Muscle-specific hybrid promoter

Abstract

The present disclosure provides a novel muscle-specific enhancer and promoter combination useful for achieving high and sustained expression in muscle tissue or muscle cells. The muscle-specific promoter element is derived from the desmin promoter. The muscle-specific enhancer element is derived from the desmin promoter and the muscle creatine kinase enhancer.

Description

Technical Field

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 077,339, filed on September 11, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence List This application is electronically submitted in ASCII format and contains a sequence listing which is hereby incorporated by reference in its entirety. The ASCII copy created on September 10, 2021 is named 85535 - 328408_SL.txt and is 48,920 bytes in size.

Background Art

[0003] Ubiquitous promoters such as CMV, EF1, or CAG do not enable target expression of gene products. This can lead to harmful side effects associated with expression in non - target tissues. For example, expression in antigen - presenting cells can result in an adverse immune response to the transgene (Weeratna RD, Wu T, Efler SM, Zhange L, Davis HL, 2001 Gene Ther. 8:1872).

[0004] In muscle tissue, the use of muscle - specific expression vectors can avoid off - target expression problems, but the low level of transgene expression from vectors containing muscle - specific promoters limits the application of these vectors in cell and gene therapy.

[0005] Therefore, there is a need for muscle - specific promoters and enhancer elements that can be incorporated into muscle - specific expression vectors for cell and gene therapy.

Summary of the Invention

[0006] This disclosure provides compositions and methods for the expression of transgenes in muscle cells using muscle-specific regulatory nucleic acid sequences.

[0007] The main objective of the present invention is to provide an expression vector optimized for high levels of transgene expression in muscle cells and muscle tissue.

[0008] The main objective of the present invention is to provide an expression vector optimized for sustained transgene expression in muscle cells and muscle tissue.

[0009] The main objective of the present invention is to provide an expression vector optimized for low transgene expression in non-muscle tissues.

[0010] The main objective of the present invention is to provide an expression vector optimized for a low CpG to GpG dinucleotide ratio.

[0011] Another object of the present invention is to provide enhancer / promoter combinations that can direct high and sustained expression levels in muscle cells and muscle tissue using a wide variety of non-viral and viral expression vector types.

[0012] These objectives are achieved by providing a hybrid promoter that drives transgene expression in muscle cells and muscle tissue by combining a desmin muscle-specific promoter and a desmin muscle-specific enhancer with an MCK muscle-specific enhancer. The resulting hybrid promoter is useful for myocyte and gene therapy. Various muscle-specific hybrid promoters of the present invention may be used for muscle-specific transgene expression in cultured cells or tissues from episomes or embedded plasmids, nanoplasmides, minicircles, Doggybone, MIDGE, adenoviruses, adeno-associated viruses (AAVs), retroviruses, and lentiviral vectors, not limited to, but as examples.

[0013] In some embodiments, a muscle-specific regulatory nucleic acid sequence is provided, comprising a mammalian desmin promoter, a mammalian desmin enhancer, and one or more mammalian muscle creatine kinase (MCK) enhancers, which are operably linked.

[0014] In some embodiments, a vector comprising the muscle-specific regulatory nucleic acid sequence of the present disclosure is provided.

[0015] In some embodiments, host cells containing the vectors of this disclosure are provided.

[0016] In some embodiments, a method for expressing an introduced gene in eukaryotic cells includes the step of transfecting eukaryotic cells with the vector of the present disclosure.

[0017] In some embodiments, a method is provided for replicating the vector of the present disclosure, comprising the steps of transforming host cells with the vector of the present disclosure and incubating the cells under conditions sufficient to replicate the vector.

[0018] For a more complete understanding of the present invention and its advantages, refer to the following description in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0019] [Figure 1A] The vector map of the NTC8685-EGFP nanoplasmid is shown. [Figure 1B] The vector map of the NTC8685-3xMCKenh-CMV-EGFP nanoplasmid is shown. [Figure 1C] The vector map of the NTC8685-3xMCKenh-MCAT-CMV-EGFP nanoplasmid is shown. [Figure 1D] The vector map of the NTC8685-C5-C12-EGFP nanoplasmid is shown. [Figure 1E] The vector map of the NTC8685-3xMCKenh-C5-C12-EGFP nanoplasmid is shown. [Figure 1F] The vector map of the NTC8685-3xMCKenh-MCAT-C5-C12-EGFP nanoplasmid is shown. [Figure 1G] The vector map of the NTC8685-3xMCKenh-MCK-EGFP nanoplasmid is shown. [Figure 1H] The vector map of the NTC8685-3xMCKenh-MCAT-MCK-EGFP nanoplasmid is shown. [Figure 1I] The vector map of the NTC8685-desmin-EGFP nanoplasmid is shown. [Figure 1J] The vector map of the pVAX1-EGFP nanoplasmid is shown. [Figure 2A] The EGFP expression results in HEK293 cells are shown. [Figure 2B] The EGFP expression results in C5-C12 myotubes are shown. [Figure 3A] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 35 and the nanoplasmid backbone has SEQ ID NO: 27. [Figure 3B] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 36 and the nanoplasmid backbone has SEQ ID NO: 27. [Figure 3C] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 35 and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 3D] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 36 and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 4A] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 35 and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 4B] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 35 and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 4C] The vector map is shown where the muscle-specific regulatory nucleic acid sequence has SEQ ID NO: 36 and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 4D]The dual promoter vector map shows that the heavy chain has the first muscle-specific regulatory nucleic acid sequence, SEQ ID NO: 35, the light chain has the second muscle-specific regulatory nucleic acid sequence, SEQ ID NO: 36, and the nanoplasmid backbone has SEQ ID NO: 28. [Figure 5A] This vector map shows a muscle-specific regulatory nucleic acid sequence having SEQ ID NO: 56 and a nanoplasmid backbone having SEQ ID NO: 27. [Figure 5B] This vector map shows a muscle-specific regulatory nucleic acid sequence having SEQ ID NO: 56 and a nanoplasmid backbone having SEQ ID NO: 28. [Figure 5C] This vector map shows a muscle-specific regulatory nucleic acid sequence having SEQ ID NO: 57 and a nanoplasmid backbone having SEQ ID NO: 28. [Figure 5D] The dual promoter vector map shows that the heavy chain has the muscle-specific regulatory nucleic acid sequence SEQ ID NO: 56, the light chain has the muscle-specific regulatory nucleic acid sequence SEQ ID NO: 58, and the nanoplasmid backbone has SEQ ID NO: 27. [Figure 6] This shows the transfection efficiency determined by luciferase expression. Administration of Nanotaxi® luciferase plasmid DNA in skeletal muscle results in high luciferase expression. Swiss mice were intramuscularly injected with 10 μg of different plasmid DNAs formulated with Nanotaxi® at D0. On day 7, injected muscles were collected and their luciferase expression was analyzed. The symbols represent individual injected muscles, and the horizontal bars represent the average and SEM values ​​for a group of 5 mice injected bilaterally. [Modes for carrying out the invention]

[0020] This disclosure provides compositions and methods for the expression of transgenes in muscle cells using muscle-specific regulatory nucleic acid sequences, and methods for replicating vectors containing said muscle-specific regulatory nucleic acid sequences.

[0021] definition As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise clearly indicated by the context.

[0022] The use of the term “or” in the claims and this disclosure shall mean “and / or” unless expressly indicated to refer only to the substitutes, or unless the substitutes are mutually exclusive.

[0023] The use of the term "approximately" when used with a number is intended to include a + / - 10% range. For example, if the number of nucleotides is specified as approximately 200, this includes 180-220 (plus or minus 10%).

[0024] As used herein, "cmv" or "CMV" refers to cytomegalovirus.

[0025] As used herein, “lentiviral vector” refers to an embedded viral vector capable of infecting dividing and non-dividing cells. It is also called a lentiviral transfer plasmid. The plasmid encodes a lentiviral LTR flanking expression unit. The transfer plasmid, along with the lentiviral envelope and packaging plasmids necessary for producing viral particles, is transfected into producing cells.

[0026] As used herein, “lentiviral envelope vector” refers to a plasmid encoding an envelope glycoprotein.

[0027] As used herein, “lentiviral packaging vector” refers to one or two plasmids that express the gag, pol, and Rev gene functions necessary for packaging a lentiviral transfer vector.

