Regulatory nucleic acid sequences

Abstract

The present invention relates to regulatory nucleic acid sequences, particularly muscle-specific promoters, elements thereof, and other such nucleic acid sequences, that can enhance muscle-specific expression of genes. In particular, the present invention relates to regulatory nucleic acid sequences that can enhance muscle-specific expression but reduce liver-specific expression. The present invention also relates to expression constructs, vectors, and cells that contain such muscle-specific regulatory nucleic acid sequences, as well as methods for their use. Regulatory nucleic acid sequences are particularly useful for gene therapy applications, but are also useful in other fields, such as bioprocessing and biotechnology.

Description

[Technical Field]

[0001] The present invention relates to regulatory nucleic acid sequences, particularly muscle-specific promoters, elements thereof, and other such nucleic acid sequences, that can enhance muscle-specific expression of genes. In particular, the present invention relates to regulatory nucleic acid sequences that can enhance muscle-specific expression but result in low expression in other tissues, such as the liver. The present invention also relates to expression constructs, vectors, and cells that contain such regulatory nucleic acid sequences, as well as methods for their use. Regulatory nucleic acid sequences are particularly useful for gene therapy applications, but are also useful in other fields, such as bioprocessing and biotechnology. [Background technology]

[0002] The following discussion is provided to aid the reader in understanding the present disclosure and does not constitute any admission of content or relevance of prior art.

[0003] In many fields, including gene therapy, it is desirable to provide regulatory nucleic acid sequences that can drive the expression of a gene to produce a protein or nucleic acid expression product in a desired cell, tissue, or organ.

[0004] Expression of therapeutic genes in muscle is attractive for gene therapy. Gene therapy in muscle has the potential to modify or enhance the expression of various muscle proteins, such as dystrophin and sarcoglycans. This can be used to treat conditions such as muscular dystrophies, e.g., Duchenne muscular dystrophy (DMD). Muscle can also be used as a platform for expressing therapeutic proteins for the treatment of other conditions.

[0005] Various vectors have been used to deliver genes to muscle cells, including adenoviruses, retroviruses, lentiviruses, and adeno-associated viruses (AAVs), as well as non-viral vectors such as plasmids. Adenoviral vectors have a relatively large cloning capacity and can efficiently transduce some cells. However, they face significant challenges due to the strong immune responses they tend to induce. Retroviral and lentiviral vectors stably integrate into the genome, which entails both advantages and disadvantages. While lentiviral vectors can transduce both dividing and non-dividing cells, most conventional retroviral vectors can only transduce dividing cells, limiting their use in non-dividing muscle cells. Plasmid DNA can be used to introduce genes into muscle cells in vitro, but their potential usefulness in clinical settings is unclear.

[0006] AAV vectors are particularly attractive for gene therapy applications in muscle. AAV vectors exhibit natural tropism for muscle cells, can promote long-term expression of therapeutic payloads, and can induce minimal immune responses. Despite the ability of some gene therapy vectors to preferentially transduce muscle cells, off-target transduction also occurs. Several phase 1 and phase 2 clinical trials using AAV serotypes 1, 2, and chimeric 2.5 have been reported for the treatment of Duchenne muscular dystrophy (DMD) and alpha-1 antitrypsin deficiency. (DE Bowles, S. WJ McPhee, C. Li, SJ Gray, JJ Samulski, AS Camp, J. Li, B. Wang, PE Monahan, JE Rabinowitz, JC Grieger, La. Govindasamy, M. Agbandje-McKenna, X. Xiao and RJ Samulski, Molecular Therapy, 20, 443~455 (2012); ML Brantly, JD Chulay, L. Wang, C. Mueller, M. Humphries, LT Spencer, F. Rouhani, TJ Conlon, R. Calcedo, MR Berts, C. Spencer, BJ Byrne, JM Wilson, TR Flotte, Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAVl-AAT gene therapy. Proceedings of the National Academy of Sciences of the United States of America 106, 16363~16368 (2009); TR Flotte, ML Brantly, LT Spencer, BJ Byrne, CT Spencer, DJ Baker, M.Humphries, Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha1 -antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Human gene therapy 15, 93~128 (2004); T. R. Flotte, B. C. Trapnell, M. Humphries, B. Carey, R. Calcedo, F. Rouhani, M. Campbell-Thompson, A. T. Yachnis, R. A. Sandhaus, N. G. McElvaney, C. Mueller, L. M. Messina, J. M. Wilson, M. Brantly, D. R. Knop, G. J. Ye, J. D. Chulay, Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing alphal -antitrypsin: interim results. Human gene therapy 22, 1239~1247 (2011); C. Mueller, J. D. Chulay, B. C. Trapnell, M. Humphries, B. Carey, R. A. Sandhaus, N. G. McElvaney, L. Messina, Q. Tang, F. N. Rouhani, M. Campbell-Thompson, A. D. Fu, A. Yachnis, D. R. Knop, G. J. Ye, M. Brantly, R. Calcedo, S. Somanathan, L. P. Richman, R. H. Vonderheide, M. A. Hulme, T. M. Brusko, J. M. Wilson, T. R.Flotte, Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression. The Journal of clinical investigation 123, 5310~5318 (2013)). .

