Novel muscle-specific promoters

Abstract

Provided herein are compositions related to genetically engineered muscle-specific promoters, including, for example, nucleic acids containing such promoters, vectors comprising such nucleic acids, and pharmaceutical compositions comprising such vectors. Also disclosed herein are methods of producing such nucleic acids and vectors, as well as methods of using such nucleic acids and vectors for, for example, gene therapy to treat muscle diseases.

Description

[0001] This application claims priority to PCT application No. PCT / CN2024 / 074031, filed on January 25, 2024, the entire contents of which are incorporated herein by reference.

[0002] 1. Electronically submitted sequence list reference This application incorporates an XML-formatted sequence list named “098A002WO02_SL”, which was created on January 16, 2025, and is 61,863 bytes in size. 2. Technical Field This invention relates to the field of biotechnology, particularly molecular biology, gene expression regulation, gene therapy, and medicine. More specifically, this article provides novel genetically engineered muscle-specific promoters and their applications.

[0004] 3. background Muscle diseases encompass a range of inherited and non-inherited conditions characterized by elevated overall morbidity and mortality due to skeletal muscle and cardiac dysfunction. To date, effective treatments for these diseases remain lacking. Gene therapy, a promising approach that delivers therapeutic genes to specific cells or tissues, holds considerable therapeutic potential. While adeno-associated virus vectors are widely used tools in gene therapy, achieving efficient delivery of transgenes to muscle remains a challenging task. Low expression levels in muscle necessitate the use of high doses, which can lead to adverse immune responses and off-target toxicity, severely limiting the application of such therapies.

[0005] Therefore, to improve the safety and efficacy of gene therapy for muscle diseases, there is an urgent need to develop muscle-specific promoters with strong transcriptional activity. The compositions and methods disclosed herein address this need and provide related advantages.

[0006] 4. summary This document provides a nucleic acid comprising a muscle-specific promoter, the promoter comprising an enhancer region operatively linked to a core promoter, wherein the enhancer region comprises at least two enhancer elements, each enhancer element being independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs, the nucleotide sequence of which shares at least 85% identity with SEQ ID NO:6-13. In some embodiments, the enhancer region comprises two, three, four, five, six, or seven enhancer elements.

[0007] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises two or three hCKM106Es. In some embodiments, the enhancer region further comprises at least one enhancer element selected from the group consisting of hDes68E, hDes78E, and sE. In some embodiments, the enhancer region comprises one, two, three, or four hDes68Es.

[0008] In some embodiments of the nucleic acids disclosed herein, the enhancer region has three hCKM106Es. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:35.

[0009] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises three hCKM106Es and one hDes68E. In some embodiments, the enhancer region comprises the following fragments operatively linked from 5' to 3': hCKM106E, hCKM106E, hCKM106E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:36.

[0010] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises three hCKM106E, one hDes68E, and one hDes78E. In some embodiments, the enhancer region comprises the following fragments operatively linked from 5' to 3': hCKM106E, hCKM106E, hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:37.

[0011] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises two hCKM106Es and one sE. In some embodiments, the enhancer region comprises the following fragments operatively linked from 5' to 3': hCKM106E, hCKM106E, and sE. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38.

[0012] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises three hCKM106E and four hDes68E. In some embodiments, the enhancer region comprises the following fragments operatively linked from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:41.

[0013] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises two hCKM106Es and four hDes68Es. In some embodiments, the enhancer region comprises the following fragments operatively linked from 5' to 3': hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:42.

[0014] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises three hCKM106E and three hDes68E. In some embodiments, the enhancer region comprises the following fragments operably linked from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43.

[0015] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises three hCKM106E and two hDes68E. In some embodiments, the enhancer region comprises the following fragments from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44.

[0016] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises two hCKM106Es and two hDes68Es. In some embodiments, the enhancer region comprises the following 5' to 3' operatively linked fragments: hCKM106E, hDes68E, hCKM106E, and hDes68E. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:45.

[0017] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises an sE and a MEF2 motif. In some embodiments, the enhancer region comprises three sEs and one MEF2 motif. In some embodiments, the enhancer region comprises the following 5' to 3' operative linker segments: sE, MEF2 motif, sE, and sE. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:39.

[0018] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises hCKM106ER or hDes68ER. In some embodiments, the enhancer region comprises three hCKM106ERs and one hDes68ER. In some embodiments, the enhancer region comprises the following 5' to 3' operative linker fragments: hDes68ER, hCKM106ER, hCKM106ER, and hCKM106ER. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:40.

[0019] In some embodiments of the nucleic acids disclosed herein, the enhancer region comprises at least two hCKM206Es. In some embodiments, the enhancer region has three hCKM206Es. In some embodiments, the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:34.

[0020] In some embodiments, the nucleic acid provided herein comprises a muscle-specific promoter including an enhancer region operatively linked to a core promoter, wherein the enhancer region is any of the enhancer regions described herein, and wherein the muscle-specific core promoter is P87 or sP86; wherein P87 has a nucleotide sequence having at least 85% identity with SEQ ID NO:1, and sP86 has a nucleotide sequence having at least 85% identity with SEQ ID NO:2. In some embodiments, the muscle-specific core promoter is P87. In some embodiments, the muscle-specific core promoter is sP86.

[0021] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs:17-30.

[0022] In some embodiments, a skeletal muscle-specific promoter is provided herein.

[0023] In some embodiments, the nucleic acids provided herein also include transgenes operatively linked to a promoter. In some embodiments, the transgene encodes a therapeutic protein for muscle-related diseases or conditions. In some embodiments, the therapeutic protein is selected from survival motor neuron (SMN), fukutin-related protein (FKRP), follistatin (FST), neurotrophin 3 (NT-3), dystrophin, tafazzin, myotubularin, merosin, α-1,4-glucosidase, calpain 3, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, poly(A)-binding protein nuclear 1 (PABPN1), and lysosome-associated membrane protein 2 isorform. B, LAMP2B).

[0024] In some embodiments, this document provides a vector comprising the nucleic acid described herein. In some embodiments, the vector is a DNA vector or an RNA vector. In some embodiments, the vector is a plasmid, a recombinant retroviral vector, a recombinant lentiviral vector, a recombinant adenovirus vector, or a recombinant adeno-associated virus (AAV) vector.

[0025] In some embodiments, this document provides an AAV vector comprising the nucleic acid described herein. In some embodiments, the AAV is a serotype of AAV1, AAV2, AAV7, AAV8, AAV9, AAV12, AAVrh74, AAVhu.32, or a mixture thereof. In some embodiments, the viral genome of the AAV comprises, from 5' to 3', a first ITR, a promoter, a transgene, a polyadenylated tail, and a second ITR.

[0026] In some embodiments, this document provides pharmaceutical compositions comprising the nucleic acids described herein or the vectors described herein and pharmaceutically acceptable vectors, wherein the nucleic acids comprise transgenes operatively linked to a promoter, and wherein the transgenes encode a therapeutic protein for muscle-related diseases or conditions.

[0027] In some embodiments, this document provides a method for increasing the expression level of a transgene in muscle cells, comprising transfecting muscle cells with an effective amount of the nucleic acid or vector described herein, wherein the nucleic acid contains a transgene operatively linked to the promoter.

[0028] In some embodiments, this document provides the use of the nucleic acids or vectors described herein in enhancing transgene expression levels in muscle cells, wherein the nucleic acids contain a transgene operatively linked to the promoter.

[0029] In some embodiments, this document provides a method for treating a subject with a muscle-related disease or condition, comprising administering to the subject a therapeutically effective amount of the nucleic acid, the vector, or the pharmaceutical composition described herein; wherein the nucleic acid comprises a transgene operably linked to the promoter, and the transgene encodes a therapeutic protein for the muscle-related disease or condition. In some embodiments, this document provides the use of the nucleic acid, the vector, or the pharmaceutical composition described herein in the treatment of a muscle-related disease or condition; wherein the nucleic acid comprises a transgene operably linked to the promoter, and the transgene encodes a therapeutic protein for the muscle-related disease or condition. In some embodiments, this document provides the use of the nucleic acid, the vector, or the pharmaceutical composition described herein in the preparation of a medicament for the treatment of a muscle-related disease or condition.

[0030] In some embodiments, the muscle-related disease or condition may be sarcopenia, muscular dystrophy (MD), congenital myopathy, distal myopathy, myotonic syndrome, ion channel disease, malignant hyperthermia, metabolic myopathy, or arthritis. In some embodiments, the muscle-related disease or condition is sarcopenia. In some embodiments, the muscle-related disease or condition is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy, myotonic dystrophy (Steiner's disease), oculopharyngeal muscular dystrophy (OMD), or limb-girdle muscular dystrophy (LGMD). In some embodiments, the muscle-related disease or condition is DMD or BMD.

[0031] In some embodiments, this document provides kits comprising the nucleic acids or vectors described herein. 5. Attached Figure Description Figure 1 Schematic diagrams of the structures of muscle-specific promoters tMCK, thCKM, thCKMs, and thMD are provided.

[0033] Figure 2 In vitro luciferase assay results were provided, demonstrating the transcriptional activity of skeletal muscle-specific promoters thCKM, thCKMs, and thMD in skeletal muscle cells.

[0034] Figure 3 In vivo imaging of mice injected with AAV carrying luciferase genes driven by promoters thCKM, thCKMs, and thMD was provided, demonstrating their transcriptional activity in vivo.

[0035] Figure 4 Quantification of luciferase activity in different skeletal muscle tissues collected from mice injected with AAV carrying luciferase genes driven by promoters thCKM, thCKMs, and thMD was provided, demonstrating the transcriptional activity of these promoters in various skeletal muscle tissues.

[0036] Figure 5 Quantification of luciferase activity in different non-skeletal muscle tissues collected from mice injected with AAV carrying luciferase genes driven by promoters thCKM, thCKMs, and thMD is provided, demonstrating the transcriptional activity of these promoters in a variety of non-skeletal muscle tissues.

[0037] Figure 6Schematic diagrams of the structures of muscle-specific promoters thMD, thMD2, hME2-sE, sE3, thMDR, L34, S24, S33, S32 and S22 are provided.

[0038] Figure 7 In vitro luciferase assay results were provided, showing the transcriptional activity of the promoters thMD, thMD2, hME2-sE, sE3, thMDR, L34, S24, S33, S32 and S22 in HEK293T (non-skeletal muscle cells) and C2C12 (skeletal muscle cells).

[0039] Figure 8 In vivo imaging of mice injected with AAV carrying luciferase genes driven by promoters thMD, thMD2, hME2-sE, sE3, thMDR, L34, S24, S33, S32, and S22 was provided, demonstrating the transcriptional activity of these promoters in vivo.

[0040] Figure 9 Quantification of luciferase activity in different skeletal muscle tissues collected from mice injected with AAV carrying luciferase genes driven by promoters thMD, thMD2, hME2-sE, sE3, thMDR, and L34 was provided, demonstrating the transcriptional activity of these promoters in various skeletal muscle tissues.

[0041] Figure 10 Quantification of luciferase activity in different non-skeletal muscle tissues collected from mice injected with AAVs carrying the luciferase gene driven by promoters thMD, thMD2, hME2-sE, sE3, thMDR, and L34 was provided, demonstrating the transcriptional activity of these promoters in various non-skeletal muscle tissues.

[0042] Figure 11 Schematic diagrams of the structures of muscle-specific promoters thM, thMD-sP86, hME2-sE, and hME2-sE-sP86 are provided.

[0043] Figure 12 In vivo imaging of mice injected with AAV carrying luciferase genes driven by promoters thMD, thMD-sP86, hME2-sE, and hME2-sE-sP86 was provided, demonstrating the transcriptional activity of these promoters in vivo.

[0044] Figure 13 Quantification of luciferase activity in different skeletal muscle tissues collected from mice injected with AAV carrying luciferase genes driven by promoters thMD and thMD-sP86 was provided, demonstrating the transcriptional activity of these promoters in various skeletal muscle tissues.

[0045] Figure 14 Quantification of luciferase activity in different non-skeletal muscle tissues collected from mice injected with AAV carrying luciferase genes driven by promoters thMD and thMD-sP86 was provided, demonstrating the transcriptional activity of these promoters in a variety of non-skeletal muscle tissues. 6. Invention Details This document provides novel genetically engineered promoters (e.g., skeletal muscle cells) with high transcriptional activity and high muscle cell specificity. It also provides expression cassettes containing such promoters, nucleic acids containing such promoters or expression cassettes, vectors containing such nucleic acids (e.g., AAV vectors), and compositions containing such nucleic acids or vectors (e.g., pharmaceutical compositions). Furthermore, this document provides methods for using the compositions disclosed herein to express transgenes specifically for muscle (e.g., a transgene encoding a protein for treating muscle-related diseases or conditions). Finally, this document provides treatments for treating muscle-related diseases or conditions (e.g., muscular dystrophy (MD)).

[0047] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments described herein, and it should also be understood that the terminology used herein is for describing specific embodiments and is not intended to be limiting.

[0048] 6.1 Definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization are well-known and commonly used in the art.

[0049] The term “a” refers to one or more of the same entity; for example, “an antibody” is understood to represent one or more antibodies.

[0050] The term “and / or” as used herein should be considered as a specific disclosure of each of the two specified features or components, whether or not the other is included. Thus, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A (alone)”, and “B (alone)”. Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0051] As used herein, the term "about" is used to indicate a value that includes a variation in the inherent error of the device, the method used to determine that value, or a variation between the objects of study. The term "about" includes the exact figures listed. In some embodiments, "about" means within ±10% of a given value or range. In some embodiments, "about" means a variation of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about". In some embodiments, "about" means a variation of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about".

[0052] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to nucleotide polymers of any length and include DNA, RNA, DNA-RNA hybrids, and DNA-RNA analogs. Such analogs can be produced using, for example, modified nucleotides or nucleotide analogs, including but not limited to inosine or triphenylmethylated bases. Nucleic acids or polynucleotides can be single-stranded or double-stranded, or contain both single-stranded and double-stranded portions, or contain a triple-stranded portion. In some embodiments, the nucleic acid provided herein is double-stranded DNA.

[0053] The terms “peptide,” “polypeptide,” and “protein” used interchangeably in this document refer to a polymer of amino acids of any length, which may be linear or branched. It may include non-natural or modified amino acids and may be interrupted by non-amino acid components. Polypeptides, peptides, polypeptide chains, peptide chains, or proteins may also be modified by, for example, the formation of disulfide bonds, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification.

[0054] In this document, the terms "identical," "percentage identity," and their grammatical equivalents, when used to describe two or more nucleotides or polypeptides, refer to the fact that when two or more identical sequences or subsequences are compared and aligned (with gaps introduced if necessary) to achieve maximum correspondence, they are identical or have a specified percentage of identical nucleotide or amino acid residues. Conserved amino acid substitutions are not considered part of sequence identity. Percentage identity can be measured by sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum full-length alignment of the compared sequences. For example, the percentage sequence identity value can be generated using the sequence comparison computer program BLAST.

[0055] In some embodiments, the two polynucleotides or polypeptides provided herein are substantially identical, meaning that when performing maximum correspondence comparison and alignment using sequence comparison algorithms or visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% nucleotide or amino acid residue identity, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, or at least about 60-80 residues, or any integer value in between. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists in a region of a nucleotide sequence having a length of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, or at least about 60-80 bases, or any integer value between these lengths. In some embodiments, identity exists in a region longer than 60-80 bases, for example, at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as nucleotide sequences encoding a target protein.

[0056] As those skilled in the art will understand, as used herein, in order to determine the percentage of sequence identity, uridine ribonucleotides in RNA molecules are considered equivalent to thymidine ribonucleotides in DNA molecules. Therefore, if the RNA equivalent and the DNA polynucleotide are different from each other only by the substitution of uridine ribonucleotides in the RNA equivalent for thymidine ribonucleotides in the DNA polynucleotide, then the RNA equivalent and the DNA polynucleotide can be considered to have 100% sequence identity.

[0057] As used herein, the term "variant" in relation to a nucleic acid or protein having specific sequence characteristics ("reference nucleic acid" or "reference protein") refers to a different nucleic acid or protein that, compared to a reference protein or reference polypeptide, has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) substitutions, deletions, and / or additions of nucleotides or amino acids. In some embodiments, the nucleic acid variant or protein variant may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the reference nucleic acid or reference protein. Variants of nucleic acids or proteins generally retain the basic structural and functional characteristics of the reference nucleic acid or reference protein.

[0058] As used herein, the term "gene" refers to a region of DNA that encodes a protein. A gene may include regulatory regions and protein-coding regions. In some embodiments, a gene includes two or more introns and three or more exons, wherein each intron forms an intermediate sequence between two exons. As used herein, the term "RNA equivalent" of a gene refers to an RNA polynucleotide corresponding to the DNA polynucleotide encoding the gene, such as an RNA transcript obtained by transcribing the DNA polynucleotide containing the gene.

[0059] As used herein, the term "transgenic" refers to a gene transferred or delivered using molecular and genetic techniques for expression in target cells. Transgenics can encode a target product, such as a therapeutic protein. Transgenics can also encode peptides, enzymes, or RNA. RNA molecules that can be encoded by transgenics include miRNA, shRNA, tRNA, dsRNA, ribosomal RNA, catalytic RNA, or antisense RNA. Transgenics can be exogenous copies of endogenous genes in the target cells. Transgenics can also be heterologous in relation to the target cells. In some embodiments, the endogenous gene in the target cells is mutated, silenced, or otherwise dysfunctionalized, and the transgenic provides a functional copy to compensate for the loss of function of the endogenous gene in the target cells. In some embodiments, transgenics can encode therapeutic proteins.

