into the genome of the cell lines. Through Figure 40, it is easy to see the regions occupied by Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM58, and Hu_M50TSS in the sequence of Hu_M242, respectively.

[0499] The fluorescence levels of GFP observed in cells into which each nucleic acid was inserted are shown in Figure 36. The Cas9 protein expression levels in cells into which each nucleic acid was inserted were confirmed through Western Blot in Experimental Example 1.7, and the Western Blot results are shown in Figure 37.

[0500] Through the results of Figures 36 and 37, it was confirmed that all cell lines inserted with Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, Hu_M50TSS, M58TSS, and M50TSS expressed GFP and Cas9 proteins. In addition, among Hu_M242, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, Hu_MM.58, and Hu_M50TSS, Hu_M50TSS showed the highest Cas9 protein expression.

[0501] Experimental Example 6.3. Trimming the wild-type MSTN promoter in canine

[0502] Experimental Example 6.3.1. Trimming Process

[0503] The sequence homologous to MP#1 in the bovine wild-type MSTN promoter sequence was extracted from the canine wild-type MSTN promoter sequence. The sequences at the homologous positions were selected based on the following criteria: 1) transcription start site (TSS); 2) E box; and 3) distance from CDS of the MSTN protein. The length of the sequence selected from the canine wild-type MSTN promoter sequence is 243 bp, and for convenience of explanation, the 243 bp sequence can be referred to as Ca_M243. The sequence of Ca_M243 is a nucleic acid sequence of SEQ ID NO: 42, and when compared with the sequence of Ca_M243 and MP#1, it shows approximately 93% homology.

[0504] The inventors of the present application performed trimming to extract sequences corresponding to M150, M100F, M100TSS, M50TSS, M58TSS, Hu_M242, Hu_M198, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, and Hu_MM.58 from the sequence of the wild-type MSTN promoter of the dog. For convenience of explanation, the sequence corresponding to M150 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M150, and the sequence of Ca_M150 is a nucleic acid sequence of SEQ ID NO: 43. For convenience of explanation, the sequence corresponding to M100F in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M100F, and the sequence of Ca_M100F is a nucleic acid sequence of SEQ ID NO: 44. For convenience of explanation, the sequence corresponding to M100TSS in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M100TSS, and the sequence of Ca_M100TSS is a nucleic acid sequence of SEQ ID NO: 45. For convenience of explanation, the sequence corresponding to M50TSS in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M50TSS, and the sequence of Ca_M50TSS is a nucleic acid sequence of SEQ ID NO: 48. When the sequence of Ca_M50TSS is compared with the sequence of M50TSS, it shows about 98% homology. For convenience of explanation, the sequence corresponding to M58TSS in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M58TSS, and the sequence of Ca_M58TSS is the nucleic acid sequence of SEQ ID NO: 56. For convenience of explanation, the sequence corresponding to Hu_M242 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M242, and the sequence of Ca_M242 is the nucleic acid sequence of SEQ ID NO: 49. For convenience of explanation, the sequence corresponding to Hu_M198 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M198, and the sequence of Ca_M198 is the nucleic acid sequence of SEQ ID NO: 50.For convenience of explanation, the sequence corresponding to Hu_M158 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M158, and the sequence of Ca_M158 is a nucleic acid sequence of SEQ ID NO: 51. For convenience of explanation, the sequence corresponding to Hu_M108 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_M108, and the sequence of Ca_M108 is a nucleic acid sequence of SEQ ID NO: 52. For convenience of explanation, the sequence corresponding to Hu_MM.192 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_MM.192, and the sequence of Ca_MM.192 is a nucleic acid sequence of SEQ ID NO: 53. For convenience of explanation, the sequence corresponding to Hu_MM.108 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_MM.108, and the sequence of Ca_MM.108 is a nucleic acid sequence of SEQ ID NO: 54. For convenience of explanation, the sequence corresponding to Hu_MM.58 in the sequence of the wild-type MSTN promoter of the dog may be referred to as Ca_MM.58, and the sequence of Ca_MM.58 is the nucleic acid sequence of SEQ ID NO: 55.

[0505] Experimental Example 6.3.2. Confirming the Effect of Trimmed Sequences

[0506] The present inventors conducted the experiment of Experimental Example 6.3.2 to confirm the effects of Ca_M243, Ca_M150, Ca_M100F, Ca_M100TSS, Ca_M50TSS, Ca_M58TSS, Ca_M242, Ca_M198, Ca_M158, Ca_M108, Ca_MM.192, Ca_MM.108, and Ca_MM.58.