[0007] One of the off-target effects consistently observed in clinical trials of AAV therapeutic products is hepatotoxicity. Recently, Audentes Therapeutics' Phase 2 gene therapy trial for X-linked myotubular myopathy was temporarily suspended by the U.S. Food and Drug Administration due to a death caused by progressive liver dysfunction (Death from High-Dose AAV Gene Therapy. Nat Biotechnol 38, 910 (2020). https: / / doi.org / 10.1038 / s41587-020-0642-9). The dose administered to these patients in the Phase 2 clinical trial was the highest ever administered to AAV-based gene therapies (3 × 10 14 Similarly, two deaths due to acute liver failure have been reported after treatment with the spinal muscular atrophy gene therapy Zolgensma (Deaths from high-dose AAV gene therapy. Nat Biotechnol 38, 910 (2020). https: / / doi.org / 10.1038 / s41587-020-0642-9).

[0008] The use of cis-acting regulatory elements has been proposed to provide muscle specificity and activity. Typically, this involves a cis-regulatory enhancer sequence, i.e., a nucleic acid sequence that acts in cis to enhance the activity of a promoter. A variety of muscle-specific promoters are known in the art, typically derived from genes predominantly expressed in muscle, such as genes encoding desmin, skeletal actin, cardiac α-actin, muscle creatine kinase (CKM), myosin heavy and light chains, and troponin T / I. The C5-12 promoter represents a known synthetic muscle promoter.

[0009] There remains a need in the art for a system that regulates gene expression in a specific manner with minimal off-target effects. In particular, there is a need for muscle-specific regulatory sequences with minimal off-target effects. Such a system has the potential to minimize the off-target effects of gene therapy vectors. This is particularly important for gene therapy vectors that have a natural tropism for tissues other than the target tissue, such as AAV vectors that have a natural tropism for non-muscle cells and tissues, such as the liver.

[0010] Furthermore, different expression profiles in different muscle tissues may be desirable in different diseases. For example, a muscle-specific promoter with higher expression in skeletal muscle may be beneficial in treating myopathy, and a muscle-specific promoter with higher expression in cardiac muscle may be beneficial in treating cardiomyopathy. For some diseases, such as DMD, high expression across different muscle groups may be desirable.

[0011] Finally, regulatory sequences of short length are desirable to minimize the proportion of the gene therapy vector that is taken up by the regulatory sequences, which is particularly important for gene therapy vectors with limited capacity (payload), such as AAV vectors. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Application No. PCT / GB2022 / 051611 [Patent Document 2] International Application No. PCT / GB2020 / 053371 [Patent Document 3] U.S. Patent No. 6,200,560 [Patent Document 4] U.S. Patent No. 6,221,349 [Patent Document 5] Local users 4,683,195

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[0014] In a first aspect of the present invention, there is provided a synthetic muscle-specific cis-regulatory module (CRM) comprising CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element (e.g., a CRE or a detargeting element).