[0060] As used herein, the term "therapeutic protein" refers to a protein that is known or engineered to have therapeutic effects on a target subject (e.g., a mammal or a human). Therapeutic proteins can be used to treat a variety of diseases or conditions in the target subject. Therapeutic proteins can be naturally occurring proteins or artificially produced through genetic engineering techniques.

[0061] As used herein, the term "encoding" and its syntactic equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid (such as a gene, cDNA, or mRNA) that serves as a template in biological processes for the synthesis of other polymers and macromolecules having specific nucleotide sequences (such as rRNA, tRNA, and mRNA) or specific amino acid sequences and the resulting biological characteristics. Thus, if the mRNA corresponding to a gene is transcribed and translated to produce a protein, then that gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences in degenerate form that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0062] As used herein, the term "promoter" is a nucleic acid sequence capable of initiating gene transcription into messenger RNA, which is initiated by binding RNA polymerase to or near the promoter. Examples of other transcriptional regulatory elements are described in Goeddel's *Gene Exposition Technology: Methods in Enzymology*, Volume 185 (Academic Press, San Diego, CA, 1990). Promoters can be constitutive or inducible. Promoters can be cell-type specific or tissue-specific, meaning that they preferably (including specifically) initiate gene transcription within a particular type of cell or tissue of the organism. The selective activity of the promoter ensures that the associated gene is expressed only in specific cells or tissues. Promoters may include a core promoter region and an enhancer region.

[0063] As used herein, a "core promoter" refers to a nucleic acid fragment (e.g., 50-100 bp) that provides a binding site for RNA polymerase and other transcription factors to initiate transcription. A core promoter may contain a transcription start site. The core promoter is a fundamental component of a promoter or promoter region, which may also contain enhancer regions.

[0064] As used herein, an "enhancer" or "enhancer element" is a regulatory nucleic acid sequence that may be located near or considerably far from the core promoter. An "enhancer region" may be a contiguous nucleic acid sequence containing one or more enhancer elements. An enhancer region may optionally include spacer sequences between enhancer elements.

[0065] As used herein, the term "operable linkage" refers to the functional interdependence of sequence elements (e.g., core promoters, enhancer elements, and coding sequences) within nucleic acid molecules. For example, if a promoter regulates the transcription of a target polynucleotide molecule in a cell, then the promoter is operably linked to the transcribed polynucleotide molecule. Furthermore, if two parts of a transcriptional regulatory element (e.g., two enhancer elements in an enhancer region) are linked together such that the transcriptional activation function of one part is not adversely affected by the presence of the other part, then they are operably linked to each other. Two transcriptional regulatory elements can be operably linked to each other via adapter nucleic acids (e.g., spacer non-coding nucleic acids), or they can be operably linked to each other in the absence of a spacer nucleotide.

[0066] As used herein, the term "expression cassette" refers to a unique and continuous component of a nucleic acid (e.g., a vector) that includes regulatory sequences that control the expression of the nucleotide sequences that the expression cassette may carry. These regulatory sequences include, for example, transcription initiation (promoter) and termination sequences, enhancers, introns, origins of replication, polyadenylation sequences, polypeptide signaling, and chromatin insulator elements. For more information on regulatory sequences, see, for example, *GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY*, Volume 185, by Goeddel. Simply put, the expression cassette directs the host cell's machinery to produce RNA and proteins encoded by the nucleotide sequences contained within it. Therefore, expression in different organisms or species, such as bacteria, yeast, plants, and mammalian cells, requires different regulatory sequences. An expression cassette can be "empty," containing a multiple cloning site (MCS) for inserting a nucleotide sequence encoding a transgenic sequence. An expression cassette can also be loaded, containing at least one transgenic sequence.

[0067] As used herein, the term "cloning site" refers to a nucleic acid sequence containing a restriction endonuclease site for restriction enzyme-mediated cloning via compatible sticky or blunt-end ligation; or a nucleic acid region serving as a primer-binding site for cloning inserted DNA via PCR-mediated homology and extension "overlap PCR splicing"; or a recombination site for insertion of a target nucleic acid via a recombinase-mediated recombination exchange reaction; or a chimeric end for target nucleic acid insertion via transposon-mediated transposition; and other techniques common in the art. As used herein and understood in the art, a "multiple cloning site" or "MCS" refers to a short segment of DNA on a vector containing multiple cloning sites to allow insertion of transgenic sequences.

[0068] As used herein, the term "vector" refers to a tool used to deliver a target gene into a host cell. Vectors can be viral vectors, such as adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, etc. Vectors can also be non-viral vectors, such as lipid-based nanoparticles, polymer nanoparticles, naked nucleic acids, etc. When the target gene encodes a foreign protein to be expressed in prokaryotic or eukaryotic cells, the vector may be called an "expression vector," examples of which are disclosed, for example, in WO1994 / 11026, the disclosure of which is incorporated herein by reference. Expression vectors described herein comprise polynucleotide sequences and additional sequence elements, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Some vectors described herein that can be used to express transgenes include plasmids containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for expressing transgenes comprise polynucleotide sequences that can improve the translation rate of these genes or improve the stability or nuclear translocation of the mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signaling sites that guide efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for screening cells containing such vectors. Examples of suitable markers include genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, or notracin.

[0069] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent intended for administration to a subject (such as a mammal, such as a human) to prevent, treat, or control a particular disease or condition that affects or may affect the subject.

[0070] As used herein, the term “pharmaceutically acceptable” means that a compound, material, composition, and / or dosage form is suitable for contact with the tissues of a subject (such as a mammal (e.g., human)) without causing excessive toxicity, irritation, allergic reactions, or other problematic complications commensurate with a reasonable benefit / risk ratio.

[0071] In this article, when associated with a disease or condition or a subject suffering from a disease or condition (e.g., muscular dystrophy), the term "treatment" refers to actions that prevent or mitigate (alleviate) undesirable physiological changes or conditions, such actions as inhibiting, eliminating, reducing, and / or improving symptoms, symptom severity, and / or symptom frequency associated with the treated disease or condition. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, whether detectable or not, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete).

[0072] As used in this article, the term "muscle-related disease or condition" refers to a pathological condition that primarily affects the body's muscles. These diseases or conditions can be congenital (hereditary) or acquired (non-hereditary) and can have a variety of causes and manifestations.

[0073] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will become the recipient of a specific treatment. Subjects can be mammals. Subjects can be humans. Subjects may suffer from a specific disease or condition.

[0074] As used herein, the term "administration" and its grammatical equivalents refer to the act or process of delivering a therapeutic agent or pharmaceutical composition into a subject body by methods described herein or known in the art. A therapeutic agent can be any compound, such as a genetically modified organism, a vector, a peptide, or a virus. Administering a therapeutic agent or pharmaceutical composition includes the process of prescribing a therapeutic agent or pharmaceutical composition to be delivered into a subject body. Example forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP) injections; transdermal dosage forms, including creams, gels, powders, or patches; buccal dosage forms; inhaled powders; sprays; suspensions, and rectal suppositories.

[0075] As used herein, the terms "effective dose," "therapeutic effective dose," and their grammatical equivalents refer to a dose (alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses) administered to a subject that produces any detectable positive effect on any symptom, aspect, or characteristic of the subject's disease, disorder, or condition. Therapeutic effective doses can be determined by measuring the associated physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the condition being treated, the clinician's judgment, etc. In any individual case, a person skilled in the art can determine the appropriate "effective dose" using routine laboratory methods.

[0076] Scope: In this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of a scope should be considered as having specifically disclosed all possible sub-scopes and the individual numerical values ​​within that scope. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0077] The example genes and peptides described herein are referenced by GenBank numbers, GI numbers, and / or SEQ ID NOS. It should be understood that those skilled in the art can easily identify homologous sequences by referring to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / GenBank / ) and EMBL (EMBL.org / ).

[0078] Suitable methods and materials for implementing and / or testing embodiments of this disclosure are described below. These methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar to or equivalent to those described herein may be used. For example, conventional methods well known in the art to which this disclosure relates have been described in various general and more specific references, such as "Sambrook..." et al ., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Sambrook et al ., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al .,Current Protocols in Molecular Biology, Greene Publishing Associates, 1992(and Supplements to 2000); Ausubel et al Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; its contents are incorporated herein by reference in full.

[0079] 6.2 Components This document provides a muscle-specific promoter comprising an enhancer region operatively linked to a core promoter. It also provides an expression cassette comprising the muscle-specific promoter, which may further comprise a transgene for muscle-specific expression. Furthermore, this document provides nucleic acids and vectors containing such expression cassettes, which can be used, for example, to deliver a target transgene to muscle cells. Finally, this document provides pharmaceutical compositions comprising the nucleic acids or vectors disclosed herein, which can be used, for example, for gene therapy of muscle-related diseases and conditions.

[0080] 6.2.1 Muscle-specific promoters The muscle-specific promoters provided herein include an enhancer region operatively linked to a core promoter, wherein the enhancer region contains at least two enhancer elements, each of which is independently selected from hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs.

[0081] 6.2.1.1 Enhanced Subregion The enhancer region contains at least two enhancer elements, each independently selected from the hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs are all muscle-specific enhancer elements.

[0082] Table I: Exemplary Enhancement Sub-Components In some embodiments, hCKM206E has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6. In some embodiments, hCKM206E has a nucleotide sequence that is at least 85% identical to SEQ ID NO:6. In some embodiments, hCKM206E has a nucleotide sequence that is at least 90% identical to SEQ ID NO:6. In some embodiments, hCKM206E has a nucleotide sequence that is at least 95% identical to SEQ ID NO:6. In some embodiments, hCKM206E has a nucleotide sequence that is at least 98% identical to SEQ ID NO:6. In some embodiments, hCKM206E has a nucleotide sequence that is at least 99% identical to SEQ ID NO:6. In some embodiments, hCKM206E has the nucleotide sequence of SEQ ID NO:6.

[0083] In some embodiments, hCKM106E has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:7. In some embodiments, hCKM106E has a nucleotide sequence that is at least 85% identical to SEQ ID NO:7. In some embodiments, hCKM106E has a nucleotide sequence that is at least 90% identical to SEQ ID NO:7. In some embodiments, hCKM106E has a nucleotide sequence that is at least 95% identical to SEQ ID NO:7. In some embodiments, hCKM106E has a nucleotide sequence that is at least 98% identical to SEQ ID NO:7. In some embodiments, hCKM106E has a nucleotide sequence that is at least 99% identical to SEQ ID NO:7. In some embodiments, hCKM106E has the nucleotide sequence of SEQ ID NO:7.

[0084] In some embodiments, hCKM106ER has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has a nucleotide sequence that is at least 85% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has a nucleotide sequence that is at least 90% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has a nucleotide sequence that is at least 95% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has a nucleotide sequence that is at least 98% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has a nucleotide sequence that is at least 99% identical to SEQ ID NO:8. In some embodiments, hCKM106ER has the nucleotide sequence of SEQ ID NO:8.

[0085] In some embodiments, hDes68E has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9. In some embodiments, hDes68E has a nucleotide sequence that is at least 85% identical to SEQ ID NO:9. In some embodiments, hDes68E has a nucleotide sequence that is at least 90% identical to SEQ ID NO:9. In some embodiments, hDes68E has a nucleotide sequence that is at least 95% identical to SEQ ID NO:9. In some embodiments, hDes68E has a nucleotide sequence that is at least 98% identical to SEQ ID NO:9. In some embodiments, hDes68E has a nucleotide sequence that is at least 99% identical to SEQ ID NO:9. In some embodiments, hDes68E has the nucleotide sequence of SEQ ID NO:9.

[0086] In some embodiments, hDes68ER has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:10. In some embodiments, hDes68ER has a nucleotide sequence that is at least 85% identical to SEQ ID NO:10. In some embodiments, hDes68ER has a nucleotide sequence that is at least 90% identical to SEQ ID NO:10. In some embodiments, hDes68ER has a nucleotide sequence that is at least 95% identical to SEQ ID NO:10. In some embodiments, hDes68ER has a nucleotide sequence that is at least 98% identical to SEQ ID NO:10. In some embodiments, hDes68ER has a nucleotide sequence that is at least 99% identical to SEQ ID NO:10. In some embodiments, hDes68ER has the nucleotide sequence of SEQ ID NO:10.

[0087] In some embodiments, hDes78E has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:11. In some embodiments, hDes78E has a nucleotide sequence that is at least 85% identical to SEQ ID NO:11. In some embodiments, hDes78E has a nucleotide sequence that is at least 90% identical to SEQ ID NO:11. In some embodiments, hDes78E has a nucleotide sequence that is at least 95% identical to SEQ ID NO:11. In some embodiments, hDes78E has a nucleotide sequence that is at least 98% identical to SEQ ID NO:11. In some embodiments, hDes78E has a nucleotide sequence that is at least 99% identical to SEQ ID NO:11. In some embodiments, hDes78E has the nucleotide sequence of SEQ ID NO:11.

[0088] In some embodiments, sE has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:12. In some embodiments, sE has a nucleotide sequence that is at least 85% identical to SEQ ID NO:12. In some embodiments, sE has a nucleotide sequence that is at least 90% identical to SEQ ID NO:12. In some embodiments, sE has a nucleotide sequence that is at least 95% identical to SEQ ID NO:12. In some embodiments, sE has a nucleotide sequence that is at least 98% identical to SEQ ID NO:12. In some embodiments, sE has a nucleotide sequence that is at least 99% identical to SEQ ID NO:12. In some embodiments, sE has the nucleotide sequence of SEQ ID NO:12.

[0089] In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 85% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 90% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 95% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 98% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has a nucleotide sequence that is at least 99% identical to SEQ ID NO:13. In some embodiments, the MEF2 motif has the nucleotide sequence of SEQ ID NO:13.

[0090] This document provides a muscle-specific promoter having a enhancer region operatively connected to a core promoter. In some embodiments, the enhancer region includes at least two enhancer elements. In some embodiments, the enhancer region includes at least two, at least three, at least four, at least five, at least six, or at least seven enhancer elements. In some embodiments, the enhancer region includes two, three, four, five, six, or seven enhancer elements. In some embodiments, the enhancer region includes two enhancer elements. In some embodiments, the enhancer region includes three enhancer elements. In some embodiments, the enhancer region includes four enhancer elements. In some embodiments, the enhancer region includes five enhancer elements. In some embodiments, the enhancer region includes six enhancer elements. In some embodiments, the enhancer region includes seven enhancer elements.

[0091] In some embodiments, each reinforcing sub-element is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motif. In some embodiments, each reinforcing sub-element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, each reinforcing sub-element is independently selected from hCKM106E, hDes68E, and hDes78E. In some embodiments, each reinforcing sub-element is independently selected from hCKM106E or hDes68E. In some embodiments, each reinforcing sub-element is independently selected from hCKM106E or sE. In some embodiments, each reinforcing sub-element is independently selected from hCKM106ER or hDes68ER. In some embodiments, the reinforcing sub-element is hCKM206E. In some embodiments, the reinforcing sub-element is hCKM106E. In some embodiments, the enhancer sub-element is hCKM106ER. In some embodiments, the enhancer sub-element is hDes68E. In some embodiments, the enhancer sub-element is hDes68ER. In some embodiments, the enhancer sub-element is hDes78E. In some embodiments, the enhancer sub-element is sE. In some embodiments, the enhancer sub-element is MEF2 motif.

[0092] In some embodiments, the enhancer subregion comprises 2, 3, 4, 5, 6, or 7 enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer subregion comprises 2 enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer subregion comprises 3 enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer subregion comprises four enhancer elements, each independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer subregion comprises five enhancer elements, each independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer subregion comprises six enhancer elements, each independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the enhancer region comprises seven enhancer elements, each of which is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs.

[0093] In some embodiments, the enhancement sub-region comprises at least two enhancement sub-elements, wherein each enhancement sub-element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises 2, 3, 4, 5, 6, or 7 enhancement sub-elements, wherein each enhancement sub-element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises 2 enhancement sub-elements, wherein each enhancement sub-element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises 3 enhancement sub-elements, wherein each enhancement sub-element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises four enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises five enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises six enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE. In some embodiments, the enhancement sub-region comprises seven enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER, and sE.

[0094] In some embodiments, the enhancement subregion comprises at least two enhancement sub-elements, each enhancement sub-element independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement subregion comprises 2, 3, 4, 5, 6, or 7 enhancement sub-elements, each enhancement sub-element independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement subregion comprises 2 enhancement sub-elements, each enhancement sub-element independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement subregion comprises 3 enhancement sub-elements, each enhancement sub-element independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement subregion comprises 4 enhancement sub-elements, each enhancement sub-element independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement sub-region comprises five enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement sub-region comprises six enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancement sub-region comprises seven enhancement sub-elements, each independently selected from the group consisting of hCKM106E, hDes68E, and hDes78E.

[0095] In some embodiments, the enhancement subregion comprises at least two enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises 2, 3, 4, 5, 6, or 7 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises 2 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises 3 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises 4 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises 5 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises six enhancement sub-elements, each of which is independently selected from hCKM106E or hDes68E. In some embodiments, the enhancement subregion comprises seven enhancement sub-elements, each of which is independently selected from hCKM106E or hDes68E.

[0096] In some embodiments, the enhancement subregion includes at least two enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 2, 3, 4, 5, 6, or 7 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 2 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 3 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 4 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 5 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion includes 6 enhancement sub-elements, each independently selected from hCKM106E or sE. In some embodiments, the enhancement subregion comprises seven enhancement sub-elements, each of which is independently selected from hCKM106E or sE.