[0028] As used herein, “minicircle” refers to a covalently bound closed cyclic plasmid derivative in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site-specific recombination or in vitro restriction digestion / ligation. Minicircle vectors are incapable of replicating in bacterial cells.

[0029] As used herein, “Nanoplasmid® vector” or “Nanoplasmid” refers to a vector that combines an RNA-selectable marker with a bacterial origin of replication, such as R6K, ColE2, or a ColE2-associated origin of replication. For example, nanoplasmid vectors may include, but are not limited to, the NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors, and the modified versions described in WO2014 / 035457.

[0030] As used herein, the “NTC8 series” refers to vectors such as the NTC8385, NTC8485, and NTC8685 plasmids, which are antibiotic-free pUC origin vectors containing short RNA (RNA-OUT) selectable markers instead of antibiotic resistance markers such as kanR. The preparation and application of these RNA-OUT-based antibiotic-free vectors are described in WO2008 / 153733.

[0031] As used herein, “retroviral vector” refers to an embedded viral vector capable of infecting dividing cells. It is also called a transfer plasmid. The plasmid encodes a retroviral LTR flanking expression unit. The transfer plasmid, along with the envelope and packaging plasmids necessary to produce viral particles, is transfected into producing cells.

[0032] As used herein, “retroviral envelope vector” refers to a plasmid encoding an envelope glycoprotein.

[0033] As used herein, “retroviral packaging vector” refers to plasmids encoding retroviral gag and pol genes necessary for packaging a retroviral transfer vector.

[0034] As used herein, “transfection” or “transformation” means methods for delivering nucleic acids to cells, as known in the art and included herein by reference [e.g., poly(lactide-coglycolide) (PLGA), ISCOM, liposomes, niosomes, visomes, block copolymers, Pluronic® block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ rapid cell deformation-mediated membrane disruption, corona plasma, plasma-assisted delivery, tissue-resistant plasma, laser microporation, shock wave energy, magnetic field, non-contact magnetic permeabilization, gene guns, microneedles, microdermabrasion, hydrodynamic delivery, high-pressure tail vein injection, etc.].

[0035] As used herein, “transgene” refers to the gene of interest that is cloned into a vector for expression in a target organism.

[0036] As used herein, “vector” refers to gene delivery vehicles, including viral (e.g., alphaviruses, poxviruses, lentiviruses, retroviruses, adenoviruses, adenovirus-associated viruses, etc.) and non-viral (e.g., plasmids, MIDGE, transcriptionally active PCR fragments, minicircles, bacteriophages, nanoplasmids®, etc.) vectors. These are well known in the art and are included herein by reference.

[0037] As used herein, "Doggybone" is a minimally closed linear DNA construct, an enzymatically produced, capped linear vector.

[0038] As referred to herein, "MIDGE" is a minimal, immunologically defined gene expression vector, a small, linear, covalently closed, dumbbell-shaped molecule.

[0039] To determine the sequence identity percentage, as understood herein, the query sequence (e.g., a nucleic acid sequence) is aligned to one or more subject sequences (global alignment) using any suitable sequence alignment program known in the art, e.g., the computer program ClustalW (version 1.83, default parameters), which enables the alignment of nucleic acid sequences to be performed over their entire length. Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In a preferred manner, the sequence alignment program (e.g., ClustalW) aligns the query sequence and one or more subject sequences so that the best match between them can be calculated and identity, similarity, and difference can be determined. Gaps of one or more nucleotides can be inserted into the query sequence, subject sequences, or both to maximize the sequence alignment.

[0040] Muscle-specific regulatory nucleic acid sequences In some embodiments, a muscle-specific regulatory nucleic acid sequence is provided, comprising a mammalian desmin promoter, a mammalian desmin enhancer, and one or more mammalian muscle creatine kinase (MCK) enhancers, which are operably linked.

[0041] In any of the embodiments described above, the mammalian desmin promoter, the mammalian desmin enhancer, and one or more MCK enhancers may be human or mouse. It should be understood that the mammalian desmin promoter, the mammalian desmin enhancer, and one or more MCK enhancers may originate from any mammalian species, so that these nucleic acid sequences can be determined using known methods and search tools. As an example, but not an limitation, the mammalian desmin promoter, the mammalian desmin enhancer, and one or more MCK enhancers may originate from human, mouse, horse, pig, cat, dog, or primate sources. It should be further understood that the origins of each of the mammalian desmin promoter, the mammalian desmin enhancer, and one or more mammalian MCK enhancers may be different or the same. If the desmin enhancer or multiple mammalian MCK enhancers are included in a muscle-specific regulatory nucleic acid sequence, it should be further understood that each of the multiple elements may come from the same or different origins. As an example, and not an limitation, the muscle-specific regulatory nucleic acid sequence may be entirely human or mouse, or any combination of human and mouse elements such as a human desmin promoter, a human desmin enhancer, and one or more mouse MCK enhancers, including a mammalian desmin promoter, a mammalian desmin enhancer, and one or more mammalian MCK enhancers. In a preferred embodiment, the muscle-specific regulatory nucleic acid sequence may include one or more mouse MCK enhancers combined with a mouse desmin enhancer and a mouse desmin promoter, or one or more mouse MCK enhancers combined with a human desmin enhancer and a human desmin promoter, and more preferably, three copies of a mouse MCK enhancer combined with a human desmin enhancer and a human desmin promoter. The mammalian desmin promoter, mammalian desmin enhancer, and one or more mammalian MCK enhancers may be full-length or cleaved, however, it should be further understood that cleavage maintains at least a portion of the function of the element, for example, that a cleaved mammalian desmin promoter still has promoter activity when assayed by the expression of a downstream transgene.

[0042] In any of the embodiments described above, if the mammalian desmin promoter is human, the mammalian desmin promoter may include a nucleic acid sequence having 80% or more identity with any of SEQ ID NOs: 7, 8, 9, and 10. As an example, but not an limitation, the mammalian desmin promoter may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NOs: 7, 8, 9, and 10. In some embodiments, the mammalian desmin promoter includes the sequence of SEQ ID NOs: 7, 8, 9, and 10.

[0043] In any of the embodiments described above, if the mammalian desmin promoter is mouse, the mammalian desmin promoter may include a nucleic acid sequence having 80% or more identity with either SEQ ID NO: 4 or SEQ ID NO: 5. As an example, but not an limitation, the mammalian desmin promoter may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with either SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the mammalian desmin promoter includes the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0044] In any of the embodiments described above, the mammalian desmin promoter may include an INR sequence. As an example, but not an limitation, the INR sequence including the initiator element may be the nucleic acid sequence of SEQ ID NO: 59. As a further example, but not an limitation, the INR sequence may include the nucleic acid sequences tataaaa and yyanwyy separated by the intervening sequence, and optionally include a downstream sequence downstream of yyanwyy. It should be understood that the initiator element may include a consensus sequence of yyanwyy, which may be, for example, tcagtcc. As a further example, but not an limitation, the intervening sequence may be about 20 to about 25 nucleotides in length, e.g., about 20, 21, 22, 23, 24, or 25 nucleotides. As a further example, but not an limitation, the downstream sequence may have any preferred length.

[0045] In any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may include two or more mammalian desmin enhancers. For example, the muscle-specific regulatory nucleic acid sequence may include one, two, three, four, five or more mammalian desmin enhancer sequences. In some embodiments, the muscle-specific regulatory nucleic acid sequence includes only one mammalian desmin enhancer, i.e., the muscle-specific regulatory nucleic acid sequence does not include two or more mammalian desmin enhancers.

[0046] In any of the embodiments described above, the mammalian desmin enhancer may include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 6. As examples, but not as an limitation, the mammalian desmin enhancer may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 3 or SEQ ID NO: 6. In some embodiments, the mammalian desmin enhancer includes the sequence of SEQ ID NO: 3. In some embodiments, the mammalian desmin enhancer includes the sequence of SEQ ID NO: 6.

[0047] In any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may include one or more mammalian MCK enhancers. As an example, but not an limitation, the muscle-specific regulatory nucleic acid sequence may include two or more mammalian MCK enhancers, one to three mammalian MCK enhancers, or one, two, three, four, or five or more mammalian MCK enhancers. It should be understood that one or more mammalian MCK enhancers may be separated by a ligation sequence, or, for example, by a desmin enhancer or desmin promoter, or by a transgene, and may have other elements between them if there are two or more. As an example, and not an limitation, one or more mammalian MCK enhancers may be isolated by 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides. It should be further understood that one or more mammalian MCK enhancers may be isolated by mammalian desmin promoters or mammalian desmin enhancers.