[0507] Nucleic acids encoding GFP are linked to each of Ca_M243, Ca_M150, Ca_M100F, Ca_M100TSS, Ca_M50TSS, Ca_M58TSS, Ca_M242, Ca_M198, Ca_M158, Ca_M108, Ca_MM.192, Ca_MM.108, and Ca_MM.58, and are inserted into the genomes of various cell lines. At this time, the transfection method disclosed in Experimental Example 1.5 is used as the genome insertion method. The target cell line for insertion is one or more of the cell lines in Table 3 or a canine-derived cell line.

[0508] The fluorescence level of GFP observed in each nucleic acid-inserted cell is observed. As a result of the observation,

[0509] Fluorescence expression was confirmed in cell lines inserted with Ca_M243, Ca_M150, Ca_M100F, Ca_M100TSS, Ca_M50TSS, Ca_M58TSS, Ca_M242, Ca_M198, Ca_M158, Ca_M108, Ca_MM.192, Ca_MM.108, and Ca_MM.58.

[0510] Experimental Example 6.4. Trimming of the wild-type MSTN promoter in porcine

[0511] Experimental Example 6.4.1. Trimming Process

[0512] The sequence homologous to MP#1 in the bovine wild-type MSTN promoter sequence was extracted from the porcine wild-type MSTN promoter sequence. The sequences at the homologous positions were selected based on the following criteria: 1) transcription start site (TSS); 2) E box; and 3) distance from CDS of the MSTN protein. The length of the sequence selected from the porcine wild-type MSTN promoter sequence is 241 bp, and for convenience of explanation, the 241-bp sequence may be referred to as Po_M241. The sequence of Po_M241 is a nucleic acid sequence of SEQ ID NO: 57, and when compared with the sequence of Po_M241 and the sequence of MP#1, it shows approximately 96% homology.

[0513] The inventors of the present application performed trimming to extract sequences corresponding to M150, M100F, M100TSS, M50TSS, M58TSS, Hu_M242, Hu_M198, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, and Hu_MM.58 from the sequence of the wild-type MSTN promoter of pigs. For convenience of explanation, the sequence corresponding to M150 in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M150, and the sequence of Po_M150 is a nucleic acid sequence of SEQ ID NO: 58. For convenience of explanation, the sequence corresponding to M100F in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M100F, and the sequence of Po_M100F is a nucleic acid sequence of SEQ ID NO: 59. For convenience of explanation, the sequence corresponding to M100TSS in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M100TSS, and the sequence of Po_M100TSS is a nucleic acid sequence of SEQ ID NO: 60. For convenience of explanation, the sequence corresponding to M50TSS in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M50TSS, and the sequence of Po_M50TSS is a nucleic acid sequence of SEQ ID NO: 63. When the sequence of Po_M50TSS is compared with the sequence of M50TSS, it shows 100% homology. For convenience of explanation, the sequence corresponding to M58TSS in the sequence of the wild-type MSTN promoter of pigs can be referred to as Po_M58TSS, and the sequence of Po_M58TSS is the nucleic acid sequence of SEQ ID NO: 71. For convenience of explanation, the sequence corresponding to Hu_M242 in the sequence of the wild-type MSTN promoter of pigs can be referred to as Po_M242, and the sequence of Po_M242 is the nucleic acid sequence of SEQ ID NO: 64. For convenience of explanation, the sequence corresponding to Hu_M198 in the sequence of the wild-type MSTN promoter of pigs can be referred to as Po_M198, and the sequence of Po_M198 is the nucleic acid sequence of SEQ ID NO: 65.For convenience of explanation, the sequence corresponding to Hu_M158 in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M158, and the sequence of Po_M158 is the nucleic acid sequence of SEQ ID NO: 66. For convenience of explanation, the sequence corresponding to Hu_M108 in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_M108, and the sequence of Po_M108 is the nucleic acid sequence of SEQ ID NO: 67. For convenience of explanation, the sequence corresponding to Hu_MM.192 in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_MM.192, and the sequence of Po_MM.192 is the nucleic acid sequence of SEQ ID NO: 68. For convenience of explanation, the sequence corresponding to Hu_MM.108 in the sequence of the wild-type MSTN promoter of a pig may be referred to as Po_MM.108, and the sequence of Po_MM.108 is the nucleic acid sequence of SEQ ID NO: 69. For convenience of explanation, the sequence corresponding to Hu_MM.58 in the sequence of the wild-type MSTN promoter of pigs may be referred to as Po_MM.58, and the sequence of Po_MM.58 is the nucleic acid sequence of SEQ ID NO: 70.

[0514] Experimental Example 6.4.2. Confirming the Effect of Trimmed Sequences

[0515] The present inventors conducted the experiment of Experimental Example 6.4.2 to confirm the effects of Po_M241, Po_M150, Po_M100F, Po_M100TSS, Po_M50TSS, Po_M58TSS, Po_M242, Po_M198, Po_M158, Po_M108, Po_MM.192, Po_MM.108, and Po_MM.58.