[0097] In some embodiments, the enhancer subregion includes at least two enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 2, 3, 4, 5, 6, or 7 enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 2 enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 3 enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 4 enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 5 enhancer elements, each of which is independently selected from either the sE or MEF2 motif. In some embodiments, the enhancer subregion includes 6 enhancer elements, each of which is independently selected from either the sE or MEF2 motif.

[0098] In some embodiments, the enhancement subregion comprises at least two enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises 2, 3, 4, 5, 6, or 7 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises 2 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises 3 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises 4 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises 5 enhancement sub-elements, each enhancement sub-element independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises six enhancement sub-elements, each of which is independently selected from hCKM106ER or hDes68ER. In some embodiments, the enhancement subregion comprises seven enhancement sub-elements, each of which is independently selected from hCKM106ER or hDes68ER.

[0099] In some embodiments, the enhancement sub-region includes at least two enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 2, 3, 4, 5, 6, or 7 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 2 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 3 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 4 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 5 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 6 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E. In some embodiments, the enhancement sub-region includes 7 enhancement sub-elements, wherein the enhancement sub-elements are hCKM206E.

[0100] In some embodiments, the enhancement sub-region includes at least two enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 2, 3, 4, 5, 6, or 7 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 2 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 3 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 4 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 5 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 6 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E. In some embodiments, the enhancement sub-region includes 7 enhancement sub-elements, wherein the enhancement sub-elements are hCKM106E.

[0101] The enhancer elements of an enhancer subregion can be operatively connected in any order. For example, in an enhancer subregion containing eight different enhancer elements, such as an hCKM206E, an hCKM106E, an hCKM106ER, an hDes68E, an hDes68ER, an hDes78E, an sE, and a MEF2 motif, the enhancer elements can be operatively connected in the 5' to 3' direction as hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motif; or the enhancer elements can be operatively connected as hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, MEF2 motif, and sE, etc., for a total of eight! different combinations.

[0102] In some embodiments of the muscle-specific promoters provided herein, the enhancer region comprises two hCKM106Es. In some embodiments, the enhancer region further comprises one hCKM106E. In some embodiments, the enhancer region further comprises an enhancer element selected from the group consisting of hDes68E, hDes78E, and sE. In some embodiments, the enhancer region further comprises an hDes68E. In some embodiments, the enhancer region further comprises an hDes78E. In some embodiments, the enhancer region further comprises an sE.

[0103] In some embodiments, the enhancer region comprises three hCKM106E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:35. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:35. The enhancer region may have the nucleotide sequence of SEQ ID NO:35.

[0104] In some embodiments, the enhancement sub-region includes two or three hCKM106Es and one to four hDes68Es. In some embodiments, the enhancement sub-region includes three hCKM106Es and four hDes68Es. In some embodiments, the enhancement sub-region includes three hCKM106Es and three hDes68Es. In some embodiments, the enhancement sub-region includes three hCKM106Es and two hDes68Es. In some embodiments, the enhancement sub-region includes three hCKM106Es and one hDes68E. In some embodiments, the enhancement sub-region includes two hCKM106Es and four hDes68Es. In some embodiments, the enhancement sub-region includes two hCKM106Es and three hDes68Es. In some embodiments, the enhancement sub-region includes two hCKM106Es and two hDes68Es. In some embodiments, the enhancement sub-region includes two hCKM106Es and one hDes68E. In some embodiments, the enhancement sub-region further includes at least one enhancement sub-element selected from the group consisting of hDes68E, hDes78E, and sE. The reinforcing sub-elements can be operably connected in different sequences from 5' to 3'.

[0105] In some embodiments, the enhancer region comprises three hCKM106Es and one hDes68E. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hCKM106E, hCKM106E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:36. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:36. The enhancer region may have the nucleotide sequence of SEQ ID NO:36.

[0106] In some embodiments, the enhancer region comprises three hCKM106E, one hDes68E, and one hDes78E. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hCKM106E, hCKM106E, hDes68E, and hDes78E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:37. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:37. The enhancer region may have the nucleotide sequence of SEQ ID NO:37.

[0107] In some embodiments, the enhancer region comprises two hCKM106Es and one sE. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hCKM106E, and sE. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:38. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:38. The enhancer region may have the nucleotide sequence of SEQ ID NO:38.

[0108] In some embodiments, the enhancer region comprises three hCKM106Es and four hDes68Es. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:41. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:41. The enhancer region may have the nucleotide sequence of SEQ ID NO:41.

[0109] In some embodiments, the enhancer region comprises two hCKM106Es and four hDes68Es. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:42. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:42. The enhancer region may have the nucleotide sequence of SEQ ID NO:42.

[0110] In some embodiments, the enhancer region includes three hCKM106E and three hDes68E. In some embodiments, the enhancer region comprises the following enhancer elements operably linked from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes69E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:43. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:43. The enhancer region may have the nucleotide sequence of SEQ ID NO:43.

[0111] In some embodiments, the enhancer region includes three hCKM106Es and two hDes68Es. In some embodiments, the enhancer region comprises the following 5' to 3' operatively linked enhancer elements: hCKM106E, hCKM106E, hDes68E, hCKM106E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:44. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:44. The enhancer region may have the nucleotide sequence of SEQ ID NO:44.

[0112] In some embodiments, the enhancer region includes two hCKM106Es and two hDes68Es. In some embodiments, the enhancer region comprises the following 5' to 3' operatively linked enhancer elements: hCKM106E, hDes68E, hCKM106E, and hDes68E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:45. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:45. The enhancer region may have the nucleotide sequence of SEQ ID NO:45.

[0113] In some embodiments, the enhancer region comprises an sE and a MEF2 motif. In some embodiments, the enhancer region comprises three sEs and one MEF2 motif. In some embodiments, the enhancer region comprises the following 5' to 3' operatively linked enhancer elements: sE, MEF2 motif, sE, and sE. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:39. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:39. The enhancer region may have the nucleotide sequence of SEQ ID NO:39.

[0114] In some embodiments, the enhancer region includes hCKM106ER or hDes68ER. In some embodiments, the enhancer region includes three hCKM106ERs and one hDes68ER. In some embodiments, the enhancer region comprises the following 5' to 3' operatively linked enhancer elements: hDes68ER, hCKM106ER, hCKM106ER, and hCKM106ER. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:40. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:40. The enhancer region may have the nucleotide sequence of SEQ ID NO:40.

[0115] In some embodiments, the enhancer region comprises three hCKM206E. In some embodiments, the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:34. The enhancer region may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:34. The enhancer region may have the nucleotide sequence of SEQ ID NO:34.

[0116] Table II: Exemplary Enhanced Subregions 6.2.1.2 Core Promoter The muscle-specific promoters provided herein include enhancer regions operatively connected to a core promoter. In some embodiments, the core promoter is P87. In some embodiments, the core promoter is sP86.

[0117] Table III: Exemplary Core Promoters In some embodiments, P87 has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1. In some embodiments, P87 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1. In some embodiments, P87 has a nucleotide sequence that is at least 90% identical to SEQ ID NO:1. In some embodiments, P87 has a nucleotide sequence that is at least 95% identical to SEQ ID NO:1. In some embodiments, P87 has a nucleotide sequence that is at least 98% identical to SEQ ID NO:1. In some embodiments, P87 has a nucleotide sequence that is at least 99% identical to SEQ ID NO:1. In some embodiments, P87 has the nucleotide sequence of SEQ ID NO:1.

[0118] In some embodiments, sP86 has a nucleotide sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2. In some embodiments, sP86 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:2. In some embodiments, sP86 has a nucleotide sequence that is at least 90% identical to SEQ ID NO:2. In some embodiments, sP86 has a nucleotide sequence that is at least 95% identical to SEQ ID NO:2. In some embodiments, sP86 has a nucleotide sequence that is at least 98% identical to SEQ ID NO:2. In some embodiments, sP86 has a nucleotide sequence that is at least 99% identical to SEQ ID NO:2. In some embodiments, sP86 has the nucleotide sequence of SEQ ID NO:2.

[0119] 6.2.1.3 Exemplary Promoters This document provides muscle-specific promoters comprising an enhancer region and a core promoter, wherein the enhancer region can be any enhancer region disclosed herein, and the core promoter can be any core promoter disclosed herein. All permutations and combinations of the disclosed enhancer regions and core promoters are explicitly considered herein. In some embodiments, the enhancer region comprises at least two enhancer elements, wherein each enhancer element is independently selected from hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motif. In some embodiments, the enhancer region is selected from thCKM, thCKMs, thMD, thMD2, hME2-sE, sE3, thMDR, L34, S24, S33, S32, and S22. In some embodiments, the enhancer region is thMD2. In some embodiments, the enhancer region is hME2-sE. In some embodiments, the enhancer region is thMDR. In some embodiments, the core promoter is P87. In some embodiments, the core promoter is sP86.

[0120] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM106E, hCKM106ER, hDes68E, hDes68ER and sE; wherein the core promoter is P87 or sP86.

[0121] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM106E, hDes68E and hDes78E; wherein the core promoter is P87 or sP86.

[0122] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, each enhancer element being independently hCKM106E or hDes68E; and the core promoter is P87 or sP86.

[0123] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, wherein each enhancer element is independently hCKM106E or sE; and wherein the core promoter is P87 or sP86.

[0124] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, each enhancer element being independently selected from sE or MEF2; and the core promoter is P87 or sP86.

[0125] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two enhancer elements, wherein each enhancer element is independently selected from hCKM106ER or hDes68ER; and wherein the core promoter is P87 or sP86.

[0126] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes at least two hCKM206E; and the core promoter is P87 or sP86. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM206E; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM206E; and the core promoter is sP86.

[0127] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region contains at least two hCKM106Es; and the core promoter is P87 or sP86. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region contains three hCKM106Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region contains three hCKM106Es; and the core promoter is sP86.

[0128] In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein: the enhancer region comprises two or three hCKM106Es and one to four hDes68Es; and the core promoter is P87 or sP86. In some embodiments, the enhancer region further comprises hDes78Es. In some embodiments, the enhancer region further comprises sEs.

[0129] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and one hDes68E; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and one hDes68E; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hCKM106E, hCKM106E, and hDes68E.

[0130] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es, one hDes68E, and one hDes78E; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es, one hDes68E, and one hDes78E; and the core promoter is sP86. In some embodiments, the enhancer region includes the following 5' to 3' operatively connected enhancer elements: hCKM106E, hCKM106E, hCKM106E, hDes68E, and hDes78E.

[0131] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and one sE; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and one sE; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hCKM106E, and sE.

[0132] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and two hDes68Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and two hDes68Es; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, and hDes68E.

[0133] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and three hDes68Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes three hCKM106Es and three hDes68Es; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, and hDes68E.

[0134] In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein: the enhancer region comprises three hCKM106Es and four hDes68Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein: the enhancer region comprises three hCKM106Es and four hDes68Es; and the core promoter is sP86. In some embodiments, the enhancer region comprises the following 5' to 3' operable connection segments: hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E.

[0135] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and four hDes68Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and four hDes68Es; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E.

[0136] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and two hDes68Es; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes two hCKM106Es and two hDes68Es; and the core promoter is sP86. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hCKM106E, hDes68E, hCKM106E, and hDes68E.

[0137] In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein: the enhancer region comprises sE and MEF2 motifs; and the core promoter is P87. In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein: the enhancer region comprises sE and MEF2 motifs; and the core promoter is sP86. In some embodiments, the enhancer region comprises three sEs and one MEF2 motif. In some embodiments, the enhancer region comprises enhancer elements operably connected from 5' to 3': sE, MEF2 motif, sE, and sE.

[0138] In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes hCKM106ER or hDes68ER; and the core promoter is P87. In some embodiments, the muscle-specific promoter includes an enhancer region and a core promoter, wherein: the enhancer region includes hCKM106ER or hDes68ER; and the core promoter is sP86. In some embodiments, the enhancer region includes three hCKM106ERs and one hDes68ER. In some embodiments, the enhancer region includes enhancer elements operatively connected from 5' to 3': hDes68ER, hCKM106ER, hCKM106ER, and hCKM106ER.

[0139] Table IV: Exemplary Promoters In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:17. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:17. The promoter may have the nucleotide sequence of SEQ ID NO:17.

[0140] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:18. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:18. The promoter may have the nucleotide sequence of SEQ ID NO:18.

[0141] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:19. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:19. The promoter may have the nucleotide sequence of SEQ ID NO:19.

[0142] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:20. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:20. The promoter may have the nucleotide sequence of SEQ ID NO:20.

[0143] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:21. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:21. The promoter may have the nucleotide sequence of SEQ ID NO:21.

[0144] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:22. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:22. The promoter may have the nucleotide sequence of SEQ ID NO:22.

[0145] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:23. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:23. The promoter may have the nucleotide sequence of SEQ ID NO:23.

[0146] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:24. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:24. The promoter may have the nucleotide sequence of SEQ ID NO:24.

[0147] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:25. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:25. The promoter may have the nucleotide sequence of SEQ ID NO:25.

[0148] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:26. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:26. The promoter may have the nucleotide sequence of SEQ ID NO:26.

[0149] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:27. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:27. The promoter may have the nucleotide sequence of SEQ ID NO:27.

[0150] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:28. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:28. The promoter may have the nucleotide sequence of SEQ ID NO:28.

[0151] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:29. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:29. The promoter may have the nucleotide sequence of SEQ ID NO:29.

[0152] In some embodiments, the muscle-specific promoters provided herein have a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 75% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 80% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 85% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 90% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 95% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 98% identical to SEQ ID NO:30. The promoter may have a nucleotide sequence that is at least 99% identical to SEQ ID NO:30. The promoter may have the nucleotide sequence of SEQ ID NO:30.

[0153] 6.2.1.4 Expression cassettes and transgenic structures This document also provides an expression cassette containing a promoter disclosed herein that is operatively linked to a transgene. The promoter may be any muscle-specific promoter disclosed herein. In some embodiments, the promoter may be a promoter disclosed in Table IV or a variant thereof. In some embodiments, the expression cassette provided herein further contains additional cis-regulatory elements, such as introns, UTRs, and / or polyadenylated (polyA) sequences.

[0154] genetically modified In some embodiments, the expression cassette provided herein further includes a promoter disclosed herein that is operatively linked to the transgene. The transgene can target muscle expression. In some embodiments, the transgene encodes a therapeutic protein for treating muscle-related diseases or conditions.

[0155] Typically, genetically modified organisms (GMOs) are used in subjects with a given disease or condition where the natural gene corresponding to the GMO in the subject is unable to produce the correct gene product or the correct amount of gene product. The GMO can provide a copy of the subject's defective gene.

[0156] Typically, subjects carry genetic mutations in corresponding natural genes, and the transgenes disclosed herein restore the function of proteins in the subjects. In some embodiments, the transgene encodes the heavy and light chains of a therapeutic antibody, or an antigen-binding fragment. In some embodiments, the transgene encodes RNA for genome engineering (e.g., genome editing via homologous recombination). In some embodiments, the transgene encodes therapeutic RNA, such as shRNA, artificial miRNA, or elements affecting splicing.

[0157] In some embodiments, the transgene encoding a therapeutic protein carried in the expression cassette disclosed herein may be a survival motor neuron (SMN), fukutin-related protein (FKRP), follistatin (FST), neurotrophin 3 (NT-3), dystrophin, tafazzin, myotubularin, merosin, α-1,4-glucosidase, calpain 3, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, or poly(A)-binding protein nuclear 1. 1. PABPN1) and lysosome-associated membrane protein 2 isorform B (LAMP2B). In some embodiments, the transgene encodes SMN. In some embodiments, the transgene encodes FKRP. In some embodiments, the transgene encodes NT-3. In some embodiments, the transgene encodes dystrophin. In some embodiments, the transgene encodes tafaxin. In some embodiments, the transgene encodes tubulin. In some embodiments, the transgene encodes Melos protein. In some embodiments, the transgene encodes α-1,4-glucosidase. In some embodiments, the transgene encodes calpain 3. In some embodiments, the transgene encodes Disferin protein. In some embodiments, the transgene encodes α-caryogam. In some embodiments, the transgene encodes β-caryogam. In some embodiments, the transgene encodes γ-caryogam. In some embodiments, the transgene encodes PABPN1. In some embodiments, the transgene encodes LAMP2B.

[0158] In some embodiments, the transgene has the same nucleotide sequence as the endogenous gene encoding the target protein (e.g., a therapeutic protein). As used herein, the term “endogenous” describes a molecule (e.g., a nucleic acid) that is naturally present in a particular organism (e.g., a human) or at a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell). In some embodiments, the transgene is codon-optimized for its target subject (e.g., a human). As used herein, “codon optimization” refers to the process of adjusting a nucleic acid sequence based on the preference for the frequency of synonymous codons (e.g., codons encoding the same amino acid) when encoding DNA across different species. This codon degeneracy allows multiple nucleotide sequences to encode the same polypeptide. Sequences modified in this manner are referred to herein as “codon-optimized.” This process can be performed on any sequence described in this specification to enhance expression or stability. Codon optimization can be performed in ways known in the art, as described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. For example, sequences around translation start sites can be converted into Kozak consensus sequences using known methods. See, for example, Kozak et al. , Nucleic Acids Res. 15 (20): 8125-8148, which is incorporated into this paper by reference in its entirety.

[0159] Introns In some embodiments, the expression cassette provided herein further comprises at least one intron or a fragment or derivative thereof. As used herein, the term "intron" refers to a region within a gene coding region whose nucleotide sequence has not been translated into the amino acid sequence of the corresponding protein. The term "intron" also refers to a corresponding region in the RNA transcribed from the gene. In some embodiments, for example, the gene may contain at least one intron forming an intermediate sequence between two exons. Introns are transcribed into pre-mRNA but are removed during processing and are not included in the mature mRNA.