[0048] In any of the embodiments described above, each of the one or more mammalian MCK enhancers may contain a nucleic acid sequence having at least 80% identity with SEQ ID NO: 11 (mouse MCK enhancer) or SEQ ID NO: 12 (human MCK enhancer). As an example, not an limitation, one or more mammalian MCK enhancers may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, each of the one or more mammalian MCK enhancers contains the sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, each of the one or more mammalian MCK enhancers may be an MCK CK7 enhancer, such as the MCK CK7 enhancer of SEQ ID NO: 1, not an limitation. As a further example, not an limitation, one or more mammalian MCK enhancers may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1.

[0049] In any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may further include one or more additional enhancers. In some embodiments, each of the one or more additional enhancers includes a nucleic acid sequence having 80% or more identity with any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. As an example, but not an limitation, each of the one or more additional enhancers may have at least 80%, 85%, 90%, 96%, 97%, 98%, or 99% or more identity with any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some embodiments, each of the one or more additional enhancers includes the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0050] In some embodiments, the muscle-specific regulatory nucleic acid sequence comprises a mammalian desmin enhancer and one or more mammalian MCK enhancers as its sole enhancer. In some embodiments, the muscle-specific regulatory nucleic acid sequence does not contain one or more additional enhancers. In some embodiments, the muscle-specific regulatory nucleic acid sequence does not contain a vertebrate troponin I IRE(FIRE) enhancer.

[0051] In any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may further include an intron. In any of the embodiments described above, the intron may be positioned 3' relative to the mammalian desmin promoter. Any suitable intron may be used. In any of the embodiments described above, the intron may include a nucleic acid sequence having 80% or more identity with SEQ ID NO: 17 or SEQ ID NO: 18. As an example, but not as an limitation, the intron may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the intron includes the sequence of SEQ ID NO: 17 or SEQ ID NO: 18. In any of the embodiments described above, the intron may have a size of about 100 to about 10,000 nucleotides. As an example, not as an limitation, introns are approximately 100-10,000 nucleotides, approximately 100-9,000 nucleotides, approximately 100-8,000 nucleotides, approximately 100-7,000 nucleotides, approximately 100-6,000 nucleotides, approximately 100-5,000 nucleotides, approximately 100-4,000 nucleotides, approximately 100-3,000 nucleotides, approximately 100-2,000 nucleotides, approximately 100-1,000 nucleotides, approximately 100-500 nucleotides, approximately 100-400 nucleotides, approximately 100-300 nucleotides, approximately 100-200 nucleotides, approximately 200-10,000 nucleotides, approximately 200-9,000 nucleotides, approximately 200-8,000 nucleotides, and approximately 20 0 to approximately 7,000 nucleotides, approximately 200 to approximately 6,000 nucleotides, approximately 200 to approximately 5,000 nucleotides, approximately 200 to approximately 4,000 nucleotides, approximately 200 to approximately 3,000 nucleotides, approximately 200 to approximately 2,000 nucleotides, approximately 200 to approximately 1,000 nucleotides, approximately 200 to approximately 500 nucleotides, approximately 200 to approximately 400 nucleotides, approximately 200 to approximately 300 nucleotides, approximately 300 to approximately 10,000 nucleotides, approximately 300 to approximately 9,000 nucleotides, approximately 300 to approximately 8,000 nucleotides, approximately 300 to approximately 7,000 nucleotides, approximately 300 to approximately 6,000 nucleotides, approximately 300 to approximately 5,000 nucleotides, approximately 300 to approximately 4,000 nucleotides, approximately 300 to approximately 3,000 nucleotides, approximately 300 to approximately 2,000 nucleotides, approximately 300-1,000 nucleotides, approximately 300-500 nucleotides, approximately 300-400 nucleotides, approximately 400-10,000 nucleotides, approximately 400-9,000 nucleotides, approximately 400-8,000 nucleotides, approximately 400-7,000 nucleotides, approximately 400-6,000 nucleotides, approximately 400-5,000 nucleotides, approximately 400-4,000 nucleotides, approximately 400-3,000 nucleotides, approximately 400-2,000 nucleotides, approximately 400-1,000 nucleotides, approximately 400-5 00 nucleotides, approximately 500-10,000 nucleotides, approximately 500-9,000 nucleotides, approximately 500-8,000 nucleotides, approximately 500-7,000 nucleotides, approximately 500-6,000 nucleotides, approximately 500-5,000 nucleotides, approximately 500-4,000 nucleotides, approximately 500-3,000 nucleotides, approximately 500-2,000 nucleotides, approximately 500-1,000 nucleotides, approximately 1,000-10,000 nucleotides, approximately 1,000-9,000 nucleotides, approximately 1,000-8,000 nucleos Tide, approximately 1,000-7,000 nucleotides, approximately 1,000-6,000 nucleotides, approximately 1,000-5,000 nucleotides, approximately 1,000-4,000 nucleotides, approximately 1,000-3,000 nucleotides, approximately 1,000-2,000 nucleotides, approximately 2,000-10,000 nucleotides, approximately 2,000-9,000 nucleotides, approximately 2,000-8,000 nucleotides, approximately 2,000-7,000 nucleotides, approximately 2,000-6,000 nucleotides, approximately 2,000-5,000 nucleotides, approximately 2,000 00-approximately 4,000 nucleotides, approximately 2,000-approximately 3,000 nucleotides, approximately 3,000-approximately 10,000 nucleotides, approximately 3,000-approximately 9,000 nucleotides, approximately 3,000-approximately 8,000 nucleotides, approximately 3,000-approximately 7,000 nucleotides, approximately 3,000-approximately 6,000 nucleotides, approximately 3,000-approximately 5,000 nucleotides, approximately 3,000-approximately 4,000 nucleotides, approximately 4,000-approximately 10,000 nucleotides, approximately 4,000-approximately 9,000 nucleotides, approximately 4,000-approximately 8,000 nucleotides, approximately 4,000-approximately 7,000 nucleotides, approximately 4,000 to 6,000 nucleotides, approximately 4,000 to 5,000 nucleotides, approximately 5,000 to 10,000 nucleotides, approximately 5,000 to 9,000 nucleotides, approximately 5,000 to 8,000 nucleotides, approximately 5,000 to 7,000 nucleotides, approximately 5,000 to 6,000 nucleotides, approximately 6,000 to 10,000 nucleotides, approximately 6,000 to 9,000 nucleotides, approximately 6,000 to 8,000 nucleotides, approximately 6,000 to 7,000 nucleos The nucleotides may have sizes of approximately 7,000 to 10,000 nucleotides, approximately 7,000 to 9,000 nucleotides, approximately 7,000 to 8,000 nucleotides, approximately 8,000 to 10,000 nucleotides, approximately 8,000 to 9,000 nucleotides, approximately 9,000 to 10,000 nucleotides, or approximately 100, 200, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 nucleotides.

[0052] In any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may further include a transgene. In any of the embodiments described above, the transgene may be positioned 3' relative to the mammalian desmin promoter. The transgene may be under the control of the mammalian desmin promoter. The transgene may be any suitable transgene, for example, a therapeutic transgene such as VEGF, a gene therapy replacement gene such as factor IX, a reverse vaccine antigen such as insulin for diabetes, or a therapeutic antibody such as Avastin. In any of the embodiments described above, the mammalian desmin promoter may be isolated from the transgene by about 500 or fewer nucleotides between the mammalian desmin promoter and the transgene, without any introns. As an example, not as an limitation, mammalian desmin promoters have approximately 0 to 1000 nucleotides, approximately 1 to 1000 nucleotides, approximately 1 to 900 nucleotides, approximately 1 to 800 nucleotides, approximately 1 to 700 nucleotides, approximately 1 to 600 nucleotides, approximately 1 to 500 nucleotides, approximately 1 to 400 nucleotides, approximately 1 to 300 nucleotides, approximately 1 to 200 nucleotides, approximately 1 to 100 nucleotides, approximately 1 to 90 nucleotides, approximately 1 to 80 nucleotides, approximately 1 to 70 nucleotides, approximately 1 to 60 nucleotides, approximately 1 to 50 nucleotides, approximately 1 to 40 nucleotides, approximately 1 to 30 nucleotides, approximately 1 to 20 nucleotides, approximately 1 to 10 nucleotides, approximately 10 to 1000 nucleotides, and approximately 10 to 900 nucleotides. Approximately 10-800 nucleotides, approximately 10-700 nucleotides, approximately 10-600 nucleotides, approximately 10-500 nucleotides, approximately 10-400 nucleotides, approximately 10-300 nucleotides, approximately 10-200 nucleotides, approximately 10-100 nucleotides, approximately 100-1000 nucleotides, approximately 100-500 nucleotides, approximately 200-1000 Transgenes can be isolated by nucleotides, approximately 200 to 500 nucleotides, approximately 500 to 1000 nucleotides, or approximately 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides.