[0516] A nucleic acid encoding GFP is linked to each of Po_M241, Po_M150, Po_M100F, Po_M100TSS, Po_M50TSS, Po_M58TSS, Po_M242, Po_M198, Po_M158, Po_M108, Po_MM.192, Po_MM.108, and Po_MM.58, and the nucleic acid encoding GFP is inserted into the genome of various cell lines. At this time, the transfection method disclosed in Experimental Example 1.5 is used as the genome insertion method. The target cell line for insertion is any one or more of the cell lines in Table 3 or a cell line of porcine origin.

[0517] The fluorescence level of GFP observed in cells into which each nucleic acid was inserted is observed. As a result of the observation, fluorescence expression is confirmed in cell lines into which Po_M241, Po_M150, Po_M100F, Po_M100TSS, Po_M50TSS, Po_M58TSS, Po_M242, Po_M198, Po_M158, Po_M108, Po_MM.192, Po_MM.108, and Po_MM.58 were inserted.

[0518] Experimental Example 6.5. Trimming the wild-type MSTN promoter in equine

[0519] Experimental Example 6.5.1. Trimming Process

[0520] In the horse wild-type MSTN promoter sequence, a sequence homologous to MP#1 in the bovine wild-type MSTN promoter sequence was extracted. The sequences at homologous positions were selected based on the following criteria: 1) transcription start site (TSS); 2) E box; and 3) distance from CDS of the MSTN protein. The length of the sequence selected from the horse wild-type MSTN promoter sequence is 243 bp, and for convenience of explanation, the 243 bp sequence may be referred to as Eq_M243. The sequence of Eq_M243 is a nucleic acid sequence of SEQ ID NO: 72, and when compared with the sequence of MP#1, it shows approximately 96% homology.

[0521] The inventors of the present application performed trimming to extract sequences corresponding to M150, M100F, M100TSS, M50TSS, M58TSS, Hu_M242, Hu_M198, Hu_M158, Hu_M108, Hu_MM.192, Hu_MM.108, and Hu_MM.58 from the sequence of the horse wild-type MSTN promoter. For convenience of explanation, the sequence corresponding to M150 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M150, and the sequence of Eq_M150 is a nucleic acid sequence of SEQ ID NO: 73. For convenience of explanation, the sequence corresponding to M100F in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M100F, and the sequence of Eq_M100F is a nucleic acid sequence of SEQ ID NO: 74. For convenience of explanation, the sequence corresponding to M100TSS in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M100TSS, and the sequence of Eq_M100TSS is a nucleic acid sequence of SEQ ID NO: 75. For convenience of explanation, the sequence corresponding to M50TSS in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M50TSS, and the sequence of Eq_M50TSS is a nucleic acid sequence of SEQ ID NO: 78. When the sequence of Eq_M50TSS is compared with the sequence of M50TSS, it shows 100% homology. For convenience of explanation, the sequence corresponding to M58TSS in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M58TSS, and the sequence of Eq_M58TSS is a nucleic acid sequence of SEQ ID NO: 86. For convenience of explanation, the sequence corresponding to Hu_M242 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M242, and the sequence of Eq_M242 is a nucleic acid sequence of SEQ ID NO: 79. For convenience of explanation, the sequence corresponding to Hu_M198 in the sequence of the wild-type MSTN promoter of a horse may be referred to as Eq_M198, and the sequence of Eq_M198 is the nucleic acid sequence of SEQ ID NO: 80.For convenience of explanation, the sequence corresponding to Hu_M158 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M158, and the sequence of Eq_M158 is a nucleic acid sequence of SEQ ID NO: 81. For convenience of explanation, the sequence corresponding to Hu_M108 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_M108, and the sequence of Eq_M108 is a nucleic acid sequence of SEQ ID NO: 82. For convenience of explanation, the sequence corresponding to Hu_MM.192 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_MM.192, and the sequence of Eq_MM.192 is a nucleic acid sequence of SEQ ID NO: 83. For convenience of explanation, the sequence corresponding to Hu_MM.108 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_MM.108, and the sequence of Eq_MM.108 is the nucleic acid sequence of SEQ ID NO: 84. For convenience of explanation, the sequence corresponding to Hu_MM.58 in the sequence of the horse wild-type MSTN promoter may be referred to as Eq_MM.58, and the sequence of Eq_MM.58 is the nucleic acid sequence of SEQ ID NO: 85.

[0522] Experimental Example 6.5.2. Confirming the Effect of Trimmed Sequences

[0523] The present inventors conducted the experiment of Experimental Example 6.5.2 to confirm the effects of Eq_M243, Eq_M150, Eq_M100F, Eq_M100TSS, Eq_M50TSS, Eq_M58TSS, Eq_M242, Eq_M198, Eq_M158, Eq_M108, Eq_MM.192, Eq_MM.108, and Eq_MM.58.