[0160] In some embodiments, at least one intron can enhance transgene expression. Non-limiting examples of introns include MVM (67-97 bps), F.IX truncated intron 1 (300 bps), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobulin splice acceptor (500 bps), SV40 late splice donor / splicing acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps).

[0161] In some embodiments, the length of an intron can be 100-500 nucleotides. The length of an intron may be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nucleotides. The length of introns can be between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500 nucleotides.

[0162] Untranslated regions (UTRs) In some embodiments, the expression cassette provided herein also includes a UTR. In some embodiments, the wild-type UTR of the gene is transcribed but not translated. Typically, the 5' UTR begins at the transcription start site and terminates at the start codon, and the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0163] Typically, features found in highly expressed genes in specific target organs can be engineered into UTRs to enhance stability and protein production. In some embodiments, the viral genome encoding the transgene described herein contains a Kozak sequence. While not wishing to be bound by theory, the wild-type 5'UTR includes features that play a role in translation initiation. The Kozak sequence is well known to be involved in the ribosomal initiation of translation of many genes and is typically contained in the 5'UTR. The Kozak sequence has a shared CCR(A / G)CCAUGG sequence, where R is a purine (adenine or guanine) at the third base upstream of the start codon (ATG), followed by another "G".

[0164] While not wishing to be bound by theory, it is known that a segment of adenosine and uridine is embedded in the wild-type 3'UTR. These AU-rich features are particularly prevalent in genes with high turnover rates. The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to regulate the stability of polynucleotides. When engineering specific polynucleotides, such as payload regions of a viral genome, one or more copies of ABE can be introduced to make the polynucleotide less stable, thereby reducing translation and the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein.

[0165] In some embodiments, the 3'UTR of the viral genome may include an oligomeric (dT) sequence used to add a poly-A tail.

[0166] Any UTR from any gene known in the art can be introduced into the viral genome of an AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as the selected gene, or their orientation or location may be altered. In some embodiments, the UTRs used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made using one or more other 5' UTRs or 3' UTRs known in the art. In some embodiments, the viral genome of the AAV particle contains at least one artificial UTR that is not a variant of a wild-type UTR. In some embodiments, the viral genome of the AAV particle contains UTRs selected from a family of transcripts that share common functions, structures, characteristics, or properties.

[0167] Polyadenylated (polyA) sequence In some embodiments, the expression cassette disclosed herein further includes at least one polyA sequence. The viral genome of the AAV particle disclosed herein includes at least one polyA sequence between the 3' end of the transgene coding sequence and the 5' end of the 3' UTR. In some embodiments, the polyA signaling region is located at the 3' end relative to the nucleic acid containing the transgene.

[0168] In some embodiments, the polyA signal region comprises a length of about 100-600 nucleotides, for example, about 100-500 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-600 nucleotides, about 200-500 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, about 300-600 nucleotides, about 300-500 nucleotides, about 300-400 nucleotides, about 400-600 nucleotides, about 400-500 nucleotides, or about 500-600 nucleotides. In some embodiments, the polyA signal region comprises about 100-150 nucleotides, for example, about 127 nucleotides. In some embodiments, the polyA signal region comprises about 450-500 nucleotides, for example, about 477 nucleotides.

[0169] 6.2.2 Nucleic Acids and Vectors This document also provides nucleic acids comprising the muscle-specific promoters disclosed herein. In some embodiments, the nucleic acid comprises a muscle-specific promoter including an enhancer region operatively linked to a core promoter, wherein the enhancer region comprises at least two enhancer elements, each enhancer element being independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDeses78E, sE, and MEF2 motifs. In some embodiments, the core promoter is P87 or sP86. In some embodiments, the nucleic acid disclosed herein comprises an expression cassette disclosed herein. In some embodiments, the expression cassette comprises a transgene operatively linked to a muscle-specific promoter. In some embodiments, the transgene encodes a target protein, for example, a protein for treating muscle-related diseases or conditions.

[0170] This document also provides vectors comprising the nucleic acids disclosed herein. In some embodiments, the vectors provided herein may comprise a muscle-specific promoter comprising an enhancer region operatively linked to a core promoter, wherein the enhancer region comprises at least two enhancer elements, each enhancer element being independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs. In some embodiments, the core promoter is P87 or sP86. In some embodiments, the nucleic acids disclosed herein comprise expression cassettes disclosed herein. In some embodiments, the expression cassette comprises a transgene operatively linked to a muscle-specific promoter. In some embodiments, the transgene encodes a target protein, for example, a protein for treating muscle-related diseases or conditions.

[0171] Vectors may contain DNA, RNA, or a combination of DNA and RNA. When a vector containing a DNA sequence is disclosed herein, a vector containing RNA equivalent to the DNA sequence is also considered to be disclosed herein. In some embodiments, the vectors provided herein are single-stranded. In some embodiments, the vectors provided herein are double-stranded. In some embodiments, viral vectors may be used to deliver transgenes to a target region. In some embodiments, the viral vectors provided herein are recombinant viral vectors. In some embodiments, the viral vectors provided herein are modified such that they are replication-deficient in human cells. Viral vectors that can be used in the methods described herein include adenoviruses, AAVs, lentiviruses, helper-dependent adenoviruses, herpes simplex viruses, poxviruses, Sendai virus (hemagglutinin virus of Japan, HVJ), alpha viruses, vaccinia virus, and retroviral vectors. In some embodiments, the viral vector is a chimeric vector, for example, an AAV vector contained within a “helpless” adenovirus vector. In some embodiments, viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus are provided herein.

[0172] In some embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. The recombinant adenovirus may be a first-generation vector with an E1 deletion, with or without an E3 deletion, and an expression cassette inserted into either deleted region. The recombinant adenovirus may be a second-generation vector containing complete or partial deletions of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeat and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3' ITR, with or without a filler sequence, to maintain the artificial genome close to the wild-type size of approximately 36 kb. For exemplary schemes for generating adenovirus vectors, see: Alba et al. , 2005, Gene Therapy 12:S18-S27, the full text of which is incorporated herein by reference.

[0173] In some embodiments, the viral vector used in the methods described herein is a lentiviral viral vector. Four plasmids are used to prepare the construct: a plasmid containing the Gag / pol sequence, a plasmid containing the Rev sequence, a plasmid encoding an envelope protein (i.e., VSV-G), and a Cis plasmid with packaging elements and transgenes. These four plasmids are co-transfected into cells (i.e., HEK293-based cells) to produce lentivirus, with polyethyleneimine or calcium phosphate used as transfection agents, etc. The lentivirus can then be harvested from the supernatant. Exemplary protocols for producing lentiviral vectors can be found at: Lesch et al. , 2011, Gene Therapy 18:531-538 and Ausubel et al., 2012, Bioprocess Int 10(2):32-43, both of which are incorporated into this paper through full citation.

[0174] In some embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In some embodiments, the viral vectors provided herein are MLV-based viral vectors. In some embodiments, the viral vectors provided herein are human immunodeficiency virus (HIV)-based vectors. In some embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In some embodiments, the viral vectors provided herein are alpha virus-based viral vectors. Alpha virus vectors include semliki forest virus (SFV) and sindbis virus (SIN). In some embodiments, the alpha virus vectors provided herein are recombinant, replication-defective alpha viruses.

[0175] In some embodiments, the vectors provided herein contain components that influence binding to or targeting cells. In some embodiments, the vectors provided herein contain components that influence the localization of polynucleotides (e.g., transgenes) taken up into cells. In some embodiments, the vectors provided herein include components that can be used as detection or screening tags for purposes such as detecting or screening cells that have taken up polynucleotides.

[0176] The selection tag may include a gene sequence or a protein or polypeptide encoded by a gene sequence expressed in the host cell, which allows for the identification, selection, and / or purification of host cells from a cell population that may or may not express the selection tag. In some embodiments, the selection tag provides resistance to enable the host cell to survive the selection process, which would otherwise kill the host cell, for example, by treatment with an antibiotic. In some embodiments, the antibiotic selection tag may include one or more antibiotic resistance factors, including but not limited to neomycin resistance (e.g., neo), hygromycin resistance, kanamycin resistance, and / or puromycin resistance.

[0177] In some embodiments, the vectors provided herein may contain screening tags, including but not limited to β-lactamases, luciferases, β-galactosidases, or any other reporter genes as understood in the art, including cell surface markers such as CD4 or truncated nerve growth factor receptor (NGFR) (for GFP, see WO96 / 23810; Heim et al., Current Biology 2:178-182 (1996); Heim et al., Proc.Natl. Acad. Sci. USA (1995); or Heim et al., Science 373:663-664 (1995); for β-lactamases, see WO 96 / 30540), the entire contents of which are incorporated herein by reference. In some embodiments, the vector may include luciferase. In some embodiments, the screening tags may include fluorescent proteins. The fluorescent proteins described herein may include any fluorescent markers, including but not limited to green, yellow, and / or red fluorescent proteins (GFP, YFP, and / or RFP). In some embodiments, the payload construct for encoding the screening label may include a human influenza hemagglutinin (HA) label.

[0178] 6.2.2.1 AAV In some embodiments, the vector provided herein is an AAV vector. As understood in the art, “adeno-associated virus” or “AAV” is a small, non-enveloped virus belonging to the Parvoviridae family. It is a single-stranded DNA virus with a genome length of approximately 4.7 kilobases, characterized by its ability to establish latent infection in host cells. The AAV genome typically contains two open reading frames encoding replication-associated proteins (Rep) and capsid structural proteins (Cap). Flanking the open reading frames are two inverted terminal repeats (ITRs), which serve as the starting point for viral genome replication. The wild-type AAV viral genome contains nucleotide sequences with two open reading frames, one encoding four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by the Rep gene) and the other encoding three capsid or structural proteins (VP1, VP2, VP3, encoded by the capsid gene or Cap gene). The Rep proteins are important for replication and packaging, while the capsid proteins assemble to form the protein coat of the AAV, or AAV capsid. Alternative splicing, alternative start codons, and promoters result in the production of four distinct Rep proteins from a single open reading frame (ORF), and three capsid proteins from the same ORF. VP1 is the full-length capsid sequence, while VP2 and VP3 are the shorter components of the entire capsid sequence. The three capsid proteins assemble together to form the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically contains a VP1:VP2:VP3 molar ratio of 1:1:10. As used herein, “AAV serotype” is primarily defined by the AAV capsid. In some cases, the ITR is also specifically described by the AAV serotype (e.g., AAV2 / 9).

[0179] In some embodiments, the AAV vector provided herein is a recombinant vector. As used herein, the terms "recombinant AAV" or "rAAV" refer to a modified or engineered version of a naturally occurring AAV.

[0180] AAV typically requires a helper (e.g., adenovirus) to carry out productive infection within cells. Without this helper function, AAV viral particles essentially only enter the host cell and establish a latent infection.

[0181] Due to certain unique characteristics, AAV vectors have been investigated for use in gene therapy delivery. Non-limiting examples of these characteristics include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host infection range, including human cells; (iii) wild-type AAVs are not associated with any disease and do not replicate in infected cells; and (iv) they do not integrate into the host chromosome, thus reducing the likelihood of long-term genetic alterations. Furthermore, AAV vector infection has a minimal effect on altering cellular gene expression patterns (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148; the entire contents of which are incorporated herein by reference).

[0182] The viral genome of the AAV particles disclosed herein may be single-stranded or self-complementary. The size of the vector genome may be small, medium, large, or maximum size. In some embodiments, the AAV vector used in this disclosure is a single-stranded vector (ssAAV). In some embodiments, the AAV vector may be a self-complementary AAV vector (scAAVs), for example, see US7,465,583. The scAAV vector contains two DNA strands that, after annealing, form double-stranded DNA. By skipping the synthesis of the second strand, scAAVs can be rapidly expressed in cells. In some embodiments, the AAV vector used in this disclosure is scAAV.

[0183] Typically, AAV vectors used to deliver transgenes are recombinant viral vectors whose viral genome lacks sequences encoding functional Rep and Cap proteins, thus being replication-deficient. In some cases, defective AAV vectors may lack most or all of the coding sequences, essentially containing only one or two AAV ITR sequences and a payload sequence.

[0184] As used herein, the term "inverted terminal repeat sequence" or "ITR" is a palindromic nucleic acid of approximately 120 to approximately 250 nucleotides in length and capable of forming a hairpin structure. The term "ITR" includes a viral genome replication site that can be recognized and bound by parvovirus proteins (e.g., Rep78 / 68). ITRs can originate from any AAV. An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term "ITR" includes wild-type ITRs and their variants (e.g., wild-type ITRs can be altered by insertion, deletion, truncation, or missense mutations, provided the ITR has functions such as mediating viral packaging, replication, integration, and / or proviral rescue). "5'ITR" refers to a parvovirus ITR located at the 5' boundary of a nucleic acid molecule; the term "3'ITR" refers to a parvovirus ITR located at the 3' boundary of a nucleic acid molecule.

[0185] In some embodiments, the viral genome includes at least one ITR region. The AAV vector disclosed herein includes a viral genome having at least one ITR region and a transgenic region. In some embodiments, the viral genome has two ITRs. These two ITRs are located at the 5' and 3' ends of the transgenic region. In some embodiments, the ITR serves as a replication initiation site, including a recognition site for replication. In some embodiments, the ITR includes sequence regions that may be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into the viral genome described herein may consist of a naturally occurring polynucleotide sequence or a recombinant-derived polynucleotide sequence.

[0186] The ITR can be of the same serotype as the capsid, selected from any known serotype. The ITR can also be of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In some embodiments, the ITRs have the same serotype as each other. In another embodiment, the ITRs have different serotypes as each other. Non-limiting examples include zero, one, or two ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0187] Independently, the length of each ITR can be from about 100 to about 150 nucleotides. In some embodiments, the length of the ITR comprises 100-180 nucleotides, for example, about 100-115, about 100-120, about 100-130, about 100-140, about 100-150, about 100-160, about 100-170, about 100-180, about 110-120, about 110-130, about 110-140, about 110-150, about 110-160, about 110-170, about 110-180, about 120-130, about 120 -140, approximately 120-150, approximately 120-160, approximately 120-170, approximately 120-180, approximately 130-140, approximately 130-150, approximately 130-160, approximately 130-170, approximately 130-180, approximately 140-150, approximately 140-160, approximately 140-170, approximately 140-180, approximately 150-160, approximately 150-170, approximately 150-180, approximately 160-170, approximately 160-180, or approximately 170-180 nucleotides. In some embodiments, the ITR comprises approximately 120-140 nucleotides, for example, approximately 130 nucleotides. In some embodiments, the ITR is 140-142 nucleotides long, for example, 141 nucleotides. In some embodiments, the ITR comprises 125-135 nucleotides, for example, 130 nucleotides. Non-limiting examples of ITR lengths are 102, 130, 140, 141, 142, and 145 nucleotides, and nucleic acids having at least 95% identity with them.

[0188] In some embodiments, the viral genome includes one or more filler sequences. The filler sequences can be wild-type sequences or engineered sequences. The filler sequences can be variants of the wild-type sequences. In some embodiments, the viral genome includes one or more filler sequences to make the length of the viral genome the optimal size for packaging. For illustrative purposes, in some embodiments, the viral genome is a single-stranded (SS) viral genome and includes one or more filler sequences, which individually or together have a length of about 0.1 kb to 3.8 kb; in some embodiments, the viral genome is a self-complementary (SC) viral genome and includes one or more filler sequences to make the length of the viral genome about 2.3 kb.

[0189] In some embodiments, the viral genome includes one or more filler sequences between one or more regions of the viral genome. In some embodiments, the filler region may precede regions such as, but not limited to, transgenic regions, ITRs, promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions. In some embodiments, the filler region may follow regions such as, but not limited to, payload regions, ITRs, promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions.

[0190] The filler sequence can be located at the 3' of the 5' ITR sequence. The filler sequence can be located at the 5' of the promoter sequence. The filler sequence can be located at the 3' of the polyadenylation signal sequence. The filler sequence can be located at the 5' of the 3' ITR sequence. The filler sequence can be located between two intron sequences. The filler sequence can be located within an intron sequence.

[0191] The filler sequence can be located after the 5' ITR. The filler sequence can be located after the promoter region. The filler sequence can be located after the transgenic region. The filler sequence can be located after the intron region. The filler sequence can be located after the enhancer region. The filler sequence can be located after the polyadenylation signal sequence region. The filler sequence can be located before the promoter region. The filler sequence can be located before the transgenic region. The filler sequence can be located before the intron region. The filler sequence can be located before the enhancer region. The filler sequence can be located before the polyadenylation signal sequence region. The filler sequence can be located before the 3' ITR.

[0192] In some embodiments, for example, the AAV vector provided herein comprises an ITR region and an expression cassette. For example, in some embodiments, the viral genome provided herein comprises two AAV ITR sequences and an expression cassette, wherein the expression cassette comprises the muscle-specific promoter and transgene disclosed herein. In some embodiments, the muscle-specific promoter comprises an enhancer region and a core promoter, wherein the enhancer region comprises at least two enhancer elements, wherein each enhancer element is independently selected from hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDeses78E, sE, and MEF2 motifs, and wherein the core promoter comprises P87 or sP86.

[0193] The expression cassette may also contain any elements known in the art, such as intron regions, Kozak sequences, etc. In some embodiments, for example, the AAV vector provided herein includes a first ITR region, a muscle-specific promoter disclosed herein, a transgene, a polyA signaling region, and a second ITR region.