[0053] In any of the embodiments described above, one or more mammalian MCK enhancers may be positioned 5' relative to a mammalian desmin enhancer, and a mammalian desmin enhancer may be positioned 5' relative to a mammalian desmin promoter. In any of the embodiments described above, it should be understood that, as already described with respect to one or more MCK enhancers, linking sequences may exist between elements of muscle-specific regulatory nucleic acid sequences, as long as the elements are operably linked. As an example, not as an limitation, a mammalian desmin promoter, a mammalian desmin enhancer, and one or more MCK enhancers, which are one or an element of a muscle-specific regulatory nucleic acid sequence, are approximately 0 to approximately 1000 nucleotides, approximately 1 to approximately 1000 nucleotides, approximately 1 to approximately 900 nucleotides, approximately 1 to approximately 800 nucleotides, approximately 1 to approximately 700 nucleotides, approximately 1 to approximately 600 nucleotides, approximately 1 to approximately 500 nucleotides, approximately 1 to approximately 400 nucleotides, approximately 1 to approximately 300 nucleotides, approximately 1 to approximately 200 nucleotides, approximately 1 to approximately 100 nucleotides, approximately 1 to approximately 90 nucleotides, approximately 1 to approximately 80 nucleotides, approximately 1 to approximately 70 nucleotides, approximately 1 to approximately 60 nucleotides, approximately 1 to approximately 50 nucleotides, approximately 1 to approximately 40 nucleotides, approximately 1 to approximately 30 nucleotides, approximately 1 to approximately 20 nucleotides, and approximately 1 to approximately 10 nucleotides. Otid, approximately 10 to approximately 1000 nucleotides, approximately 10 to approximately 900 nucleotides, approximately 10 to approximately 800 nucleotides, approximately 10 to approximately 700 nucleotides, approximately 10 to approximately 600 nucleotides, approximately 10 to approximately 500 nucleotides, approximately 10 to approximately 400 nucleotides, approximately 10 to approximately 300 nucleotides, approximately 10 to approximately 200 nucleotides, approximately 10 to approximately 100 nucleotides, approximately 100 to approximately 1000 nucleotides, approximately 100 It can be separated by approximately 500 nucleotides, approximately 200 to approximately 1000 nucleotides, approximately 200 to approximately 500 nucleotides, approximately 500 to approximately 1000 nucleotides, or approximately 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides.

[0054] Preferred sequences for muscle-specific regulatory nucleic acid sequences may include SEQ ID NOs: 31, 32, 33, 34, 35, 36, 55, 56, 57, and 58. As an example, but not an limitation, muscle-specific regulatory nucleic acid sequences may include nucleic acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 55, 56, 57, and 58.

[0055] Details of these preferred sequences are provided below.

[0056] MCK CK7 enhancer, h-desmin enhancer, h-desmin promoter, MVM intron (SEQ ID NO: 31).

[0057] MCKCK7 enhancer, h-desmin enhancer, h-desmin INR promoter, MVM intron (SEQ ID NO: 32).

[0058] MCK CK7 enhancer, hDesmin enhancer, hDesmin S promoter, MVM intron (SEQ ID NO: 33).

[0059] MCK CK7 enhancer, hDesmin enhancer, hDesmin S INR promoter, MVM intron (SEQ ID NO: 34).

[0060] MCK CK7 enhancer, h desmin enhancer, h desmin INR promoter, pCI intron (Figures 3A, 3C, 4A, 4B, and 4D, Sequence ID 35)

[0061] MCK CK7 enhancer, h desmin enhancer, h desmin S INR promoter, pCI intron (Figures 3B, 3D, 4C, and 4D, Sequence ID 36).

[0062] MCK CK7 enhancer, m-desmin enhancer, m-desmin promoter, MVM intron (SEQ ID NO: 55).

[0063] MCK CK7 enhancer, mdesmin enhancer, mdesmin INR promoter, MVM intron (Figures 5A, 5B, and 5D, Sequence ID 56).

[0064] MCK CK7 enhancer, mdesmin enhancer, mdesmin INR promoter, pCI intron (Figure 5C, (SEQ ID NO: 57)).

[0065] MCK CK7 enhancer, m-desmin enhancer, m-desmin promoter, pCI intron (Figure 5D, Sequence ID 58).

[0066] A preferred configuration, not as an limitation but as an example, may include the following:

[0067] 5'-1 or more mammalian MCK enhancers, mammalian desmin enhancers, mammalian desmin promoters-3'.

[0068] Other configurations of muscle-specific regulatory nucleic acid sequences may include, but are not limited to, the following:

[0069] One or more mammalian MCK enhancers, mammalian desmin enhancers, one or more mammalian MCK enhancers, mammalian desmin promoters.

[0070] Mammalian desmin enhancer, one or more mammalian MCK enhancers, mammalian desmin promoter.

[0071] A mammalian desmin promoter, one or more mammalian MCK enhancers, and a mammalian desmin enhancer.

[0072] Mammalian desmin promoter, one or more mammalian MCK enhancers, mammalian desmin enhancer, one or more mammalian MCK enhancers.

[0073] Mammalian desmin promoter, mammalian desmin enhancer, one or more mammalian MCK enhancers.

[0074] In any of the other configurations described above, introns may be inserted into muscle-specific regulatory nucleic acid sequences. Similarly, in any of the configurations described above, the transgene may be located downstream of the mammalian desmin promoter, possibly between additional elements, or between the desmin promoter and additional elements. It should also be understood that multiple mammalian desmin enhancers may exist, and that any combination of a mammalian desmin enhancer and one or more mammalian MCK enhancers can be constructed with respect to the order of the elements.

[0075] vector In some embodiments, a vector is provided that includes a muscle-specific regulatory nucleic acid sequence from any of the embodiments described above.

[0076] The vector can be any suitable vector for transfecting cells with a muscle-specific regulatory nucleic acid sequence. As an example, but not an limitation, the vector may be a plasmid, minicircle, Doggybone, MIDGE, nanoplasmid, or viral vector. As a further example, but not an limitation, the vector may be an episome non-replicating expression vector, an episome replicating expression vector, a transposon-integrated vector, a viral-integrated vector, or a homologous recombination repair vector.

[0077] In some embodiments, when the vector is a viral vector, the viral vector may be an adenovirus, an adeno-associated virus (AAV), a lentivirus, or a retrovirus.

[0078] In some embodiments, the vector may contain two or more muscle-specific regulatory nucleic acid sequences.

[0079] In some embodiments, the vector may be a double promoter vector.

[0080] If the vector is a nanoplasmid, it should be understood that the nanoplasmid may include a eukaryotic region having 5' and 3' ends and potentially containing muscle-specific regulatory nucleic acid sequences and transgenes, and a spacer region of less than 500 base pairs linking the 5' and 3' ends of the eukaryotic region, and may include, as an example but not an limitation, bacterial origins of replication and RNA selectable markers such as R6K or ColE2. Non-limiting exemplary R6K origins are provided in SEQ ID NOs. 19-23, and exemplary RNA selectable markers are provided in SEQ ID NOs. 24 and 26. If the bacterial origin is any one of the R6K origins in SEQ ID NOs. 19-23, it should be understood that each bacterial origin may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs. It should be further understood that if the RNA-selectable marker is one of SEQ ID NOs. 24 or 26, the RNA-selectable marker may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with either SEQ ID NOs. 24 or 26, respectively. Nanoplasmid vectors are also described in International Patent Application Publication No. 2014 / 077866 and U.S. Patent Application No. 2010 / 0184158, each of which is incorporated herein by reference in whole.