[0015] In some embodiments, the at least one additional regulatory element is selected from the following group: a CRE, a CRM, an inducible or repressible element, a boundary control element, an insulator, a locus control region, a response element, a binding site, a segment of a terminal repeat, a response site, a stabilizing element, a destabilizing element, a detargeting element, a liver detargeting element, an intron, a UTR, and a splicing element, etc., provided that they do not render the CRM non-functional.

[0016] In some embodiments, the at least one additional regulatory element is a CRE, an inducible or repressible element, or a detargeting element. In some embodiments, the at least one additional regulatory element is a CRE. In some embodiments, the at least one additional regulatory element is a detargeting element. In some embodiments, the at least one detargeting element is a liver detargeting element.

[0017] The additional regulatory elements may be the additional regulatory elements disclosed herein, or they may be other additional regulatory elements. In some embodiments, the additional regulatory elements may be selected from CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), liver detargeting sequence 1 (SEQ ID NO: 13), liver detargeting sequence 2 (SEQ ID NO: 14), liver detargeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA targeting sequence 1 (SEQ ID NO: 16), Mir122 miRNA targeting sequence 2 (SEQ ID NO: 19), tMCK SA / SD intron (SEQ ID NO: 29), and MVM cleavage intron (SEQ ID NO: 30). In some embodiments, the additional regulatory element may be selected from CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), liver detargeting sequence 1 (SEQ ID NO: 13), liver detargeting sequence 2 (SEQ ID NO: 14), liver detargeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA targeting sequence 1 (SEQ ID NO: 16), and Mir122 miRNA targeting sequence 2 (SEQ ID NO: 19).

[0018] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element are operably linked.

[0019] In some embodiments, the at least one additional regulatory element has a length of 20 nucleotides or more, preferably 30 nucleotides or more, more preferably 40 nucleotides or more, even more preferably 50 nucleotides or more, and most preferably 60 nucleotides or more.

[0020] In some embodiments, a CRM according to the first aspect of the invention retains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of a CRM consisting of SEQ ID NO: 8. The activity of a CRM consisting of SEQ ID NO: 8 is shown in International Application PCT / GB2022 / 051611, particularly on page 9, lines 1-11, and in Figures 5, 6, 17, and 18, which is incorporated herein by reference.

[0021] In some embodiments, a CRM according to the first aspect of the invention comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.

[0022] In some embodiments, the at least one additional regulatory element is located 5' relative to CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof. In some embodiments, the at least one additional regulatory element is located 3' relative to CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof. In some embodiments, the at least one additional regulatory element is located between CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof.

[0023] In some preferred embodiments, functional variants of CRE0145 and DES_MT_enhancer_48bp comprise sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0024] In some embodiments, the synthetic muscle-specific cis-regulatory module (CRM) comprises CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element, wherein the additional regulatory element is - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof is a liver detargeting element selected from the group consisting of:

[0025] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and an additional regulatory element are operably linked.

[0026] The regulatory elements may be those disclosed herein, or they may be other regulatory elements. In some embodiments, the regulatory elements or functional variants thereof referred to herein may be selected from CRE0145, DES_MT_enhancer_48bp, liver detargeting sequence 1, liver detargeting sequence 2, liver detargeting sequence 3, ZBTB20 binding site, Mir122 miRNA targeting sequence 1, and Mir122 miRNA targeting sequence 2.

[0027] In some embodiments, the synthetic muscle-specific CRM comprises: - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 1; - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 2; - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 3; - CRE0145, liver detargeting sequence 3, DES_MT_enhancer_48bp, and liver detargeting sequence 3; - CRE0145, DES_MT_enhancer_48bp, and ZBTB20 binding sites; and - CRE0145, ZBTB20 binding site, DES_MT_enhancer_48bp, and ZBTB20 binding site The present invention comprises a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0028] In some embodiments, the regulatory elements are present in the CRM in the order listed and are adjacent to each other. In some embodiments, the regulatory elements are operably linked.

[0029] In any of the combinations of regulatory elements or functional variants thereof disclosed herein, the listed regulatory elements can be present in any order, hi some preferred embodiments, the regulatory elements are present in the listed order (i.e., in upstream to downstream order with reference to their position relative to the operably linked promoter element or gene).