[0194] In some embodiments, the AAV vectors disclosed herein can be introduced into mammalian cells. In some embodiments, the AAV vectors can be modified to improve delivery efficiency. Such modified AAV vectors of this disclosure can be efficiently packaged and can be used to successfully infect target cells with high frequency and minimal toxicity. Methods for preparing and / or modifying AAV vectors, such as pseudotyped AAV vectors, are disclosed in the art (International Patent Publications WO200028004; WO200123001; WO2004112727; WO2005005610 and WO2005072364, the entire contents of which are incorporated herein by reference).

[0195] 6.2.2.2 AAV serotypes The AAV vector of this disclosure may comprise or be derived from any natural or recombinant AAV serotype. According to this disclosure, AAV particles may utilize or be serotype-based, or comprise polypeptides selected from any of the following serotypes: AAV1, AAV2, AAV2.5, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV13, and any other AAV now known or hereafter discovered. For example, see Fields et al. VIROLOGY, 4th ed. Lippincott-Raven Publishers, Philadelphia, 1996; other AAV serotypes and clades have recently been discovered. For example, see Gao et al. J. Virol. 78:6381 (2004); Moris et al. Virol. 33:375 (2004).

[0196] In addition, AAV particles can also utilize or be based on serotypes, or include peptides selected from the following serotypes: VOY101, VOY201, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN,AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP,AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12,AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV16.3,AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4,AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23,AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4,AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62,AAV2-3 / rh.61,AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54,AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3,AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3,AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3,AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3,AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5,AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVcy.5R3. AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5,AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16,AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32,AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42,AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46,AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51,AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19,AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13,AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21,AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33,AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39,AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49,AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58,AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74,AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV,bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16,AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4,AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1,AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13,AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAVShuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAVShuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAVSM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62,AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53,AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10,Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBR-B7.3, AAVCBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAVCBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3,AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9,AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7,AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAVCKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1,AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6,AAV CLg-F7, AAVCLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV-18-1, AAV Clv, AAV Clv CLv-2, AAVCLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3,AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-DAV-1, AAV-CLv-1, AAV-CLv-1 AAVCLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-MAV, AAV-CLv-AAV7 CLv-M8,AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAVCLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CLv-1, AAV AVCS-10, AAV AVCSp CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAVCSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8, AAV CSp-8. CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4,AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14,AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4,AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and their variants.

[0197] The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITR, Rep protein, and capsid subunit, are known in the art. These sequences can be found in literature or public databases (such as GenBank). For example, see NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562. NC_001358, NC_001540, AF513851, AF513852 and AY530579; the public information thereof is incorporated herein by reference for the purpose of studying AAV nucleic acid and amino acid sequences. See also, for example, Bantel-Schaal et al. J. Virol. 73:939 (1999); Chiorini et al. J. Virol. 71:6823 (1997); Chiorini et al. J. Virol. 73:1309 (1999); Gao et al. Proc. Nat. Acad. Sci. USA 99:11854 (2002); Moris et al. Virol. 33:375 (2004); Muramatsu et al. Virol. 221:208 (1996); Ruffing et al. J. Gen. Virol. 75:3385 (1994); Rutledge et al. J. Virol. 72:309 (1998); Schmidt et al. J. Virol. 82:8911 (2008); Shade et al. J. Virol. 58:921 (1986); Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); Pulicherla et al., Molecular Therapy, 19(6):1070-1078 (2011); US 6,156,303; US20030138772; US20150159173; US 7,198,951; US9,475,845; US20140359799; US9,233,131; US20150376607; US9,163,261; US20150376240; US20160017295; US20150238550; US20150315612; US9,238,800; US9,193,769; US7,427,396; US9,624,274; S20150159173; US20160017005; US8,734,809; WO 00 / 28061,WO 99 / 61601, WO 98 / 11244; WO1998011244; WO2014144229; WO2005033321;WO2015168666; WO2015121501; WO2015038958; WO2016049230; WO2016065001;WO2017100671; WO2017058892; the contents of these publications are incorporated herein by reference for the purpose of studying AAV nucleic acid and amino acid sequences.

[0198] In some embodiments, AAVs of serotypes AAV1, AAV2, AAV7, AAV8, AAV9, AAV12, AAVrh74, AAVhu.32, or mixtures thereof can be used to deliver transgenes to the target region. In some embodiments, AAV9 can be used. In some embodiments, AAV1 can be used. In some embodiments, AAV2 can be used. In some embodiments, AAV8 can be used. In some embodiments, AAVrh74 can be used.

[0199] 6.2.3 Production Method The nucleic acids and vectors (e.g., AAV) disclosed herein can be produced using any method known in the art. The AAV or other expression vectors used in the gene therapy methods disclosed herein can be produced using any method known in the art.

[0200] General virus production process Cells used for producing AAV (such as rAAV) may, in some embodiments, include mammalian cells (e.g., HEK293 cells) and / or insect cells (e.g., Sf9 cells). In various embodiments, AAV production includes processes and methods for producing AAV vectors that can contact target cells to deliver transgenes. In some embodiments, the viral vector is an AAV vector, such as a recombinant AAV vector.

[0201] In some embodiments, this document discloses a vector comprising the viral genome of this disclosure. In some embodiments, this document discloses a cell comprising the viral genome of this disclosure. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., HEK293 cell), or an insect cell (e.g., Sf9 cell).

[0202] In some embodiments, this document discloses a method for preparing a viral genome. The method includes providing a nucleic acid encoding the viral genome described herein and a backbone region adapted for replication of the viral genome in a cell (e.g., a bacterial cell) (e.g., where the backbone region contains one or both of a bacterial origin of replication and a selection tag), and cutting the viral genome from the backbone region, for example by cutting nucleic acid molecules upstream and downstream of the viral genome. In some embodiments, the viral genome includes a promoter operatively linked to a nucleic acid containing a transgene, the viral genome being introduced into generated AAV particles in the cell. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., HEK293 cells), or an insect cell (e.g., Sf9 cells). In some embodiments, this document discloses a method for preparing recombinant AAV particles of the present disclosure, the method comprising (i) providing a host cell containing the viral genome described herein, and incubating the host cell under conditions suitable for encapsulating the viral genome in a capsid protein, thereby preparing recombinant AAV particles. In some embodiments, the method comprises introducing a first nucleic acid containing the viral genome into the cell prior to step (i). In some embodiments, the host cell contains a second nucleic acid encoding a capsid protein. In some embodiments, the second nucleic acid is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. In some embodiments, the host cell is a bacterial cell, a mammalian cell (e.g., HEK293 cell), or an insect cell (e.g., Sf9 cell).

[0203] In various embodiments, this document provides methods for producing AAV vectors by: (a) contacting virus-producing cells with one or more viral packaging constructs encoding at least one AAV capsid protein and one or more expression constructs encoding transgenes and regulatory nucleic acids; (b) culturing virus-producing cells under conditions capable of producing at least one AAV particle or vector; and (c) isolating AAV particles or vectors from the production process.

[0204] In these methods, the viral packaging construct may encode at least one structural protein and / or at least one non-structural protein. The structural protein may include any natural or wild-type capsid protein VP1, VP2, and / or VP3, or chimeric proteins thereof. The non-structural protein may include any natural or wild-type Rep78, Rep68, Rep52, and / or Rep40 protein, or chimeric proteins thereof.

[0205] In some embodiments, contact occurs via transient transfection, viral transduction, and / or electroporation.

[0206] In some embodiments, the virus-producing cells are selected from mammalian cells and insect cells. In some embodiments, the insect cells include fall armyworm cells. In some embodiments, the insect cells include Sf9 insect cells. In some embodiments, the insect cells include Sf21 insect cells. AAV vectors prepared according to the methods described herein are also provided.

[0207] In various embodiments, the AAV carrier of this disclosure can be formulated into a pharmaceutical composition containing one or more acceptable excipients.

[0208] In some embodiments, AAV particles can be produced by contacting virus-producing cells (e.g., insect or mammalian cells) with at least one viral packaging construct encoding at least one capsid protein and at least one transgenic expression construct. The virus-producing cells can be contacted via transient transfection, viral transduction, and / or electroporation. The virus-producing cells can be cultured under conditions that generate, isolate (e.g., using temperature-induced lysis, mechanical lysis, and / or chemical lysis), and / or purify (e.g., by filtration, chromatography, and / or immunoaffinity purification) at least one AAV particle or vector.

[0209] In some embodiments, AAV particles are produced in insect cells (e.g., fall armyworm (Sf9) cells) using the methods described herein. As a non-limiting example, insect cells can be treated with viral transduction, which may include baculovirus transduction.

[0210] In some embodiments, AAV particles are produced in mammalian cells (e.g., HEK293 cells) using the methods described herein. As a non-limiting example, mammalian cells can be treated with multi-plasmid transient transfection (e.g., three-plasmid transient transfection).

[0211] In some embodiments, the method of this disclosure includes producing viral particles in viral production cells using a viral production system comprising at least one viral packaging construct and at least one transgenic expression construct. The at least one viral packaging construct and the at least one transgenic expression construct can be introduced into the viral production cells via co-transfection (e.g., two-plasmid transfection, three-plasmid transfection). Transfection is performed using standard molecular biology techniques known and routinely performed by those skilled in the art. The viral production cells provide cellular apparatus for expressing proteins and other biological materials required for the production of AAV particles, including the Rep protein for replicating the payload construct and the Cap protein for assembling a capsid encapsulating the replicated payload construct. The produced AAV particles are extracted from the viral production cells and can be processed into pharmaceutical formulations for drug delivery.

[0212] In various embodiments, once administered, the AAV particles disclosed herein can contact and enter target cells (e.g., enter endosomes) without being constrained by theoretical limitations. The AAV particles (e.g., AAV particles released from endosomes) can then contact the nucleus of the target cell to deliver a transgenic construct. The transgenic construct can be delivered into the nucleus of the target cell, where the transgene can be expressed.

[0213] In some embodiments, the production process of virus particles utilizes a seed culture of virus production cells containing one or more baculoviruses (e.g., a baculoviral expression vector (BEV) that has been transfected with a virus packaging construct and a transgenic expression construct, or baculovirus-infected insect cell (BIIC)).

[0214] In some embodiments, large-scale production of AAV particles utilizes a bioreactor. Without being constrained by theory, the use of a bioreactor allows for the precise measurement and / or control of variables supporting cell growth and activity for virus production, such as mass, temperature, mixing conditions (impeller speed or oscillating motion), CO2 concentration, O2 concentration, gas injection rate and volume, gas coverage rate and volume, pH, viable cell density (VCD), cell viability, cell diameter, and / or optical density (OD). In some embodiments, the bioreactor is used for batch production, harvesting the entire culture at experimentally determined time points for AAV particle purification. In some embodiments, the bioreactor is used for continuous production, harvesting a portion of the culture at experimentally determined time points, purifying AAV particles, and adding fresh culture medium components to refresh the remaining culture in the bioreactor.

[0215] In various embodiments, AAV viral particles can be extracted from virus-producing cells, a process that includes cell lysis, clarification, sterilization, and purification. Cell lysis includes any process that disrupts the structure of virus-producing cells to release AAV particles. In some embodiments, cell lysis may include heat shock, chemical, or mechanical lysis methods. Clarification may include crude purification of a mixture of lysed cells, culture medium components, and AAV particles. In some embodiments, clarification includes centrifugation and / or filtration, including but not limited to depth filtration, tangential flow filtration, and / or hollow fiber filtration.

[0216] In various embodiments, the final product of virus production is purified AAV particles, which consist of two components: (1) a transgenic expression construct (e.g., a recombinant AAV vector genome construct) and (2) a viral capsid.

[0217] In some embodiments, the virus production system or process of this disclosure includes the step of producing baculovirus-infected insect cells (BIICs) using viral production cells (VPCs) and plasmid constructs. In some embodiments, the virus production system or process of this disclosure includes the step of producing AAV particles using viral production cells (VPCs) and baculovirus-infected insect cells (BIICs).

[0218] Viral packaging constructs In various embodiments, the viral production system of this disclosure includes one or more viral packaging constructs that can be transfected / transduced into viral production cells. In some embodiments, the viral packaging construct or transgenic expression construct of this disclosure may be a baculovirus, also known as a baculovirus plasmid or recombinant baculovirus genome. In some embodiments, the viral expression construct includes a protein-coding nucleotide sequence and at least one expression control sequence for expression in viral production cells. In some embodiments, the viral expression construct includes a protein-coding nucleotide sequence operatively linked to at least one expression control sequence for expression in viral production cells. In some embodiments, the viral packaging construct comprises a parvovirus gene controlled by one or more promoters. The parvovirus gene may include a nucleotide sequence encoding a non-structural AAV replication protein, such as the Rep gene encoding Rep52, Rep40, Rep68, or Rep78 proteins. The parvovirus gene may include a nucleotide sequence encoding a structural AAV protein, such as the Cap gene encoding VP1, VP2, and VP3 proteins.

[0219] The viral packaging constructs disclosed herein may include any compound or formulation, whether biological or chemical, to facilitate the transformation, transfection, or transduction of nucleic acids into cells. Exemplary biological viral packaging constructs include plasmids, linear nucleic acid molecules, and recombinant viruses, including baculoviruses. Exemplary chemical vectors include lipid complexes. According to the present disclosure, viral packaging constructs are used to integrate nucleic acid sequences into viral replication cells. O' Reilly et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL. Oxford University Press, 1994; Maniatis et al., eds. MOLECULAR CLONING. CSH Laboratory, NY, NY (1982); and Philiport and Scluber, eds. LIPOSOMES AS TOOLS IN BASIC RESEARCHAND INDUSTRY. CRC Press, Ann Arbor, Mich. (1995); Each item relating to viral packaging constructs is incorporated herein by reference in its entirety.

[0220] In some embodiments, the viral packaging construct is an AAV expression construct that includes one or more nucleotide sequences encoding a non-structural AAV replication protein, a structural AAV capsid protein, or a combination thereof.

[0221] In some embodiments, the viral packaging construct of this disclosure may be a plasmid vector. In some embodiments, the viral packaging construct of this disclosure may be a rod-shaped virus construct.

[0222] This disclosure is not limited to the number of viral packaging constructs used to produce AAV particles or viral vectors. In some embodiments, according to this disclosure, AAV particles can be produced in virus-producing cells using one, two, three, four, five, six, or more viral packaging constructs. In some embodiments of this disclosure, the viral packaging constructs can be used to produce AAV particles in insect cells. In some embodiments, the wild-type AAV sequence of the capsid and / or rep gene can be modified, for example, to improve the properties of the viral particles, such as increasing infectivity or specificity, or increasing production yield.

[0223] In some embodiments, the VP coding region encodes one or more AAV capsid proteins of a specific AAV serotype. The VP coding regions for AAV serotypes may be the same or different. In some embodiments, the VP coding region may be codon-optimized. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for mammalian cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for insect cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for fall armyworm cells. In some embodiments, the VP coding region or nucleotide sequence may be codon-optimized for Sf9 or Sf21 cell lines.

[0224] This disclosure describes the process and method for producing AAV particles or viral vectors that are contacted with target cells to deliver a transgenic expression construct, such as a recombinant AAV particle or viral construct containing target gene nucleotides. The virus production cells can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells (e.g., insect cells, yeast cells, and mammalian cells).

[0225] mammalian cells In some embodiments, the AAV particles of this disclosure can be produced in virus-producing cells comprising mammalian cells. Virus-producing cells include mammalian cells such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293T (293T), Saos, C2C12, L cells, HT1080, Huh7, HepG2, C127, 3T3, CHO, HeLa cells, KB cells, BHK, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals. Virus-producing cells may include cells derived from any mammalian species, including but not limited to humans, monkeys, mice, rats, rabbits, and hamsters, or cell types including but not limited to fibroblasts, hepatocytes, tumor cells, transformed cell lines, etc.

[0226] AAV virus production cells commonly used for producing recombinant AAV particles include, but are not limited to, U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 6,428,988, and 5,688,676; U.S. Patent Application No. 2002 / 0081721; and International Patent Publications Nos. WO 00 / 47757, WO 00 / 24916, and WO 96 / 17947. The contents of these publications are incorporated herein by reference in their entirety unless they conflict with this disclosure.

[0227] In some embodiments, AAV virus production cells are trans-complementary packaging cell lines that can provide functionality removed from replication-defective helper viruses, such as HEK293 cells or other Ea trans-complementary cells. In some embodiments, packaging cell line 293-10-3 (ATCC accession number PTA-2361) can be used to produce AAV particles, as described in U.S. Patent No. 6,281,010, the contents of which relate to the 293-10-3 packaging cell line and its uses are incorporated herein by reference in their entirety. In some embodiments, cell lines of this disclosure, such as the HeLA cell line, trans-complementary adenoviral vectors that encode adenovirus Ela and adenovirus under the control of the phosphoglycerate kinase (PGK) promoter, can be used to produce AAV particles, as described in U.S. Patent No. 6,365,394, the contents of which relate to the HeLa cell line and its uses are incorporated herein by reference in their entirety.

[0228] In some embodiments, AAV particles can be produced in mammalian cells using a multi-plasmid transient transfection method (such as a three-plasmid transient transfection). In some embodiments, the multi-plasmid transient transfection method includes transfecting three different constructs: (i) a transgenic expression construct, (ii) a Rep / Cap construct (parvovirus Rep and parvovirus Cap), and (iii) a helper construct. In some embodiments, a three-component triple transfection method for AAV production can be used to produce small batches of virus for assays including transduction efficiency, target tissue (tropism) assessment, and stability. In some embodiments, a three-component triple transfection method for AAV production can be used to produce large quantities of material for clinical or commercial applications.