[0081] In some embodiments, if the vector is a nanoplasmid, the vector may contain any of the sequences of SEQ ID NOs. 27, 28, 29, and 30. SEQ ID NOs. 27 and 29 are six- and seven-R6K originating iterone versions of the NTC9385R backbone, and SEQ ID NOs. 28 and 30 are six- and seven-R6K originating iterone versions of the NTC9385R(3xCpG) backbone.

[0082] Nanoplasmid vector maps using these preferred sequences, including the transgene (EGFP), are shown as examples in Figures 3A-3D and 5A-5D. Nanoplasmid vector maps using these preferred sequences, including the monoclonal antibody light chain (mAB LC) or heavy chain (mAB HC) or both LC and HC transgenes, are shown as examples in Figures 4A-4D and 5A-5D. These vectors can be used for passive immunotherapy, for example, for the in vivo expression of virus-neutralizing antibodies (Bakker JM, Bleeker WK, Parren PWHI. 2004. Mol Ther 10:1525; Tjelle TE, Corthay A, Lunde E, Sandlie I, Michaelsen TE, Mathiesen I, Bogen B. 2004. Mol Ther 9:328; Hollevoet K, Declerck PJ. 2017. J Transl Med 15:131). The antibody light and heavy chains may be expressed in different vectors, or both may be expressed in a dual promoter vector. A dual promoter vector, as an example of expressing both the LC and HC transgenes from a preferred muscle promoter in a single vector, is shown in Figures 4D and 5D.

[0083] In some preferred embodiments, the vector may have a CpG-to-GpG ratio of less than 0.7. As examples, but not as limitations, the vector may have a CpG-to-GpG ratio of 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, or less than 0.3. As further examples, but not as limitations, the vector may have a CpG-to-GpG ratio of about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.4 to about 0.5, about 0.25, about 0.3, about 0.35, about 0.4, about 0.5, about 0.55, or about 0.6.

[0084] In any of the embodiments described above, the vector may comprise two or more muscle-specific regulatory nucleic acid sequences according to any of the embodiments described above. As an example, but not as an limitation, the vector may comprise a first transgene comprising a first muscle-specific regulatory nucleic acid sequence under the control of a mammalian desmin promoter of the first muscle-specific regulatory nucleic acid sequence, and a second transgene comprising a second muscle-specific regulatory nucleic acid sequence under the control of a mammalian desmin promoter of the second muscle-specific regulatory nucleic acid sequence. In such embodiments, the first and second transgenes may encode the same or different products. As a further example, but not as an limitation, the first transgene may encode an antibody heavy chain, and the second transgene may encode an antibody light chain.

[0085] It should be understood that in any of the embodiments described above, the muscle-specific regulatory nucleic acid sequence may be located at the 5' or 3' end of the transgene. As an example, and not an limitation, in a nanoplasmid, the muscle-specific regulatory nucleic acid sequence may be located at the 5' end or the 3' end of the eukaryotic region, for example, at the 5' or 3' end of the transgene, because the transgene may still be under the control of the mammalian desmin promoter throughout the spacer region.

[0086] Similarly, it should be understood that the mammalian MCK enhancer and desmin enhancer of this disclosure may be positioned downstream of the transgene.

[0087] host cell In some embodiments, transformed host cells containing the vector of the Disclosure are provided. The host cells may be any suitable bacterial cells, such as DH5α. In some embodiments, transfected eukaryotic cells containing the vector of the Disclosure are provided. The eukaryotic cells may be human muscle cells, myotubes, or myoblasts, not as an example but as an limitation. It should be understood that the human muscle cells may be skeletal muscle cells, cardiomyocytes, and diaphragmatic muscle cells, not as an example but as an limitation.

[0088] Method for producing the vector of this disclosure In some embodiments, a method for preparing a muscle-specific expression vector may include providing a vector containing a non-muscle-specific promoter or a non-desmin promoter, and modifying the vector so that the non-muscle-specific promoter or non-desmin promoter is replaced by a muscle-specific regulatory nucleic acid sequence of the Disclosure. The vector and the muscle-specific regulatory nucleic acid sequence may be as described in any of the above embodiments of the Disclosure.

[0089] Methods for replication or expression In some embodiments, a method for expressing a transgene in eukaryotic cells includes the step of transfecting eukaryotic cells with the vector of the Disclosure. It should be understood that the transfection can be performed under conditions sufficient for the vector to express the transgene in eukaryotic cells. In some embodiments, the eukaryotic cells are muscle cells. It should be understood that human muscle cells may be, but not limited to, skeletal muscle cells, cardiomyocytes, or diaphragmatic muscle cells. As, but not limited to, a method for transforming host cells with the vector of the Disclosure may include administering the vector of the Disclosure to a subject. As, but not limited to, a further example, the subject may be human.

[0090] In some embodiments, methods are provided for replicating the vectors of the present disclosure, comprising the steps of transforming host cells with the vectors of the present disclosure and incubating the cells under conditions sufficient to replicate the vectors. Methods for transfecting host cells and conditions for incubating host cells under conditions sufficient to replicate the vectors are known to those skilled in the art. [Examples]

[0091] In the following examples, cloning for creating vectors containing various transgenes, muscle promoters, 5'UTR introns, etc., as described herein was constructed using standard restriction fragment ligation-mediated cloning. All constructs were validated by restriction digestion and sequencing.

[0092] In the following examples, nanoplasmid vectors were cloned and amplified in the R6K-based "copy cutter" host cell lines NTC1050811-HF and NTC1050811-HFdcm, which were created and disclosed in Williams 2019 VIRAL AND NON-VIRAL NANOPLASMID VECTORS WITH IMPROVED PRODUCTION, International Patent Application Publication No. 2019 / 183248 (incorporated herein by reference). The pVAX1 vector was amplified in DH5α cells.

[0093] In the following examples, a proprietary plasmid + shaking culture medium was used for shaking flask production. Seed culture was started from glycerol stock or colonies and streaked onto LB agar plates containing 50 μg / mL of antibiotic (kanR-selective pVAX1 plasmid) or 6% sucrose (RNA-OUT-selective NTC8 plasmid and NTC9 nanoplasmid). The plates were grown at 30-32°C, and the cells were resuspended in the culture medium at approximately 2.5 OD. 600 The inoculum was used to prepare a 500 mL plasmid + shaking flask containing either 50 μg / mL of antibiotic for the kanR-selective pVAX1 plasmid, or 0.5% sucrose for selection of RNA-OUT plasmids and nanoplasmides. The flask was grown with shaking to saturate at the growth temperature as indicated. Low-endotoxin nanoplasmid DNA was purified using Nucleobond AX2,000 or AX10,000 columns (Macherey Nagel, Duren, Germany).

[0094] Table 1 summarizes the various muscle-specific promoters described in the art. Native muscle promoters such as human or mouse desmin, or mouse muscle creatine kinase (MCK), have relatively low expression levels compared to CMV. Many hybrid muscle promoters, which combine enhancers and promoters from different muscle-specific regulatory elements, also have relatively low expression compared to CMV. For example, Souza and Armentano's WO2002 / 095006 obtained a relatively weak promoter by combining the MCK enhancer with the h-desmin promoter (Table 1; DC310 and DC311). This teaches that combining the MCK enhancer and the h-desmin promoter is an inhibitor of obtaining a potent muscle promoter. Other promoter-enhancer combinations, such as tMCK and Sk-CRM4-Des, have been shown to create hybrid muscle promoters with activity exceeding that of the CMV promoter (Table 1). [Table 1-1] [Table 1-2]

[0095] [Example 1: EGFP reporter expression in various promoter-enhancer constructs] Various native and hybrid muscle promoter versions of the NTC8685 vector (containing a pUC origin and an antibiotic-free RNA-OUT sucrose selector cassette) were constructed (Luke, JM, Vincent JM, Du, SX, Gerdemann U, Leen AM, Whalen RG, Hodgson CP Williams JA. 2011. Gene Ther 18:334), and their expression levels were determined in C2C12 myotubes, A549, and HEK293 cells.

[0096] Vector maps of the tested structures are provided in Figures 1A to 1H. Individual arrangements of specific elements in the vectors are listed and described in Table 2 below. [Table 2]

[0097] Adherent HEK293 (human embryonic kidney), A549 (human lung cancer), and C2C12 (mouse muscle) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were propagated in Dulbecco's modified Eagle medium / F12 containing 10% fetal bovine serum and divided using Invitrogen (Carlsbad, CA, USA) reagent and conventional methods (0.25% trypsin-EDTA).