[0030] In any of the combinations of regulatory elements or functional variants thereof disclosed herein, some or all of the listed regulatory elements may be suitably located adjacent to one another in the CRM (i.e., without intervening regulatory elements). The regulatory elements may be contiguous or non-contiguous (i.e., they may be located directly adjacent to one another, or they may be separated by a spacer or other sequence). In some preferred embodiments, the regulatory elements or functional variants thereof are provided in the order listed and adjacent to one another. For example, a synthetic muscle-specific CRM may contain CRE0145 immediately upstream of DES_MT_enhancer_48bp, immediately upstream of liver detargeting sequence 1, etc. In some embodiments, it is preferred that some or all of the regulatory elements be contiguous.

[0031] In some embodiments of the invention, the synthetic muscle-specific CRM comprises a CRM selected from the group consisting of CRM_SP0525 (SEQ ID NO: 20), CRM_SP0526 (SEQ ID NO: 21), CRM_SP0527 (SEQ ID NO: 22), and CRM_SP0528 (SEQ ID NO: 9), or a functional variant of any thereof. Suitably, a functional variant of any of the CRMs comprises a sequence that is at least 70% identical to the reference synthetic muscle-specific CRM, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference synthetic muscle-specific CRM (SEQ ID NOs: 20, 21, 22, and 9).

[0032] In a second aspect of the present invention there is provided a synthetic muscle-specific promoter comprising a CRM according to the first aspect.

[0033] In some preferred embodiments, the CRM is operably linked to a promoter element, hi some preferred embodiments, the promoter element is SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0034] In some embodiments, the synthetic muscle-specific promoter comprises a synthetic muscle-specific CRM comprising CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, operably linked to promoter elements SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof, at least one additional regulatory element, and optionally a liver-detargeting element, wherein the liver-detargeting element is - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; and - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; is selected from the group consisting of:

[0035] In some embodiments, the synthetic muscle-specific cis-regulatory module (CRM) comprises CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof.

[0036] In some embodiments, a promoter element operably linked to a promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof, - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 1; - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 2; - CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 3; - CRE0145, DES_MT_enhancer_48bp, and ZBTB20 binding sites; CRE0145, liver detargeting sequence 3, DES_MT_enhancer_48bp, and liver detargeting sequence 3; and - CRE0145, ZBTB20 binding site, DES_MT_enhancer_48bp, and ZBTB20 binding site; A synthetic muscle-specific promoter is provided comprising a synthetic muscle-specific CRM comprising a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0037] In some embodiments, the regulatory elements are present in the CRM in the order listed and are adjacent to each other.

[0038] In any of the combinations of regulatory elements or functional variants thereof disclosed herein, the listed regulatory elements can be present in any order, hi some preferred embodiments, the regulatory elements are present in the listed order (i.e., in upstream to downstream order with reference to their position relative to the operably linked promoter element or gene).

[0039] In some embodiments, a synthetic muscle-specific promoter is provided that comprises or consists of a cis-regulatory module (CRM) consisting of or comprising CRM_SP0525 (SEQ ID NO:20), CRM_SP0526 (SEQ ID NO:21), CRM_SP0527 (SEQ ID NO:22), or CRM_SP0528 (SEQ ID NO:9), or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a CRM consisting of or comprising CRM_SP0525 (SEQ ID NO:20), CRM_SP0526 (SEQ ID NO:21), CRM_SP0527 (SEQ ID NO:22), or CRM_SP0528 (SEQ ID NO:9), or a functional variant thereof, operably linked to a promoter element. The promoter element can be a minimal or proximal promoter. The proximal promoter is preferably a muscle-specific proximal promoter. The promoter element can be a promoter element that is broadly active in various tissues and cell types. In some embodiments, the promoter element is SCP1 (SEQ ID NO:12). The promoter element can be a minimal promoter. In some embodiments, the promoter element is CRE0053 (SEQ ID NO: 26).