[0229] Insect cells In some embodiments, the AAV particles or viral vectors of this disclosure can be produced in virus-producing cells comprising insect cells. Growth conditions for culturing insect cells and the production of heterologous products in cultured insect cells are well known in the art, see U.S. Patent No. 6,204,059, which relates to insect cell growth and its application in virus production, and is incorporated herein by reference in its entirety.

[0230] According to this disclosure, any insect cell that allows parvovirus replication and can be cultured and maintained may be used. AAV virus production cells commonly used for producing recombinant AAV particles include, but are not limited to, fall armyworm cell lines (including but not limited to Sf9 or Sf21 cell lines), Drosophila cell lines, or mosquito cell lines (such as Aedes albopictus-derived cell lines). Methods for expressing heterologous proteins using insect cells, such as methods for introducing nucleic acids (e.g., vectors, e.g., insect cell-compatible vectors) into insect cells, and methods for culturing and maintaining such cells, are well documented. See, for example, METHODS INMOLECULAR BIOLOGY, ed. Richard, Humana Press, NJ (1995); O' Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al. al., Proc.Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30(1992); Kimbauer et al., Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93(2000); and Samulski et al., US Pat. No. 6,204,059, of which the content concerning the use of insect cells in virus production is incorporated herein by reference in its entirety.

[0231] Baculovirus Production System In some embodiments, the methods of this disclosure may include a method for producing AAV particles or viral vectors using viral packaging constructs and transgenic expression constructs in a baculovirus system. In some embodiments, the baculovirus system includes baculovirus expression vectors (BEVs) and / or baculovirus-infected insect cells (BIICs). In some embodiments, the viral packaging constructs or transgenic expression constructs of this disclosure may be baculoviruses, also known as baculovirus plasmids or recombinant baculovirus genomes. In some embodiments, the viral packaging constructs or transgenic expression constructs of this disclosure may be polynucleotides introduced into baculovirus plasmids via homologous recombination (transposon donor / recipient system) using standard molecular biology techniques known and practiced by those skilled in the art. Transfection of a separate viral replication cell population yields two or more sets (e.g., two or three sets) of baculoviruses (BEVs), wherein one or more sets of baculoviruses may include viral packaging constructs (expression BEVs), and one or more sets may include payload constructs (payload BEVs). Baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors.

[0232] In some embodiments, the method includes transfecting a single viral replication cell population to produce a single set of baculoviruses (BEVs), which includes viral packaging constructs and payload constructs. These baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors. In some embodiments, the BEVs are produced using a baculovirus transfection agent such as PromegaFuGENE®HD, WFI water, or ThermoFisher Cellfectin® II Reagent. In some embodiments, the BEVs are produced and amplified in virus-producing cells, such as insect cells.

[0233] In some embodiments, the method utilizes a seed culture of virus-producing cells comprising one or more BEVs, including baculovirus-infected insect cells (BIICs). Seed BIICs have been transfected / transduced / infected using expression BEVs including viral packaging constructs and transgenic expression BEVs including transgenic expression constructs. BEVs used to generate AAV particles in insect cells (including, but not limited to, fall armyworm (Sf9) cells) can provide high-titer viral vector products.

[0234] In some embodiments, a genetically stable baculovirus can produce one or more components for generating AAV particles in invertebrate cells. In some embodiments, a defective baculovirus expression vector exists as a free organism in insect cells. In these embodiments, the corresponding baculovirus vector is engineered with replication control elements, including but not limited to promoters, enhancers, and / or cell cycle-regulating replication elements. In some embodiments, virus-producing stable cells that allow baculovirus infection are engineered to stably integrate at least one copy of the elements required for AAV replication and vector production, including but not limited to the entire AAV genome, Rep and Cap genes, Rep gene, Cap gene, a separate transcript cassette for each Rep protein, a separate transcript cassette for each VP protein, AAP (assembly activation protein), or at least one of baculovirus helper genes having a natural or non-natural promoter.

[0235] In some embodiments, the AAV particles of this disclosure can be produced in insect cells (e.g., Sf9 cells). In some embodiments, the AAV particles of this disclosure can be produced in mammalian cells. In some embodiments, the AAV particles of this disclosure can be produced using triple transfection. In some embodiments, the AAV particles of this disclosure can be produced using triple transfection of mammalian cells. In some embodiments, the AAV particles of this disclosure can be produced by triple transfection in HEK293 cells.

[0236] 6.3 Drug components This disclosure also provides pharmaceutical compositions for delivering the transgenic substances described herein to subjects, including human subjects. In some embodiments, the composition comprises any nucleic acid or vector described herein. In some embodiments, the pharmaceutical compositions disclosed herein comprise any vector disclosed herein and one or more pharmaceutically acceptable vectors. In some embodiments, the composition comprises any AAV vector described herein. In some embodiments, the pharmaceutical compositions disclosed herein comprise any AAV vector disclosed herein and one or more pharmaceutically acceptable vectors.

[0237] Although the pharmaceutical compositions provided herein, such as AAV carriers, are primarily intended for the preparation of pharmaceutical compositions suitable for human administration, those skilled in the art will understand that such compositions are generally suitable for any other animal, for example, non-human animals, such as non-human mammals. It is widely understood that modifying pharmaceutical compositions suitable for human administration to make them suitable for administration to different animals is a modification that can be designed and / or carried out by veterinary pharmacologists of ordinary skill level in a conventional or experimental manner. Subjects to administration of the pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0238] In some embodiments, the composition is applied to humans.

[0239] According to this disclosure, pharmaceutical compositions can be prepared in bulk form, prepared in a single unit dose and / or in multiple single unit doses, packaged, and / or sold. As used herein, a “unit dose” refers to an independent dose of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be given to a subject and / or an appropriate fraction of that dose, such as half or one-third of the dose. Examples of unit dose forms include ampoules, vials, pre-filled syringes, or cartridges.

[0240] The pharmaceutical compositions described herein can be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, followed by, if necessary and / or required, the product being fractionated, shaped, and / or packaged into the desired single or multiple dosage units.

[0241] According to this disclosure, the relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the pharmaceutical composition will vary depending on the identity, size, and / or condition of the subject being treated and the route of administration of the composition.

[0242] The AAV particles disclosed herein can be formulated using one or more excipients to: (1) increase stability; (2) promote cell transfection or transduction; (3) allow sustained or delayed release; (4) alter biodistribution (e.g., target viral particles to specific tissues or cell types); (5) increase the translation of encoded proteins in vivo; (6) alter the release profile of encoded proteins in vivo and / or (7) allow for the regulated expression of the payload.

[0243] The pharmaceutical compositions disclosed herein may include, but are not limited to, saline, lipids, liposomes, liposome nanoparticles, polymers, cationic liposomes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, and combinations thereof. Furthermore, the viral vectors disclosed herein may be formulated using self-assembled nucleic acid nanoparticles.

[0244] The pharmaceutical compositions disclosed herein may include one or more excipients, the amount of each excipient collectively increasing the stability of AAV particles, transfection or transduction of cells by viral particles, expression of proteins encoded by viral particles, and / or altering the release profile of proteins encoded by viral particles. In some embodiments, the purity of the pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipients are approved for human and veterinary use.

[0245] As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid carriers, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., suitable for the desired particular dosage form. Various excipients used to formulate pharmaceutical compositions and techniques for preparing such compositions are known in the art (see: REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Edition, ARGennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; the contents of which are incorporated herein by reference in their entirety). Conventional excipient media may be considered within the scope of this disclosure unless any conventional excipient media may be incompatible with a substance or its derivatives, for example, may produce any adverse biological effects, or may otherwise interact with any other component of the pharmaceutical composition in a harmful manner.

[0246] The pharmaceutical compositions of AAV particles disclosed herein may include cationic or anionic compounds. In some embodiments, the formulation includes metal cations, such as, but not limited to, Zn. 2+ Ca 2+ Cu 2+ Mg 2+ Or a combination thereof. In some embodiments, the pharmaceutical composition may include a polymer or polynucleotide complexed with a metal cation (see, for example, U.S. Patent Nos. 6,265,389 and 6,555,525, the contents of which are incorporated herein by reference in their entirety).

[0247] In some embodiments, the pharmaceutical compositions provided herein are liquid compositions. In some embodiments, the pharmaceutical compositions are frozen compositions. In some embodiments, the pharmaceutical compositions are lyophilized compositions or reconstituted lyophilized compositions. In some embodiments, the pharmaceutical compositions provided herein can be formulated into various dosage forms for administration, such as intra-articular administration.

[0248] 6.4 Methods and Applications The nucleic acids, vectors (e.g., AAV vectors), or pharmaceutical compositions provided herein can be introduced into muscle cells in vivo or in vivo to express transgenes. In some embodiments, this disclosure provides a method of delivering any of the aforementioned nucleic acids or vectors (e.g., AAV vectors) to muscle cells or tissue, including contacting the cells or tissue with the nucleic acid or vector (e.g., AAV vector), or contacting the cells or tissue with any of the said compositions, including pharmaceutical compositions. The method of delivering AAV vectors to cells or tissue can be performed in vitro, ex vivo, or in vivo.

[0249] In some embodiments, this disclosure provides methods for administering and / or delivering nucleic acids and vectors (e.g., AAV vectors) encoding the genetic material disclosed herein to increase the expression level of the genetic material, particularly in muscle. In some embodiments, this disclosure provides methods for administering and / or delivering vectors (e.g., AAV vectors) encoding the genetic material disclosed herein for the prevention, treatment, or improvement of diseases or conditions. In some embodiments, this disclosure provides methods for administering and / or delivering vectors (e.g., AAV vectors) encoding the genetic material disclosed herein for the treatment, prevention, or improvement of muscle-related diseases or conditions. Exemplary muscle-related diseases and conditions treatable by the methods disclosed herein include sarcopenia, muscular dystrophy (MD), congenital myopathy, distal myopathy, myotonia syndrome, ion channel disorders, malignant hyperthermia, metabolic myopathy, and arthritis. In some embodiments, the methods provided herein can be used to treat sarcopenia. In some embodiments, the methods provided herein can be used to treat MD. Exemplary MDs include, for example, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy, myotonic dystrophy (Steiner's disease), oculopharyngeal muscular dystrophy (OMD), or limb-girdle muscular dystrophy (LGMD). In some embodiments, the methods provided herein can treat DMD. In some embodiments, the methods provided herein can be used to treat BMD.

[0250] In some embodiments, the transgene delivered by the methods disclosed herein may encode molecules that are beneficial for the treatment of muscle-related diseases or conditions (such as those disclosed above). In some embodiments, the transgene can be survival motor neuron (SMN), fukutin-related protein (FKRP), follistatin (FST), neurotrophin 3 (NT-3), dystrophin, tafazzin, myotubularin, merosin, α-1,4-glucosidase, calpain 3, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, poly(A)-binding protein nuclear 1 (PABPN1), and lysosome-associated membrane protein 2 isorform. B, LAMP2B). In some embodiments, the transgene encodes SMN. In some embodiments, the transgene encodes FKRP. In some embodiments, the transgene encodes NT-3. In some embodiments, the transgene encodes dystrophin. In some embodiments, the transgene encodes tafacinin. In some embodiments, the transgene encodes tubulin. In some embodiments, the transgene encodes Melos protein. In some embodiments, the transgene encodes α-1,4-glucosidase. In some embodiments, the transgene encodes calpain 3. In some embodiments, the transgene encodes disforin. In some embodiments, the transgene encodes α-caryogam. In some embodiments, the transgene encodes β-caryogam. In some embodiments, the transgene encodes γ-caryogam. In some embodiments, the transgene encodes PABPN1. In some embodiments, the transgene encodes LAMP2B.

[0251] Following transduction of target cells, this muscle-specific expression cassette enhanced protein product expression. This enhancement can be measured by methods not limited to the following, such as 1) protein titer, measured by assays known to those skilled in the art, including but not limited to sandwich ELISA, Western blotting, histological staining, and liquid chromatography-mass spectrometry (LC-MS / MS); 2) protein activity, measured by binding assays, functional assays, enzyme assays, and / or substrate assays; and / or 3) serum half-life or long-term expression; and / or 4) detection of transgenic mRNA encoding therapeutic proteins. Enhanced transgenic expression can be determined to be effective and applicable for human treatment (Hintze). et al. , Biomarker Insights 2011:6 69-78). The assessment of the quantitative and functional properties of transgenes through such in vitro and in vivo cell, blood, and tissue studies has been shown to be associated with the efficacy of certain therapies and has been used to evaluate the therapeutic response of transgenes to gene therapy using the vectors described herein.

[0252] The nucleic acid, vector (e.g., AAV), and pharmaceutical compositions disclosed herein can also facilitate delivery, particularly targeted delivery, of transgenes operatively linked to the regulatory sequences described herein into target cells or tissues. The delivered contents include, but are not limited to, oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, target cells, or antibodies. The nucleic acid, vector (e.g., AAV), and pharmaceutical compositions disclosed herein can also facilitate delivery, particularly targeted delivery, of non-coding DNA, RNA, or oligonucleotides into target tissues.

[0253] In some embodiments, this disclosure provides a method for delivering any of the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions to a subject, including mammalian subjects. In some embodiments, systemic delivery is required. In some embodiments, the methods provided herein include intravenous administration of the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions. In some embodiments, the methods provided herein include subcutaneous administration of the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions. In some embodiments, the methods provided herein include intramuscular administration of the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions. In some embodiments, the methods provided herein include intraperitoneal administration of the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions. In some embodiments, local delivery to a target area is required. In some embodiments, the above-described nucleic acids, carriers (e.g., AAV), or pharmaceutical compositions may be delivered to a target area (e.g., muscle). This document provides methods for administering and / or delivering the AAV carriers described herein to a target area. Administration methods also include, but are not limited to, intradermal, epidural, and absorption via epithelial or mucosal skin or mucosal linings (e.g., intranasal, oral mucosa, rectal and intestinal mucosa, etc.).

[0254] The amount of an effective therapeutic agent (e.g., rAAV carrying a transgene encoding a therapeutic protein) can be determined using standard clinical techniques. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model experimental systems. For any reagent used in the methods disclosed herein, the therapeutically effective dose can initially be estimated from cell culture experiments. Doses can be designed in animal models to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the concentration of the test compound at which half-maximal inhibition of symptoms is achieved) determined in cell culture. This information can be used to more accurately determine the useful dose in humans. For example, plasma levels can be measured using high-performance liquid chromatography (HPLC).

[0255] Dosage and frequency typically vary depending on individual patient factors, such as the specific therapeutic or preventative medication being administered, the severity and type of disease, the route of administration, and the patient's age, weight, response, and medical history. They should be determined based on the judgment of a professional and the individual patient's circumstances. Those skilled in the art can select an appropriate regimen by considering these factors and following, for example, the dosages reported in the literature and those recommended in *The Physician's Desk Reference* (56th edition, 2002).

[0256] In some embodiments, the carrier or pharmaceutical composition is applied to the muscle tissue of a subject in need. In some embodiments, the carrier or pharmaceutical composition is applied to the skeletal muscle tissue of a subject in need. In some embodiments, the carrier or pharmaceutical composition is applied to the gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, quadriceps femoris, triceps brachii, and diaphragm of a subject in need. The subject may have a muscle-related disease or condition. The subject may be at risk of developing a muscle-related disease or condition. The subject may be a human. The subject may be a non-human animal. The subject may be a non-human mammal, such as a dog or cat.

[0257] In some embodiments, the nucleic acids, vectors (e.g., AAV), or pharmaceutical compositions disclosed herein are administered co-administered with secondary treatment. In some embodiments, secondary treatment includes a therapeutic agent for muscle-related diseases (e.g., sarcopenia, muscular dystrophy, congenital muscular dystrophy, congenital myopathy, distal myopathy, myotonia syndrome, ion channel disorders, malignant hyperthermia, metabolic myopathy, other muscle diseases, or arthritis) or any other appropriate therapy for treating symptoms of such diseases. Any convenient treatment method may be employed.

[0258] 6.5 Reagent Kits and Equipment In some embodiments, this disclosure provides various kits to facilitate and / or efficiently implement the methods of this disclosure. Typically, the kits will include sufficient quantities and / or numbers of components to allow a user to administer multiple treatments and / or conduct multiple experiments on a subject.

[0259] Any composition or carrier disclosed herein may be included in the kit. In some embodiments, the kit may also include reagents and / or instructions for generating and / or synthesizing the compounds and / or pharmaceutical compositions disclosed herein. In some embodiments, the kit may also include one or more buffer solutions. In some embodiments, the kit of this disclosure may include components for preparing protein or nucleic acid arrays or libraries, and therefore may include, for example, a solid support.

[0260] In some embodiments, kit components may be packaged in an aqueous medium or in lyophilized form. The kit's container device typically includes at least one vial, test tube, flask, bottle, syringe, or other container device in which components can be placed and appropriately aliquoted. If there are multiple kit components (labeled reagents and labels may be packaged together), the kit may also include second, third, or other additional containers for individually holding additional components. In some embodiments, the kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, one or more vials may contain different combinations of components. The kits of this disclosure may also typically include a device for containing compounds and / or pharmaceutical compositions of this disclosure, such as proteins, nucleic acids, and any other closed reagent containers intended for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are retained.

[0261] In some embodiments, the kit components are provided in the form of one and / or more liquid solutions. In some embodiments, the liquid solution is an aqueous solution, particularly a sterile aqueous solution. In some embodiments, the kit components may be provided in the form of a dry powder. When reagents and / or components are provided in dry powder form, these powders can be reconstituted by adding an appropriate volume of solvent. In some embodiments, it is conceivable that the solvent may also be provided in another container device.

[0262] In some embodiments, the kit may include instructions for using kit components and any other reagents not included in the kit. The instructions may include feasible modification options.