[0098] Cells were plated onto 24-well tissue culture dishes for transfection. Plasmids and nanoplasmids were transfected into cell lines using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). For HEK293 and A549 cells, 0.1 ug of test plasmid per well was used for transfection, and expression was measured 2-3 days after transfection. For C2C12 cells, 0.4 ug of test plasmid per well was used for transfection, and the cells were then differentiated into myotubes by adding DMEM F12 + 2% horse serum (differentiation medium). Expression in the differentiated myotube structures was measured at T=6-7 days.

[0099] Whole cell lysates for EGFP determination were prepared by resuspending cells in cell lysis buffer (CelLytic M, Sigma, St Louis, MO, USA), incubating at 37°C for 30 minutes, and then lysing the cells by freeze-thaw cycles at -80°C. The lysed cells were clarified by centrifugation, and the supernatant was assayed for EGFP using an FLX800 microplate fluorescence reader (Bio-Tek, Winooski, VT, USA).

[0100] The EGFP expression levels obtained for HEK293 cells and C5-C12 myoblasts are shown in Figures 2A and 2B.

[0101] As shown in Figures 2A and 2B, the C5-12 randomly assembled synthetic promoter (Li X, Eastman EM, Schwartz RJ, Draghia-Akli RD 1999. Nature Biotech 17:241) was the most potent muscle promoter, followed by γ-desmin, and then mMCK (combination of three copies of the CK7 enhancer SEQ ID NO: 1 with the mouse MCK promoter -357- + SEQ ID NO: 40). Interestingly, attempts to create hybrid promoters with improved activity by modifying the CMV, C5-12, and MCK promoters and instead adding muscle-specific enhancer elements resulted in reduced promoter strength. Adding three copies of the CK7 MCK enhancer SEQ ID NO: 1 upstream of the CMV or C5-12 promoter dramatically reduced muscle expression. Similarly, muscle-specific expression was further reduced by adding two copies (SEQ ID NO: 47) of M-CAT (a muscle-CAT motif containing a 5'CATTCCT-3'TEF-1 binding site; Li et al., see above, 1999) (SEQ ID NO: 46) between the CK7 MCK enhancer (SEQ ID NO: 1) and the C5-12 and MCK promoters. This is consistent with the teachings of Souza and Armentano, WO2002 / 095006, who obtained a relatively weak promoter by combining the MCK enhancer with the h-desmin promoter. In summary, these results suggest that the CK7 MCK enhancer and M-CAT motif reduce muscle-specific expression when cloned upstream of heterologous promoters. Consistent with this, all potent promoters in Table 1 that use the MCK enhancer incorporate them upstream of the MCK promoter.

[0102] Other promoter-enhancer combinations, such as tMCK (Wang B, Li J, Fu FH, Chen C, Zhu X, Zhou L, Jiang X, Xiao X. 2008. Gene Ther 15:1489) and Sk-CRM4-Des (Sarcar S, Tualamba W, et al. 2019. Nat Comm 10:492), have been shown to create hybrid muscle promoters with activity exceeding that of the CMV promoter (Table 1). These promoters were tested using linear AAV vectors and may not function similarly in superhelical plasmids or nanoplasmid DNA templates. To test this, several muscle promoter expression vectors were created using pVAX1 (pUC-derived kanR vector; Invitrogen) and the NTC9385R nanoplasmid backbone. This allowed for the evaluation of 1) the muscle promoter itself and 2) the effects of the vector backbone on myocyte expression. The expression results are shown in Table 3. All vectors have the same bovine growth hormone-derived polyadenylation signal. [Table 3-1] [Table 3-2]

[0103] The vector backbone had a dramatic effect on expression. pVAX1 expression was 10–20 times lower than with the NTC9385R with CMV and the Sk-CRM4-Des promoter, and 3 times lower with the novel hybrid Sk-CRM4-tMCK promoter. This teaches that the nanoplasmid backbone dramatically improves expression compared to pVAX1.

[0104] When pVAX1 CMV is used as the baseline for CMV promoter expression, all muscle-specific nanoplasmid vectors exhibit improved expression in muscle cells compared to CMV.

[0105] However, when compared to the nanoplasmid CMV as a reference, only the Sk-CRM4-Des promoter is improved as compared to CMV.

[0106] The tMCK promoter expression was much lower than that of the Sk-CRM4-Des promoter. As observed in FIGS. 2A-2B using the CK7 MCK enhancer and the M-CAT motif, the addition of the muscle enhancer to the tMCK promoter (intron MCK SIE enhancer SEQ ID NO: 41; Tai PWL, Fisher-Aylor KI, Himeda CL, Smith CL, MacKenzie AP, Helterline DL, Agnello JC, Welikson RE <Wold BH, Hauschka SD. 2011. Skeletal Muscle1:25) decreased the expression (Table 3). The intron MCK SIE enhancer also reduced the expression from the Sk-CRM4-Des promoter by 2-fold and slightly reduced the expression from the tMCKE-m desmin promoter.

[0107] The mMCK 2RS5 enhancer SEQ ID NO: 38 (three copies of SEQ ID NO: 37; Wang et al., supra, 2008) was tested in combination with the m desmin enhancer and promoter (tMCKE-m desmin) to determine whether the MCK enhancer could serve as an alternative to Sk-CRM4 in the Sk-CRM4-Des promoter. However, NTC9385R-tMCKE-m desmin had 2-fold lower activity in myocytes than NTC9385 R-CRM-m desmin.

[0108] Collectively, these data suggest that the MCK enhancer may not improve expression in myocytes when cloned upstream of the CMV, C5-12, and desmin promoters, and that most combinations of muscle-specific promoter elements are not beneficial but harmful to the expression levels from these promoters.

[0109] [Example 2: Evaluation of a novel MCK desmin muscle promoter in pVAX1 and nanoplasmid backbone] Of the promoters tested from Example 1, the Sk-CRM4-Des promoter showed the most potent expression in the nanoplasmid vector backbone. Surprisingly, contrary to the above results, replacing the Sk-CRM4-Des promoter in NTC9385R-CRM-m desmin with one or three copies of the MCK CK7 enhancer (SEQ ID NO: 1) created a novel NTC9385R-MCK CK7 E vector with muscle-specific expression equivalent to that of the Sk-CRM4-Des promoter, as shown in Table 4 below. This additional data was generated using the same method as used in Example 1. All vectors have the same bovine growth hormone-derived polyadenylation signal. [Table 4-1] [Table 4-2]

[0110] The NTC9385R-MCK CK7 E-Vector is, For a) and b), 1 to 3 copies of the MCK CK7 enhancer located at 5', For b)c), the m-desmin enhancer located at 5', For c)d), the m-desmin promoter located at 5' (with or without INR), d) Having a hybrid muscle promoter, including an MVM intron.

[0111] The mDesmin INR promoter was constructed similarly to the MCK INR disclosed in Salva et al. 2007. Mol Ther 15:320. Alterations to the INR (Initiator-Core Promoter Element) increase the activity of the TATA box, thereby increasing transcription initiation. While INR alterations slightly increase promoter expression in non-muscle cells (Table 5; HEK293 and A549 cells), the INR-containing promoter remains highly specific to muscle cells.

[0112] Table 5 below provides the EGFP expression levels of various constructs. All vectors have the same bovine growth hormone-derived polyadenylation signal. The enhancer, promoter, and intron sequences are as shown in Table 4. [Table 5]

[0113] Substitution of three copies of the MCK CK7 enhancer with three copies of the MCK2R enhancer resulted in a twofold reduction in expression (Table 6: NTC9385R-MCK 3x MCK2R m-desmin-pCI vs. NTC9385R-MCK 3x CK7 E m-desmin-pCI). This suggests that the 2R modification that alters the left E-box to match the right E-box (Hauser et al 2000. Mol Ther 2: 16) is detrimental to expression with the desmin promoter in this context.

[0114] We constructed the NTC9385R nanoplasmid MCK-desmin promoter vector and replaced the mouse desmin enhancer promoter with the human desmin enhancer promoter (with or without INR and short and long versions; the short version removes the negative region within the promoter as reported in Li, Z and Paulin D. 1991. J Biol Chem 266:-6562). Similarly, we constructed MVM intron and pCI intron versions of both the MCK m-desmin and MCK h-desmin promoters and tested their expression in muscle cells. The results are given in Tables 6 and 7. [Table 6-1] [Table 6-2] [Table 7]

[0115] The data demonstrate that MCK h desmin exhibits superior expression in myotubes compared to MCK m desmin. Both MCK h desmin S and MCK h desmin show similarly high expression in myotubes, with the inclusion of INR providing improved expression in myotubes. The pCI intron demonstrates improved expression compared to the MVM intron in myotubes, and the nanoplasmid vector shows a significant improvement in expression compared to the pVAX1 vector in myotubes.