[0040] In some embodiments, a synthetic muscle-specific promoter is provided that comprises or consists of a sequence according to any one of SEQ ID NOs: 1-4, or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a promoter selected from the group consisting of SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), and SP0528 (SEQ ID NO: 4), or a functional variant of any thereof. Suitably, a functional variant of any of the above promoters comprises a sequence that is at least 70% identical to the reference synthetic muscle-specific promoter, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference synthetic muscle-specific promoter (SEQ ID NOs: 1-4).

[0041] In a third aspect, - CRE0145 or a functional variant thereof, - DES_MT_enhancer_48bp or a functional variant thereof, - SCP1 or a functional variant thereof or CRE0053 or a functional variant thereof; and - at least one additional regulatory element, for example a detargeting element, optionally a liver detargeting element; A synthetic muscle-specific promoter is provided, comprising or consisting of:

[0042] In some embodiments, the at least one additional regulatory element is selected from the following group: a CRE, a CRM, an inducible or repressible element, a boundary control element, an insulator, a locus control region, a response element, a binding site, a segment of terminal repeat, a response site, a stabilizing element, a destabilizing element, a detargeting element, a liver detargeting element, an intron, a UTR, and a splicing element, etc., provided that they do not render the synthetic muscle-specific promoter non-functional.

[0043] In some embodiments, the at least one additional regulatory element is selected from the following group: a CRE, a CRM, an inducible or repressible element, a detargeting element, a UTR (e.g., a 5' and / or 3' UTR), and an intron. In some embodiments, the at least one additional regulatory element is selected from the following group: a CRE, a CRM, an inducible or repressible element, a detargeting element, a UTR (e.g., a 5' and / or 3' UTR), and an intron, provided that they do not render the synthetic muscle-specific promoter non-functional.

[0044] The additional regulatory element may be a CRE or a detargeting element. In some embodiments, the detargeting element is a liver detargeting element. In some embodiments, the liver detargeting element is one of the following: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof is selected from the group consisting of:

[0045] In some embodiments, the synthetic muscle-specific promoter comprises SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), or a functional variant of any of them. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0046] In some embodiments, the at least one additional regulatory element may be selected from CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), liver detargeting sequence 1 (SEQ ID NO: 13), liver detargeting sequence 2 (SEQ ID NO: 14), liver detargeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA targeting sequence 1 (SEQ ID NO: 16), Mir122 miRNA targeting sequence 2 (SEQ ID NO: 19), tMCK SA / SD intron (SEQ ID NO: 29), MVM cleavage intron (SEQ ID NO: 30).

[0047] In some embodiments, the at least one additional regulatory element can be a UTR (e.g., 5' and / or 3' UTR) or an intron. In some embodiments, the at least one additional regulatory element can be an intron. In some embodiments, the intron is the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncation intron (SEQ ID NO: 30).

[0048] In some embodiments, the synthetic muscle-specific promoter comprises SP0530 (SEQ ID NO:27) or SP0531 (SEQ ID NO:28), or a functional variant of either thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of 27-28.

[0049] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, SCP1 or a functional variant thereof (or CRE0053 or a functional variant thereof), and at least one additional regulatory element are operably linked.

[0050] In some embodiments, the at least one additional regulatory element has a length of 20 nucleotides or more, preferably 30 nucleotides or more, more preferably 40 nucleotides or more, even more preferably 50 nucleotides or more, and most preferably 60 nucleotides or more.

[0051] In some embodiments, the synthetic muscle-specific promoter according to the third aspect of the present invention retains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the activity of the synthetic promoter consisting of SEQ ID NO: 7. The activity of the synthetic muscle-specific promoter consisting of SEQ ID NO: 7 is shown in International Application PCT / GB2022 / 051611, particularly on page 9, lines 7-11, and Figures 5, 6, and 18, which is incorporated herein by reference.

[0052] In some embodiments, the synthetic muscle-specific promoter according to the third aspect of the invention comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the synthetic muscle-specific promoter consisting of SEQ ID NO:7.

[0053] In some embodiments, the at least one additional regulatory element is located 5' relative to other regulatory elements. In some embodiments, the at least one additional regulatory element is located 3' relative to other regulatory elements. In some embodiments, the at least one additional regulatory element is located between other regulatory elements.