[0263] 6.6 Exemplary Implementation Scheme Implementation Scheme 1: A nucleic acid comprising a muscle-specific promoter, the promoter comprising an enhancer region operatively linked to a core promoter, wherein the enhancer region comprises at least two enhancer elements, wherein each enhancer element is independently selected from hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs, and the nucleotide sequence of each of them is at least 85% identical to SEQ ID NO:6-13.

[0264] Implementation Scheme 2: The nucleic acid of Implementation Scheme 1, wherein the enhancer region contains two, three, four, five, six or seven enhancer elements.

[0265] Implementation Plan 3: The nucleic acid of Implementation Plan 1, wherein the enhancer region contains two or three hCKM106E.

[0266] Implementation Scheme 4: The nucleic acid of Implementation Scheme 3, wherein the enhancer region further comprises at least one enhancer element selected from hDes68E, hDes78E and sE.

[0267] Implementation Scheme 5: The nucleic acid of Implementation Scheme 4, wherein the enhancer region contains one, two, three or four hDes68E.

[0268] Implementation Scheme 6: The nucleic acid of Implementation Scheme 3, wherein the enhancer region has three hCKM106E.

[0269] Implementation Scheme 7: The nucleic acid of Implementation Scheme 6, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:35.

[0270] Implementation Scheme 8: The nucleic acid of Implementation Scheme 5, wherein the enhancer region contains three hCKM106E and one hDes68E.

[0271] Implementation Scheme 9: The nucleic acid of Implementation Scheme 8, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E, hCKM106E and hDes68E.

[0272] Implementation Scheme 10: The nucleic acid of Implementation Scheme 9, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:36.

[0273] Implementation Scheme 11: The nucleic acid of Implementation Scheme 5, wherein the enhancer region contains three hCKM106E, one hDes68E and one hDes78E.

[0274] Implementation Scheme 12: The nucleic acid of Implementation Scheme 11, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E, hCKM106E, hDes68E and hDes78E.

[0275] Implementation Scheme 13: The nucleic acid of Implementation Scheme 12, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:37.

[0276] Implementation Scheme 14: The nucleic acid of Implementation Scheme 4, wherein the enhancer region contains two hCKM106E and one sE.

[0277] Implementation Scheme 15: The nucleic acid of Implementation Scheme 14, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E and sE.

[0278] Implementation Scheme 16: The nucleic acid of Implementation Scheme 15, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38.

[0279] Implementation Scheme 17: Nucleic acid from Implementation Scheme 5, wherein the enhancer region contains three hCKM106E and four hDes68E.

[0280] Implementation Scheme 18: The nucleic acid of Implementation Scheme 17, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E and hDes68E.

[0281] Implementation Scheme 19: The nucleic acid of Implementation Scheme 18, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:41.

[0282] Implementation Scheme 20: The nucleic acid of Implementation Scheme 5, wherein the enhancer region contains two hCKM106E and four hDes68E.

[0283] Implementation Scheme 21: The nucleic acid of Implementation Scheme 20, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E and hDes68E.

[0284] Implementation Scheme 22: The nucleic acid of Implementation Scheme 21, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:42.

[0285] Implementation Scheme 23: The nucleic acid of Implementation Scheme 5, wherein the enhancer region contains three hCKM106E and three hDes68E.

[0286] Implementation Scheme 24: The nucleic acid of Implementation Scheme 23, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E and hDes68E.

[0287] Implementation Scheme 25: The nucleic acid of Implementation Scheme 24, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43.

[0288] Implementation Scheme 26: Nucleic acid from Implementation Scheme 5, wherein the enhancer region contains three hCKM106E and two hDes68E.

[0289] Implementation Scheme 27: The nucleic acid of Implementation Scheme 26, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E and hDes68E.

[0290] Implementation Scheme 28: The nucleic acid of Implementation Scheme 27, wherein the enhancer region contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44.

[0291] Implementation Scheme 29: The nucleic acid of Implementation Scheme 5, wherein the enhancer region contains two hCKM106E and two hDes68E.

[0292] Implementation Scheme 30: The nucleic acid of Implementation Scheme 29, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hCKM106E, hDes68E, hCKM106E, and hDes68E.

[0293] Implementation Scheme 31: The nucleic acid of Implementation Scheme 30, wherein the enhancer region contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:45.

[0294] Implementation Scheme 32: Nucleic acid of Implementation Scheme 1, wherein the enhancer region contains sE and MEF2 motifs.

[0295] Implementation Scheme 33: The nucleic acid of Implementation Scheme 32, wherein the enhancer region contains three sE and one MEF2 motif.

[0296] Implementation Scheme 34: The nucleic acid of Implementation Scheme 33, wherein the enhancer region contains the following 5' to 3' operative linker fragments: sE, MEF2 motif, sE, and sE.

[0297] Implementation Scheme 35: The nucleic acid of Implementation Scheme 34, wherein the enhancer region contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:39.

[0298] Implementation Scheme 36: The nucleic acid of Implementation Scheme 1, wherein the enhancer region contains hCKM106ER or hDes68ER.

[0299] Implementation Scheme 37: The nucleic acid of Implementation Scheme 36, wherein the enhancer region contains three hCKM106ER and one hDes68ER.

[0300] Implementation Scheme 38: The nucleic acid of Implementation Scheme 37, wherein the enhancer region contains the following 5' to 3' operative linker fragments: hDes68ER, hCKM106ER, hCKM106ER and hCKM106ER.

[0301] Implementation Scheme 39: The nucleic acid of Implementation Scheme 38, wherein the enhancer region contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:40.

[0302] Implementation Scheme 40: The nucleic acid of Implementation Scheme 1, wherein the enhancer region contains at least two hCKM206E.

[0303] Implementation Scheme 41: The nucleic acid of Implementation Scheme 40, wherein the enhancer region has three hCKM206E.

[0304] Implementation Scheme 42: The nucleic acid of Implementation Scheme 41, wherein the enhancer region contains a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:34.

[0305] Implementation Scheme 43: A nucleic acid of any one of Implementation Schemes 1 to 42, wherein the core promoter is P87 or sP86; wherein P87 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1, and sP86 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:2.

[0306] Implementation Scheme 44: The nucleic acid of Implementation Scheme 43, in which the core promoter is P87.

[0307] Implementation Scheme 45: Nucleic acid of Implementation Scheme 43, wherein the core promoter is sP86.

[0308] Implementation Scheme 46: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:17.

[0309] Implementation Scheme 47: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:18.

[0310] Implementation Scheme 48: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:19.

[0311] Implementation Scheme 49: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:20.

[0312] Implementation Scheme 50: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:21.

[0313] Implementation Scheme 51: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22.

[0314] Implementation Scheme 52: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:23.

[0315] Implementation Scheme 53: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:24.

[0316] Implementation Scheme 54: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:25.

[0317] Implementation Scheme 55: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:26.

[0318] Implementation Scheme 56: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:27.

[0319] Implementation Scheme 57: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:28.

[0320] Implementation Scheme 58: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:29.

[0321] Implementation Scheme 59: The nucleic acid of Implementation Scheme 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:30.

[0322] Implementation Scheme 60: Nucleic acid of any one of Implementation Schemes 1 to 59, wherein the promoter is a skeletal muscle-specific promoter.

[0323] Implementation Scheme 61: The nucleic acid of any of Implementation Schemes 1 to 60, further comprising a transgene operatively linked to a promoter.

[0324] Implementation Scheme 62: The nucleic acid of Implementation Scheme 61, wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

[0325] Implementation Scheme 63: The nucleic acid of Implementation Scheme 62, wherein the therapeutic protein is selected from survival motor neuron (SMN), fukutin-related protein (FKRP), follistatin (FST), neurotrophin 3 (NT-3), dystrophin, tafazzin, myotubularin, merosin, α-1,4-glucosidase, calpain 3, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, poly(A)-binding protein nuclear 1 (PABPN1), and lysosome-associated membrane protein 2B. membrane protein 2 isorform B, LAMP2B).

[0326] Implementation Scheme 64: A vector comprising the nucleic acid of any one of Implementation Schemes 1 to 63.

[0327] Implementation Scheme 65: The vector of Implementation Scheme 64, wherein the vector is a DNA vector or an RNA vector.

[0328] Implementation Scheme 66: The vector of Implementation Scheme 64, wherein the vector is a plasmid, a recombinant retroviral vector, a recombinant lentiviral vector, a recombinant adenovirus vector, or a recombinant adeno-associated AAV vector.

[0329] Implementation Scheme 67: The carrier of Implementation Scheme 66, wherein the carrier is an AAV carrier.

[0330] Implementation Scheme 68: The vector of Implementation Scheme 67, wherein the AAV serotype is AAV1, AAV2, AAV7, AAV8, AAV9, AAV12, AAVrh74, AAVhu.32 or a mixture thereof.

[0331] Implementation Scheme 69: The vector of Implementation Scheme 67 or 68, wherein the viral genome of AAV contains 5' to 3': a first ITR, a promoter, a transgene, a poly A tail, and a second ITR.

[0332] Implementation Scheme 70: A pharmaceutical composition comprising a nucleic acid of any one of Implementation Schemes 1 to 62 or a vector of any one of Implementation Schemes 64 to 69, and a pharmaceutically acceptable vector; wherein the nucleic acid comprises a transgene operably linked to a promoter, and wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

[0333] Implementation Scheme 71: A method for enhancing the expression level of a transgene in muscle cells, comprising transfecting muscle cells with an effective amount of a nucleic acid of any one of Implementation Schemes 1 to 60 or a vector of any one of Implementation Schemes 64 to 69, wherein the nucleic acid contains a transgene operatively linked to a promoter.

[0334] Implementation Scheme 72: Use of any nucleic acid of Implementation Scheme 1 to 60 or any vector of Implementation Scheme 64 to 69 in enhancing the expression level of transgenes in muscle cells, wherein the nucleic acid comprises a transgene operatively linked to a promoter.

[0335] Implementation Scheme 73: A method for treating a subject with a muscle-related disease or condition, comprising administering to the subject a therapeutically effective amount of a nucleic acid of any one of Implementation Schemes 1 to 60, a vector of any one of Implementation Schemes 64 to 69, or a pharmaceutical composition of Implementation Scheme 70; wherein the nucleic acid comprises a transgene operatively linked to a promoter, and the transgene encodes a therapeutic protein for the muscle-related disease or condition.

[0336] Implementation Scheme 74: Use of any one of the nucleic acids of Implementation Schemes 1 to 60, any one of the vectors of Implementation Schemes 64 to 69, or the pharmaceutical composition of Implementation Scheme 70 in the treatment of muscle-related diseases or conditions; wherein the nucleic acid comprises a transgene operably linked to a promoter, and wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

[0337] Implementation Scheme 75: Use of the nucleic acid of any of Implementation Schemes 1 to 60, the carrier of any of Implementation Schemes 64 to 69, or the pharmaceutical composition of Implementation Scheme 70 for the preparation of a medicament for the treatment of muscle-related diseases or conditions.

[0338] Implementation Scheme 76: The method of use of any one of Implementation Schemes 73 to 75, wherein the muscle-related disease or condition is sarcopenia, muscular dystrophy (MD), congenital myopathy, distal myopathy, myotonic syndrome, ion channel disease, malignant hyperthermia, metabolic myopathy, or arthritis.

[0339] Implementation Plan 77: The method of Implementation Plan 76, wherein the muscle-related disease or condition is sarcopenia.

[0340] Implementation scheme 78: The method of implementation scheme 76, wherein the muscle-related disease or condition is MD, where MD is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy, myotonic dystrophy (Steiner's disease), oculopharyngeal muscular dystrophy (OMD) or limb-girdle muscular dystrophy (LGMD).

[0341] Implementation Plan 79: The method of Implementation Plan 78, wherein the muscle-related disease or condition is DMD or BMD.

[0342] Implementation Plan 80: A kit containing any one of the nucleic acids in Implementation Plans 1 to 60 or any one of the vectors in Implementation Plans 64 to 69.

[0343] Implementation scheme 81: The kit of implementation scheme 80, and also includes instructions for using the kit.

[0344] 6.7 Implementation Plan The examples provided below are for illustrative purposes only and are not restrictive unless otherwise stated. Therefore, the invention should not be construed in any way as limited to the examples below, but should be interpreted as covering any and all variations that may appear as taught herein.

[0345] 6.7.1 Implementation Plan 1: Screening for Humanized Skeletal Muscle-Specific Promoters method: Step 1. To construct expression vectors containing muscle-specific promoters. In order to prepare expression vectors with... Figure 1 The expression vector for the muscle-specific promoter shown was used to synthesize a polynucleotide containing the relevant promoter element, which was then digested and ligated into an ssAAV plasmid expressing luciferase. Sequencing confirmed the presence of the inserted promoter in the cloned plasmid.

[0346] Step 2. In vitro screening of skeletal muscle-specific promoters was conducted. The cloning plasmid prepared in step 1 was transfected into the mouse myoblast cell line C2C12. After transfection, the cells were cultured for 24 hours, and luciferase expression was collected and measured. The EGFP expression plasmid driven by the CB promoter was used as a negative control.

[0347] Step 3. Assess the role of skeletal muscle-specific promoters in vivo: 1) Package the cloning plasmid prepared in step 1 into ssAAV9 and quantify its titer.

[0348] 2) The viral vector from 1) was injected into the tail vein of 6-8 week old wild-type male BALB / c mice at a dose of 2E+13 vg / kg.

[0349] 3) In vivo imaging was performed at 2, 4 and 6 weeks post-injection (pi) to measure luciferase expression.

[0350] 4) Six weeks after injection, mice were sacrificed, and the following organs or tissues were collected: gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, quadriceps femoris, diaphragm, triceps surae, liver, heart, spleen, lung, kidney, stomach, intestine, brain, and testis. Total protein was extracted, and the expression level of luciferase in the isolated tissues and organs was measured.

[0351] result: 1. Design of humanized skeletal muscle-specific promoters Creatine kinase (MCK or CKM) mRNA is the second most abundant mRNA in skeletal muscle cells. The MCK promoter consists of a core promoter and an upstream enhancer. (See Table 1 and...) Figure 1 As shown, a variety of muscle-specific promoters with enhanced transcriptional activity and specificity were designed and constructed, some of which contain elements of the MCK promoter.

[0352] A 725 bp thCKM promoter was formed by combining the human MCK core promoter (87 bp, P87) and the human MCK enhancer hCKM206E (206 bp). A 426 bp thCKMs promoter was formed by combining the human MCK core promoter (87 bp) and a truncated human MCK enhancer (106 bp). A 497 bp thMD promoter was formed by combining the human MCK core promoter (87 bp), a truncated human desmin enhancer (68 bp), and a truncated human MCK enhancer (106 bp). A 723 bp tMCK promoter, consisting of the mouse MCK core promoter (87 bp) and a modified mouse MCK enhancer 2RS5 (206 bp), was used as a control.

[0353] Table 1: Structure of skeletal muscle-specific promoters 2RS5: Modified mouse MCK enhancer hCKM206E: Human MCK enhancer hCKM106E: Truncated human MCK enhancer hDes68E: Truncated human desmin enhancer Note: Information about the adapter sequence (e.g., restriction enzyme site) has been omitted.

[0354] 2. In vitro transcriptional activity of humanized skeletal muscle-specific promoters like Figure 2 As shown, transfection Figure 1 Following the application of the ssAAV plasmid, luciferase expression was detected in the mouse myoblast cell line C2C12, but not in the vector control group, demonstrating the skeletal muscle-specific transcriptional activity of these promoters. Furthermore, in C2C12 cells, both thCKM and thMD showed stronger transcriptional activity than tMCK, with thCKM exhibiting the strongest transcriptional activity. Figure 2 ).

[0355] 3. In vivo transcriptional activity of humanized skeletal muscle-specific promoters 1) For example Figure 3 As shown, two weeks or more after injection, in patients receiving tail vein injections from... Figure 1 Significant luciferase expression was detected in mice using the ssAAV9 vector driven by the four promoters shown, but not in mice receiving PBS control injections, demonstrating the in vivo transcriptional activity of these promoters. The thMD promoter exhibited the strongest transcriptional activity at weeks 2, 4, and 6 post-injection. Figure 3 ).

[0356] 2) For example Figure 4 As shown, compared with the control group, Figure 1 All four promoters described in the study activated luciferase expression in seven skeletal muscle tissues, including the gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, quadriceps femoris, triceps brachii, and diaphragm, demonstrating their transcriptional activity in these skeletal muscle lineage cells. Compared to the tMCK promoter, the thCKM promoter produced higher levels of luciferase in the soleus and diaphragm. Of the four promoters, the thMD promoter produced the highest levels of luciferase and the highest levels of total luciferase expression in all seven skeletal muscle tissues except the diaphragm. Figure 4 ).

[0357] 3) For example Figure 5 As shown, Figure 1 All four promoters shown activated luciferase expression in the heart, but at much lower levels compared to those detected in skeletal muscle cells. The thCKM promoter also activated relatively low levels of expression in the liver. Compared to the control group, the thMD promoter activated only minimal expression (if any) in the liver, spleen, lung, kidney, stomach, intestine, brain, and testis.

[0358] In summary, the overall relative expression levels of thCKM and thMD promoter activation were higher than those of the tMCK promoter in skeletal muscle cells. The thMD promoter showed minimal transcriptional activity in non-skeletal muscle tissues (such as liver, spleen, lung, kidney, stomach, intestine, brain, and testis), low activity in cardiomyocytes, and high activity in skeletal muscle tissues, demonstrating that thMD is an effective and specific skeletal muscle-specific expression promoter.