[0116] The muscle-specific regulatory nucleic acid sequences and vectors of this disclosure may also preferably have a low CpG to GpG ratio (Table 8). A lower CpG to GpG ratio correlates with reduced transgene immunogenicity, hypothesized by GpG competition with CpG for TLR9 binding (Gottlieb P, Utz PJ, Robinson W, Steinman L. 2013. Clin Immunol 149:297). Nanoplasmid vectors incorporating the muscle promoters of the present invention also preferably have a low CpG to GpG ratio, particularly compared to existing CMV promoter vectors such as pVAX1 (Table 9). This may lead to a reduction in the immune response to target transgenes, which would be highly beneficial for gene therapy and passive immunotherapy applications where the immune response is a concern (Weeratna et al, see above, 2001; Hollevoet and Declerck, see above, 2017). [Table 8] [Table 9]

[0117] [Example 3: In vivo evaluation of a novel MCK desmin muscle promoter in a nanoplasmid vector scaffold] The MCK desmin muscle promoter nanoplasmid vector can be evaluated in vivo for improved expression compared to the pVAX1 CMV control. For example, a luciferase transgene version of pVAX1, as well as the nanoplasmid MCK-h desmin vectors shown in Figures 3A-3D and 5A-5D, and the nanoplasmid MCK-m desmin vectors shown in Figures 5A-5D, were prepared, and purified DNA was delivered in vivo to mouse muscle. Delivery methods included IM delivery by electroporation, IM delivery by block polymers such as X-shaped poloxamines = Tetronic 304, 704, 904, 908 from BASF (Ludwigshafen, Germany), and IM delivery by lipids. Luciferase expression was determined at various time points in the injected muscle. Nanoplasmid MCK desmin muscle promoter expression levels 1-2 logs better than pVAX1 CMV are expected.

[0118] For example, in vivo block copolymer delivery to mouse muscle of the luciferase-transformed version of the MCK-h desmin nanoplasmid [NTC9385R(3xCpG)-MCK h desmin-Luc CpG-free BGH pA(4099bp)] in Figure 3C and the MCK-m desmin nanoplasmid [NTC9385R(3xCpG)-MCK m desmin-Luc CpG-free BGH pA(3948bp)] in Figure 5B demonstrated more than one log improvement in expression compared to the pVAX1 plasmid [pVAX1-Luc(4613bp)], as described below (Figure 6).

[0119] Three DNA preparations using amphiphilic block copolymer (ABC) (Nanotaxi; In-Cell-Art, Nantes, France) were prepared by mixing equal volumes of ABC stock aqueous solution and plasmid DNA solution in a buffer solution at the desired concentration.

[0120] Animal experiments were conducted in accordance with institutional and national ethical guidelines. Mice were anesthetized with isoflurane and then injected with the ABC / DNA solution. Mouse luciferase gene expression experiments were performed using a group of 6-week-old female Swiss mice (Janvier, Le Genest Saint Isle, France). The ABC / DNA preparation was intramuscularly injected bilaterally into both shaved tibialis anterior muscles. The injected muscles were harvested 7 days after injection, frozen in liquid nitrogen, and stored at -80°C until assayed for luciferase activity.

[0121] Luciferase activity in the injected muscle was analyzed as described in Pitard B, et al. 2002. Human Gene Therapy 13:1767-75. The results are shown in Figure 6.

[0122] [Example 4: Evaluation of a novel MCK desmin muscle promoter in a nanoplasmid vector scaffold] Extending from the surprising observation disclosed herein that one or three copies of the MCK CK7 enhancer can substitute for SK-CRM4 in the Sk-CRM4-Des promoter (Table 1: Sarcar et al., see above 2019), it is predicted that the incorporation of the CRE02 (SEQ ID NO: 15) and CRE64 (SEQ ID NO: 16) enhancers, which improve expression from the Sk-CRM4-Des promoter (Table 1: Chuah and Vanderdriessche WO2018 / 178067), will also improve expression from the MCK-desmin promoter of this disclosure. It is further intended that adding the Sk-CRM4 enhancer (SEQ ID NO: 14) upstream of the MCK-desmin promoter disclosed herein may also improve expression from the MCK-desmin promoter of this disclosure. It is further intended that adding an αMHC E enhancer (SEQ ID NO: 13) upstream of the MCK-desmin promoter disclosed herein may improve expression from the MCK promoter, similar to the improvement of expression from the MCK promoter when positioned upstream of the CK7 enhancer in the MHCK7 promoter (Table 1). These intended promoters were tested as described in Examples 1 and 3. a) Mammalian desmin promoter element, b) Mammalian desmin enhancer element, c) Mammalian MCK enhancer, d) The configuration will include a mammalian enhancer element selected from the group consisting of Sequence ID No. 13, Sequence ID No. 14, Sequence ID No. 15, and Sequence ID No. 16, wherein the promoter and the enhancer element will be operably connected.

[0123] All references, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein in whole, to the extent that each reference is individually and specifically indicated as being incorporated herein by reference.

[0124] The terms “includes,” “possesses,” “contains,” and “contains” should be interpreted as open-ended terms (i.e., “includes but not limited to”) unless otherwise stated herein. The enumeration of value ranges herein is intended solely as a simplification of the individual values ​​included within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any and all examples or exemplary language provided herein (e.g., “such as”) is intended solely to better illustrate the invention and does not limit the scope of the invention unless otherwise requested. No language herein should be interpreted as indicating that any unclaimed element is essential to the practice of the invention.