[0524] Nucleic acids encoding GFP are linked to each of Eq_M243, Eq_M150, Eq_M100F, Eq_M100TSS, Eq_M50TSS, Eq_M58TSS, Eq_M242, Eq_M198, Eq_M158, Eq_M108, Eq_MM.192, Eq_MM.108, and Eq_MM.58, and are inserted into the genomes of various cell lines. At this time, the transfection method disclosed in Experimental Example 1.5 is used as the genome insertion method. The target cell line for insertion is any one or more of the cell lines in Table 3 or a cell line derived from equine.

[0525] The fluorescence level of GFP observed in each nucleic acid-inserted cell is observed. As a result of the observation,

[0526] Fluorescence expression is confirmed in cell lines into which Eq_M243, Eq_M150, Eq_M100F, Eq_M100TSS, Eq_M50TSS, Eq_M58TSS, Eq_M242, Eq_M198, Eq_M158, Eq_M108, Eq_MM.192, Eq_MM.108, and Eq_MM.58 are inserted.

Claims

1. A nucleic acid having promoter activity, The sequence of the nucleic acid having the above promoter activity comprises the nucleic acid sequence of SEQ ID NO: 19, The length of the nucleic acid having the above promoter activity is 50 bp to 243 bp.

2. In paragraph 1, A nucleic acid having promoter activity, wherein the sequence of the nucleic acid having the above promoter activity is identical to a part of the sequence of SEQ ID NO:

7.

3. In paragraph 1, A nucleic acid having promoter activity, wherein the sequence of the nucleic acid having the above promoter activity is any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

4. In paragraph 1, A nucleic acid having promoter activity, characterized in that the nucleic acid having the above promoter activity functions as a promoter for a desired nucleic acid encoding a desired protein.

5. In paragraph 4, A nucleic acid having promoter activity, characterized in that when the nucleic acid functioning as the above promoter is used to express the above desired protein from the above desired nucleic acid, the 3' end of the nucleic acid functioning as the promoter can be operably linked to the 5' end of the desired nucleic acid.

6. A nucleic acid for expressing a target protein comprising the following: A first nucleic acid having promoter activity; and A second nucleic acid encoding the target protein, At this time, the sequence of the first nucleic acid having the promoter activity includes the nucleic acid sequence of sequence number 19, and the length of the first nucleic acid having the promoter activity is 50 bp to 243 bp, The second nucleic acid encoding the target protein is a nucleic acid encoding the target protein to be expressed in the cell, The second nucleic acid is characterized in that it is operably linked to the 3' direction of the first nucleic acid.

7. In paragraph 6, A nucleic acid for expressing a target protein, wherein the sequence of the first nucleic acid having the above promoter activity is identical to a part of the sequence of SEQ ID NO:

7.

8. In paragraph 6, A nucleic acid for expressing a target protein, wherein the sequence of the first nucleic acid having the above promoter activity is any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

9. A method for expressing a target protein in a cell, the method comprising a step of treating a vector into a target cell, At this time, the vector comprises a first nucleic acid having promoter activity and a second nucleic acid encoding a target protein, The sequence of the first nucleic acid having the above promoter activity includes the nucleic acid sequence of SEQ ID NO: 19, and the length of the first nucleic acid having the above promoter activity is 50 bp to 243 bp, The second nucleic acid encoding the target protein is a nucleic acid encoding the target protein to be expressed in the cell, The second nucleic acid is characterized in that it is operably linked to the 3' direction of the first nucleic acid.

10. In paragraph 9, A method for expressing a target protein in a cell, wherein the sequence of the first nucleic acid having the above promoter activity is identical to a part of the sequence of SEQ ID NO:

7.

11. In paragraph 9, A method for expressing a target protein in a cell, wherein the sequence of the first nucleic acid having the above promoter activity is any one of SEQ ID NOs: 7, 19, 22, 23, and 24.

12. In paragraph 9, A method for expressing a target protein in a cell, characterized in that the vector is a viral vector or a non-viral vector.

13. In paragraph 12, A method for expressing a target protein in a cell, characterized in that the viral vector is one selected from the group consisting of a retroviral (retrovirus) vector, a lentiviral (lentivirus) vector, an adenovirus (adenovirus) vector, an adeno-associated viral (adeno-associated virus; AAV) vector, a vaccinia viral (vaccinia virus) vector, a poxviral (poxvirus) vector, and a herpes simplex viral (herpes simplex virus) vector.

14. In paragraph 12, A method for expressing a target protein in a cell, characterized in that the non-viral vector is a plasmid, phage, naked DNA, DNA complex, mRNA (transcript), or PCR amplicon.

15. In paragraph 9, A method for expressing a target protein in a cell, characterized in that the step of treating the target cell with the vector is performed in vitro, in vivo or ex vivo.

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