[0359] 6.7.2 Implementation Scheme 2: Optimizing the enhancer of skeletal muscle-specific promoters method: 1. Construct expression vectors containing muscle-specific promoters: Synthesis with Figure 6 The polynucleotide of the promoter element shown was digested and ligated into the ssAAV plasmid expressing luciferase. Sequencing confirmed the presence of the inserted promoter in the cloned plasmid.

[0360] 2. Measuring the in vitro activity of skeletal muscle-specific promoters: Plasmids cloned in the previous steps were transfected into HEK293T or C2C12 cells, and cell samples were collected 24 hours after transfection. Luciferase expression was detected.

[0361] 3. Evaluation of the in vivo effects of skeletal muscle-specific promoters: 1) Package the cloned plasmid from the previous step into ssAAV9 and quantify its titer.

[0362] 2) The viral vector from 1) was injected into the tail vein of 6-8 week old wild-type male BALB / c mice at a dose of 2E+13 vg / kg. The 10 rAAV vectors were divided into two groups for testing. The first group consisted of thMD, thMD2, hME2-sE, sE3, thMDR, and L34, while the second group consisted of L34, S24, S33, S32, and S22.

[0363] 3) In vivo imaging was performed at 2, 4 and 6 weeks post-injection (pi) to measure luciferase expression.

[0364] 4) Six weeks after injection, mice were sacrificed, and the following organs or tissues were collected: gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, quadriceps femoris, diaphragm, triceps surae, liver, heart, spleen, lung, kidney, stomach, intestine, brain, and testis. Total protein was extracted, and the expression level of luciferase in the isolated tissues and organs was measured.

[0365] result: 1. Optimization of reinforcing sub-components: Based on thMD, new promoters are designed by combining the core promoter with other alternative enhancer sequences (see Table 2).

[0366] Table 2: Structure of Enhancers hCKM106E: truncated human MCK enhancer; hDes68E: truncated human desmin enhancer; hDes78E: truncated human desmin enhancer; sE: synthetic enhancer; hDes68ER: reverse complementary sequence of hDes68E; hCKM106ER: reverse complementary sequence of hCKM106ER.

[0367] Note: Information about the adapter sequence (e.g., restriction enzyme site) has been omitted.

[0368] 2. Optimize the in vitro transcriptional activity of the promoter. Will Figure 6 The plasmid shown was transfected into non-muscle lineage HEK293T cells and mouse myoblast line C2C12, and luciferase expression was measured. Figure 7 As shown, compared with thMD, the promoters thMD2, hME2-sE, sE3, L34, S24, S33, and S32 induced lower levels of luciferase expression in HEK293T cells, indicating improved muscle specificity of these promoters. Meanwhile, in C2C12 cells, compared with thMD, the promoters sE3, thMDR, L34, S24, S33, S32, and S22 induced higher levels of luciferase expression, indicating improved transcriptional activity of these promoters in muscle cells.

[0369] 3. In vivo transcriptional activity of the promoter 1) such as Figure 8 As shown, compared with the control group, luciferase driven by ten different promoters was effectively expressed 2 weeks after tail vein injection, indicating that these promoters have transcriptional activity in mice. The hME2-sE and sE3 promoters showed higher transcriptional activity than the thMD promoter at weeks 2, 4, and 6, while the L34 promoter showed higher transcriptional activity than thMD at weeks 2 and 4.

[0370] 2) Figure 9The quantitative analysis of luciferase activity in different skeletal muscle tissues was presented. As shown in the figure, in the seven skeletal muscle tissues tested, the sE3 promoter expressed slightly higher levels of luciferase than the thMD promoter; in some tested muscle tissues (including gastrocnemius, tibialis anterior, extensor digitorum longus, triceps brachii, and diaphragm), the hME2-sE promoter expressed higher levels of luciferase than the thMD promoter; and in some tested muscle tissues (including the diaphragm, tibialis anterior, and soleus), the L34 promoter also expressed higher levels of luciferase than the thMD promoter. Overall, the hME2-sE, sE3, thMDR, and L34 promoters showed higher in vivo transcriptional activity in skeletal muscle tissues than the thMD promoter.

[0371] 3) Figure 10 The quantification of luciferase activity in different non-skeletal muscle tissues was shown. As shown in the figure, overall, the hME2-sE promoter and thMDR promoters exhibited similarly low transcriptional activity in non-skeletal muscle cells compared to the thMD promoter.

[0372] In summary, skeletal muscle-specific promoters were further improved after optimizing enhancer elements. For example, compared with thMD, the hME2-sE and thMDR promoters showed higher transcriptional activity and relatively higher specificity in skeletal muscle tissue.

[0373] 6.7.3 Implementation Scheme 3: Optimization of Core Promoter Elements of Skeletal Muscle-Specific Promoters method: 1. Construct expression vectors containing muscle-specific promoters: Synthesis with Figure 11 The polynucleotides of the promoter elements shown were digested and ligated to the ssAAV plasmid expressing luciferase. The cloned plasmid was confirmed by sequencing. The sP86 promoter is a synthetic sequence derived from the core promoter P87, comprising a 28 bp MyoD1 binding motif from the first human gene DES from the 5' to 3' ends, a 13 bp MyoD1 binding motif from the second human gene CKM, and a 45 bp fragment from 43 to 87 nt in the third P87 sequence.

[0374] 2. In vivo effects of skeletal muscle-specific promoters: 1) Package the cloned plasmid from the previous step into ssAAV9 and quantify its titer.

[0375] 2) The viral vector from 1) was injected into the tail vein of 6-8 week old wild-type male BALB / c mice at a dose of 2E+13 vg / kg.

[0376] 3) In vivo imaging was performed at 2, 4 and 6 weeks post-injection (pi) to measure luciferase expression.

[0377] 4) Six weeks after injection, mice were sacrificed, and the following organs or tissues were collected: gastrocnemius, soleus, tibialis anterior, extensor digitorum longus, quadriceps femoris, diaphragm, triceps surae, liver, heart, spleen, lung, kidney, stomach, intestine, brain, and testis. Total protein was extracted, and the expression of luciferase in the isolated tissues and organs was measured.

[0378] result: In vivo transcriptional activity: 1) such as Figure 12 As shown, compared with the control group, luciferase expression driven by the thMD, thMD-sP86, hME2-sE, and hME2-sE-sP86 promoters was detected from week 2. At weeks 2, 4, and 6 post-injection, luciferase expression driven by the thMD-sP86 promoter was higher than that driven by the thMD promoter at week 2, and comparable at weeks 4 and 6.

[0379] 2) Figure 13 The quantification of luciferase activity in different skeletal muscle tissues was shown. As shown in the figure, compared with the thMD promoter, the thMD-sP86 promoter expressed higher levels of luciferase in some tested tissues, such as the soleus and triceps muscles, and showed higher overall skeletal muscle luciferase expression.

[0380] 3) Figure 14 Quantification of luciferase activity in several non-skeletal muscle tissues was shown. As shown in the figure, the expression of the thMD-sP86 promoter in tissues such as liver, spleen, lung, kidney, stomach, intestine, brain, and testis was comparable to that of the thMD promoter. Overall, the luciferase level expressed by the thMD-sP86 promoter in non-skeletal muscle cells was slightly higher than that of the thMD promoter.

[0381] In summary, skeletal muscle-specific promoters have been further improved after optimizing the core promoter elements. For example, compared with thMD, the thMD-sP86 promoter exhibits higher transcriptional activity and relatively higher specificity in skeletal muscle tissue.

[0382] Although the invention has been described in detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. Unless the context otherwise requires, the various features described herein may be used in any combination. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0383] Therefore, the foregoing only illustrates the principles of the invention. It is understood that those skilled in the art can devise various arrangements, although these arrangements are not explicitly described or shown herein, but embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language herein are primarily intended to help the reader understand the principles of the invention and the concepts contributed by the inventors to the further development of the art, and should be interpreted as not being limited to these specifically referenced examples and conditions. Moreover, all statements herein regarding the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Furthermore, such equivalents include both currently known equivalents and future development equivalents; that is, any element developed with the same function, regardless of its structure, falls within the scope of the equivalent. Furthermore, whether expressly stated in the claims, nothing disclosed herein is intended to be made public.

[0384] All publications and patents referenced in this specification are incorporated herein by reference, as if each individual publication or patent were specifically and individually indicated to be incorporated herein by reference, and incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials relating to the cited publication. Reference to any publication is solely for the purpose of disclosing its content prior to the filing date and should not be construed as an admission that the present invention is not entitled to be considered prior to that publication by virtue of any prior invention.

Claims

1. A nucleic acid comprising a muscle-specific promoter, the muscle-specific promoter comprising an enhancer region operatively linked to a core promoter, wherein the enhancer region comprises at least two enhancer elements, wherein each enhancer element is independently selected from the group consisting of hCKM206E, hCKM106E, hCKM106ER, hDes68E, hDes68ER, hDes78E, sE, and MEF2 motifs, each enhancer element having a nucleotide sequence having at least 85% identity with SEQ ID NO:6-13.

2. The nucleic acid according to claim 1, wherein the enhancer region comprises two, three, four, five, six or seven enhancer elements.

3. The nucleic acid according to claim 1, wherein the enhancer region comprises two or three hCKM106E.

4. The nucleic acid according to claim 3, wherein the enhancer region further comprises at least one enhancer element selected from the group consisting of hDes68E, hDes78E and sE.

5. The nucleic acid according to claim 4, wherein the enhancer region comprises one, two, three or four hDes68E.

6. The nucleic acid according to claim 3, wherein the enhancer region has three hCKM106E.

7. The nucleic acid according to claim 6, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

35.

8. The nucleic acid according to claim 5, wherein the enhancer region comprises three hCKM106E and one hDes68E.

9. The nucleic acid of claim 8, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, hCKM106E and hDes68E.

10. The nucleic acid according to claim 9, wherein the enhancer region has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical nucleotide sequence to SEQ ID NO:

36.

11. The nucleic acid according to claim 5, wherein the enhancer region comprises three hCKM106E, one hDes68E, and one hDes78E.

12. The nucleic acid of claim 11, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, hCKM106E, hDes68E, and hDes78E.

13. The nucleic acid according to claim 12, wherein the enhancer region has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the same nucleotide sequence as SEQ ID NO:

37.

14. The nucleic acid according to claim 4, wherein the enhancer region comprises two hCKM106E and one sE.

15. The nucleic acid of claim 14, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, and sE.

16. The nucleic acid according to claim 15, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

38.

17. The nucleic acid according to claim 5, wherein the enhancer region comprises three hCKM106E and four hDes68E.

18. The nucleic acid of claim 17, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E and hDes68E.

19. The nucleic acid according to claim 18, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:

41.

20. The nucleic acid according to claim 5, wherein the enhancer region comprises two hCKM106E and four hDes68E.

21. The nucleic acid of claim 20, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hDes68E, hCKM106E, hDes68E, hDes68E, and hDes68E.

22. The nucleic acid according to claim 21, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

42.

23. The nucleic acid according to claim 5, wherein the enhancer region comprises three hCKM106E and three hDes68E.

24. The nucleic acid of claim 23, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E, hDes68E, and hDes68E.

25. The nucleic acid according to claim 24, wherein the enhancer region has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

43.

26. The nucleic acid according to claim 5, wherein the enhancer region comprises three hCKM106E and two hDes68E.

27. The nucleic acid of claim 26, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hCKM106E, hDes68E, hCKM106E, and hDes68E.

28. The nucleic acid of claim 27, wherein the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

44.

29. The nucleic acid according to claim 5, wherein the enhancer region comprises two hCKM106E and two hDes68E.

30. The nucleic acid of claim 29, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hCKM106E, hDes68E, hCKM106E, and hDes68E.

31. The nucleic acid of claim 30, wherein the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

45.

32. The nucleic acid according to claim 1, wherein the enhancer region comprises the sE and MEF2 motifs.

33. The nucleic acid of claim 32, wherein the enhancer region comprises three sEs and one MEF2 motif.

34. The nucleic acid of claim 33, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: sE, MEF2 motif, sE, and sE.

35. The nucleic acid of claim 34, wherein the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

39.

36. The nucleic acid according to claim 1, wherein the enhancer region comprises hCKM106ER or hDes68ER.

37. The nucleic acid of claim 36, wherein the enhancer region comprises three hCKM106ERs and one hDes68ER.

38. The nucleic acid of claim 37, wherein the enhancer region comprises the following 5' to 3' operable linker fragments: hDes68ER, hCKM106ER, hCKM106ER, and hCKM106ER.

39. The nucleic acid of claim 38, wherein the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:

40.

40. The nucleic acid according to claim 1, wherein the enhancer region comprises at least two hCKM206E.

41. The nucleic acid of claim 40, wherein the enhancer region has three hCKM206E.

42. The nucleic acid of claim 41, wherein the enhancer region comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:

34.

43. The nucleic acid according to any one of claims 1 to 42, wherein the core promoter is P87 or sP86; wherein P87 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1, and sP86 has a nucleotide sequence that is at least 85% identical to SEQ ID NO:

2.

44. The nucleic acid according to claim 43, wherein the core promoter is P87.

45. The nucleic acid according to claim 43, wherein the core promoter is sP86.

46. ​​The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

17.

47. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

18.

48. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

19.

49. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

20.

50. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

21.

51. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

22.

52. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

23.

53. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

24.

54. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

25.

55. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

26.

56. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

27.

57. The nucleic acid according to claim 1, wherein the muscle-specific promoter has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotide sequence that is identical to SEQ ID NO:

28.

58. The nucleic acid according to claim 1, wherein the muscle-specific promoter has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotide sequence that is identical to SEQ ID NO:

29.

59. The nucleic acid according to claim 1, wherein the muscle-specific promoter has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

30.

60. The nucleic acid of any one of claims 1 to 59, wherein the muscle-specific promoter is a skeletal muscle-specific promoter.

61. The nucleic acid according to any one of claims 1 to 60, further comprising a transgene operatively linked to a promoter.

62. The nucleic acid of claim 61, wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

63. The nucleic acid according to claim 62, wherein the therapeutic protein is selected from survival motor neuron (SMN), fukutin-related protein (FKRP), follistatin (FST), neurotrophin 3 (NT-3), dystrophin, tafazzin, myotubularin, merosin, α-1,4-glucosidase, calpain 3, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, poly(A)-binding protein nuclear 1 (PABPN1), and lysosome-associated membrane protein 2B. protein 2 isorform B, LAMP2B).

64. A vector comprising the nucleic acid of any one of claims 1 to 63.

65. The vector of claim 64, wherein the vector is a DNA vector or an RNA vector.

66. The vector of claim 64, wherein the vector is a plasmid, a recombinant retroviral vector, a recombinant lentiviral vector, a recombinant adenovirus vector, or a recombinant adeno-associated virus (AAV) vector.

67. The carrier of claim 66, wherein the carrier is an AAV carrier.

68. The vector of claim 67, wherein the serotype of the AAV vector is AAV1, AAV2, AAV7, AAV8, AAV9, AAV12, AAVrh74, AAVhu.32 or a mixture thereof.

69. The vector of claim 67 or 68, wherein the viral genome of the AAV vector comprises 5' to 3': a first ITR, a promoter, a transgene, a poly A tail, and a second ITR.

70. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 62 or a vector according to any one of claims 64 to 69, and a pharmaceutically acceptable vector; wherein the nucleic acid comprises a transgene operatively linked to a promoter, and wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

71. A method for enhancing transgene expression levels in muscle cells, comprising transfecting muscle cells with an effective amount of a nucleic acid according to any one of claims 1 to 60 or a vector according to any one of claims 64 to 69, wherein the nucleic acid comprises a transgene operatively linked to a promoter.

72. Using the nucleic acid of any one of claims 1 to 60 or the vector of any one of claims 64 to 69 to enhance the expression level of a transgene in muscle cells, wherein the nucleic acid comprises a transgene operatively linked to a promoter.

73. A method for treating a subject suffering from a muscle-related disease or condition, comprising administering to the subject a therapeutically effective amount of a nucleic acid of any one of claims 1 to 60, a vector of any one of claims 64 to 69, or a pharmaceutical composition of claim 70; wherein the nucleic acid comprises a transgene operatively linked to a promoter, and wherein the transgene encodes a therapeutic protein for the muscle-related disease or condition.

74. Use of the nucleic acid of any one of claims 1 to 60, the vector of any one of claims 64 to 69, or the pharmaceutical composition of claim 70 for the treatment of muscle-related diseases or conditions; wherein the nucleic acid comprises a transgene operatively linked to a promoter, and wherein the transgene encodes a therapeutic protein for muscle-related diseases or conditions.

75. Use of the nucleic acid of any one of claims 1 to 60, the carrier of any one of claims 64 to 69, or the pharmaceutical composition of claim 70 in the preparation of a medicament for treating muscle-related diseases or conditions.

76. The method of use according to any one of claims 73 to 75, wherein the muscle-related disease or condition is sarcopenia, muscular dystrophy (MD), congenital myopathy, distal myopathy, myotonic syndrome, ion channel disease, malignant hyperthermia, metabolic myopathy, or arthritis.

77. The method of claim 76, wherein the muscle-related disease or condition is sarcopenia.

78. The method of claim 76, wherein the muscle-related disease or condition is MD, wherein the MD is Duchenne MD (DMD), Becker MD (BMD), congenital muscular dystrophy, myotonic dystrophy (Steiner's disease), oculopharyngeal muscular dystrophy (OMD), or limb-girdle muscular dystrophy (LGMD).

79. The method of claim 78, wherein the muscle-related disease or condition is DMD or BMD.

80. A kit comprising the nucleic acid of any one of claims 1 to 60 or the vector of any one of claims 64 to 69.

81. The kit of claim 80, further comprising instructions for using the kit.

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