[0125] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the foregoing description. The inventors expect that such variations will be appropriately used by those skilled in the art, and they intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein or unless it is clearly inconsistent with the context, any combination of the elements described above in all possible variations is encompassed by the Invention. This disclosure provides the following: 1. Recombinant muscle-specific regulatory nucleic acid sequences, Mammalian desmin promoter and Mammalian desmin enhancers, It comprises one or more mammalian muscle creatine kinase (MCK) enhancers, A recombinant muscle-specific regulatory nucleic acid sequence in which the mammalian desmin promoter, mammalian desmin enhancer, and mammalian MCK enhancer are operably linked. 2. The recombinant muscle-specific regulatory nucleic acid sequence according to 1 above, wherein the mammalian desmin promoter is the human desmin promoter. 3. The recombinant muscle-specific regulatory nucleic acid sequence according to 1, wherein the mammalian desmin promoter includes a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. 4. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein the mammalian desmin promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. 5. The recombinant muscle-specific regulatory nucleic acid sequence according to item 1, wherein the mammalian desmin promoter is the mouse desmin promoter. 6. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein the mammalian desmin promoter includes a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. 7. The recombinant muscle-specific regulatory nucleic acid sequence according to 1, wherein the mammalian desmin promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. 8. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 7 above, wherein the mammalian desmin enhancer is a human desmin enhancer. 9. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of 1 to 7 above, wherein the mammalian desmin enhancer includes a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of Sequence ID No. 6. 10. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of 1 to 7 above, wherein the mammalian desmin enhancer comprises the nucleic acid sequence of SEQ ID NO: 6. 11. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 7 above, wherein the mammalian desmin enhancer is a mouse desmin enhancer. 12. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of 1 to 7 above, wherein the mammalian desmin enhancer includes a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of Sequence ID No. 3. 13. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of 1 to 7 above, wherein the mammalian desmin enhancer includes the nucleic acid sequence of SEQ ID NO: 3. 14. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of items 1 to 13 above, wherein each of the one or more mammalian MCK enhancers is an MCK CK7 enhancer. 15. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of items 1 to 14 above, wherein each of the one or more mammalian MCK enhancers is a human MCK enhancer. 16. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of items 1 to 14 above, wherein each of the one or more mammalian MCK enhancers is a mouse MCK enhancer. 17. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of 1 to 13 above, wherein each of the one or more mammalian MCK enhancers has a nucleic acid sequence that has at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12. 18. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of 1 to 13 above, wherein each of the one or more mammalian MCK enhancers comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12. 19. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of 1 to 14 above, wherein each of the one or more mammalian MCK enhancers has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of Sequence ID No. 1. 20. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of 1 to 14 above, wherein each of the one or more mammalian MCK enhancers contains the nucleic acid sequence of Sequence ID No. 1. 21. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 20 above, wherein the sole enhancer of the recombinant muscle-specific regulatory nucleic acid sequence is the mammalian desmin enhancer and the one or more mammalian MCK enhancers. 22. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 20 above, wherein the recombinant muscle-specific regulatory nucleic acid sequence does not include any additional enhancers other than the mammalian desmin enhancer and the one or more mammalian MCK enhancers. 23. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 20 above, wherein the recombinant muscle-specific nucleic acid sequence does not contain the vertebrate troponin I IRE(FIRE) enhancer. 24. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of items 1 to 20 above, further comprising one or more additional enhancers. 25. Recombinant muscle-specific regulatory nucleus sequence according to any one of items 1 to 20 above, further comprising one or more additional enhancers having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. 26. Recombinant muscle-specific regulatory nucleus sequence according to any one of items 1 to 20 above, further comprising one or more additional enhancers comprising nucleic acid sequences selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. 27. The recombinant muscle-specific regulatory nucleic acid sequence according to any of the preceding descriptions, wherein the muscle-specific regulatory nucleic acid sequence comprises two or more mammalian MCK enhancers. 28. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of 1 to 27 above, wherein the muscle-specific regulatory nucleic acid sequence comprises 1 to 3 of the mammalian MCK enhancers. 29. Recombinant muscle-specific regulatory nucleic acid sequences according to any of the preceding descriptions, further comprising one or more copies of introns. 30. The recombinant muscle-specific regulatory nucleic acid sequence according to 29, wherein one or more copies of the intron each contain a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18. 31. The recombinant muscle-specific regulatory nucleic acid sequence according to 29, wherein one or more copies of the intron each contain a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18. 32. Recombinant muscle-specific regulatory nucleic acid sequences according to any one of 29 to 31, wherein one or more copies of the intron are positioned 3' relative to the mammalian desmin promoter. 33. The recombinant muscle-specific regulatory nucleic acid sequence according to any of the above prior art, wherein the mammalian desmin enhancer and the mammalian MCK enhancer are positioned 5' relative to the mammalian desmin promoter. 34. The recombinant muscle-specific regulatory nucleic acid sequence according to 33, wherein the mammalian MCK enhancer is positioned 5' relative to the mammalian desmin enhancer. 35. Recombinant muscle-specific regulatory nucleic acid sequences according to any of the above prior arts, further comprising a transgene. 36. The recombinant muscle-specific regulatory nucleic acid sequence described in 35, wherein the introduced gene is under the control of the mammalian desmin promoter. 37. The recombinant muscle-specific regulatory nucleic acid sequence according to 35, wherein the introduced gene is positioned at 3' of the mammalian desmin promoter. 38. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 37 above, wherein the muscle-specific regulatory nucleic acid sequence comprises only one mammalian desmin enhancer. 39. The recombinant muscle-specific regulatory nucleic acid sequence according to any one of items 1 to 37 above, wherein the muscle-specific regulatory nucleic acid sequence does not contain two or more mammalian desmin enhancers. 40. Recombinant muscle-specific regulatory nucleic acid sequences comprising a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 55, 56, 57, and 58. 41. Recombinant muscle-specific regulatory nucleic acid sequences comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 55, 56, 57, and 58. 42. A vector comprising a muscle-specific regulatory nucleic acid sequence as described in any of the preceding paragraphs. 43. The vector according to 42 above, wherein the vector is selected from the group consisting of plasmids, minicircles, Doggybone, MIDGE, nanoplasmides, and viral vectors. 44. The vector according to 42 above, wherein the vector is selected from the group consisting of an episome non-replicating expression vector, an episome replicating expression vector, a transposon-integrated vector, a viral vector-integrated vector, and an identity-directed repair-integrated vector. 45. The vector according to 42, wherein the vector is a viral vector, and the viral vector is selected from the group consisting of adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. 46. ​​The vector according to 42, wherein the vector is a nanoplasmid, and the nanoplasmid contains a nucleic acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. 47. A host cell containing a muscle-specific regulatory nucleic acid sequence as described in any of items 1 to 41 above, or a vector as described in any of items 42 to 46 above. 48. A method for expressing an introduced gene in eukaryotic muscle cells or muscle cells, A method comprising transfecting eukaryotic muscle cells or muscle cells with a vector described in any of the above 42 to 46. 49. A method for replicating a vector containing an introduced gene in cells, A method comprising transforming host cells with a vector described in any of 42 to 46 above, and incubating the cells under conditions sufficient to replicate the vector. 50. It is a vector, i) A eukaryotic region sequence comprising a muscle-specific regulatory nucleic acid sequence described in any of the above 35 to 37, having 5' and 3' ends, ii) A vector comprising: a spacer region having less than 500 base pairs in length, which ligates the 5' and 3' ends of the eukaryotic region sequence and contains a bacterial replication origin and an RNA selectable marker. 51. The vector according to 50, wherein the muscle-specific regulatory nucleic acid sequence comprises only one mammalian desmin enhancer. 52. The vector according to 50, wherein the muscle-specific regulatory nucleic acid sequence does not contain two or more mammalian desmin enhancers. 53. The vector according to any one of 50 to 52 above, wherein the muscle-specific regulatory nucleic acid sequence comprises three mammalian MCK enhancers. 54. A vector according to any one of the above 50-53, wherein the bacterial replication origin is an R6K bacterial replication origin. 55. The vector according to 54, wherein the bacterial replication origin contains a sequence having at least 80% identity with any one of sequence numbers 19 to 23. 56. The vector according to any one of 50 to 55 above, wherein the RNA-selectable marker includes a sequence having at least 80% identity with SEQ ID NO: 24 or SEQ ID NO: 26. 57. A method for producing a muscle-specific vector, To provide a vector containing a non-muscle-specific promoter or a non-desmin promoter, A method comprising modifying the vector to replace the non-muscle-specific promoter or non-desmin promoter with a muscle-specific regulatory nucleic acid sequence described in any of items 1 to 41 above. 58. The method according to 57 above, wherein the vector is a nanoplasmid. 59. The method according to 58 above, wherein the bacterial replication origin is an R6K bacterial replication origin. 60. The method according to 59, wherein the bacterial replication origin comprises a sequence having at least 80% identity with any one of sequence numbers 19 to 23. 61. The method according to any one of the above 57 to 60, wherein the RNA-selectable marker includes a sequence having at least 80% identity with SEQ ID NO: 24 or SEQ ID NO: 26.

Claims

1. Recombinant muscle-specific regulatory nucleic acid sequences, A mammalian desmin promoter comprising a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and SEQ ID NOs: 10, A mammalian desmin enhancer comprising a nucleic acid sequence having at least 90% identity with the nucleic acid sequence of Sequence ID No. 6, It contains three mammalian muscle creatine kinase (MCK) enhancers, A recombinant muscle-specific regulatory nucleic acid sequence in which the mammalian desmin promoter, the mammalian desmin enhancer, and three mammalian MCK enhancers are operably linked.

2. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein the mammalian desmin enhancer comprises the nucleic acid sequence of SEQ ID NO:

6.

3. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein each of the three mammalian MCK enhancers is an MCK CK7 enhancer.

4. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein each of the three mammalian MCK enhancers has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of Sequence ID No.

1.

5. The recombinant muscle-specific regulatory nucleic acid sequence according to claim 1, wherein the nucleic acid sequence containing the three mammalian MCK enhancers includes the nucleic acid sequence of SEQ ID NO:

2.

6. It is a vector, i) A eukaryotic region sequence comprising a recombinant muscle-specific regulatory nucleic acid sequence according to any one of claims 1 to 5, ii) The 5' and 3' ends of the eukaryotic region sequence are linked, and a spacer region containing a bacterial replication origin and an RNA selectable marker is provided. A vector containing...

7. The vector according to claim 6, wherein the bacterial replication origin is an R6K bacterial replication origin.

8. The vector according to claim 7, wherein the bacterial replication origin includes a sequence having at least 90% identity with any one of sequence numbers 19 to 23.

9. The vector according to claim 6, wherein the RNA-selectable marker includes a sequence having at least 90% identity with SEQ ID NO: 24 or SEQ ID NO: 26.

Images