[0054] In some embodiments, a synthetic muscle-specific promoter is provided comprising CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, optionally operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof, wherein the additional regulatory element is a liver detargeting element, and at least one additional regulatory element: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof, or the additional regulatory element is an intron and is selected from the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncation intron (SEQ ID NO: 30).

[0055] In some embodiments, the synthetic muscle-specific promoter comprises SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0530 (SEQ ID NO: 27), or SP0531 (SEQ ID NO: 28), or a functional variant of any of them. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, 27-28.

[0056] In some embodiments, the muscle-specific promoter is operably linked to one or more additional regulatory elements. The additional regulatory elements can enhance expression, for example, compared to a muscle-specific promoter that is not operably linked to the additional regulatory elements. Generally, it is preferred that the additional regulatory elements do not substantially reduce the specificity of the muscle-specific promoter.

[0057] For example, a synthetic muscle-specific promoter according to the present invention can be operably linked to a sequence encoding a UTR (eg, 5' and / or 3' UTR), an intron, or the like.

[0058] In a fourth aspect of the present invention, there is provided a synthetic muscle-specific promoter SP0524 (SEQ ID NO:7) operably linked to an intron. In some embodiments, the intron is the tMCK SA / SD intron (SEQ ID NO:29) or the MVM truncated intron (SEQ ID NO:30). In some embodiments, the synthetic muscle-specific promoter comprises or consists of SP0530 (SEQ ID NO:27) or SP0531 (SEQ ID NO:28), or a functional variant of either thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:27-28.

[0059] In a fifth aspect of the present invention, a synthetic muscle-specific promoter is provided that comprises a liver detargeting element. In some embodiments, a synthetic muscle-specific promoter is provided that comprises a minimal ZBTB20 binding site, preferably comprising SEQ ID NO: 15 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic muscle-specific promoter that includes a liver-detargeting element reduces expression in the liver by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to a comparable synthetic muscle-specific promoter that does not include the liver-detargeting element. In some embodiments, the addition of a liver-detargeting element to a synthetic muscle-specific promoter does not affect its muscle-specific expression.In some preferred embodiments, the muscle-specific promoter consists of or comprises SEQ ID NOs: 1-137, 342-367, 424-453, and 478-509 of International Application PCT / GB2020 / 053371, which is incorporated herein by reference, or functional variants thereof. In some preferred embodiments, the muscle-specific promoter consists of or comprises SEQ ID NOs: 1-137, 342-367, 424-453, and 478-509 of International Application PCT / GB2020 / 053371, or functional variants thereof, and: a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15; a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14; or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 consisting of or comprising:

[0060] In some preferred embodiments, the muscle-specific promoter consists of or comprises SEQ ID NOs: 1-29, 66 of International Application No. PCT / GB2022 / 051611, which is incorporated herein by reference, or a functional variant thereof. In some preferred embodiments, the muscle-specific promoter consists of or comprises SEQ ID NOs: 1-29, 66 of International Application No. PCT / GB2022 / 051611, or a functional variant thereof, and: a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15; a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14; or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 consisting of or comprising:

[0061] In a sixth aspect of the present invention, a synthetic CNS-specific promoter comprising a liver detargeting element is provided. In some embodiments, a synthetic CNS-specific promoter comprising a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof is provided. In some embodiments, the synthetic CNS-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic CNS-specific promoter that includes a liver-detargeting element reduces expression in the liver by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to a comparable synthetic CNS-specific promoter that does not include the liver-detargeting element. In some embodiments, the addition of a liver-detargeting element to a synthetic CNS-specific promoter does not affect its CNS-specific expression.

[0062] In a seventh aspect of the present invention, a synthetic kidney-specific promoter comprising a liver detargeting element is provided. In some embodiments, a synthetic kidney-specific promoter comprising a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof is provided. In some embodiments, the synthetic kidney-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic kidney-specific promoter that includes a liver-detargeting element reduces expression in the liver by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to a comparable synthetic kidney-specific promoter that does not include the liver-detargeting element. In some embodiments, the addition of a liver-detargeting element to a synthetic kidney-specific promoter does not affect its kidney-specific expression.

[0063] In an eighth aspect of the present invention, a synthetic lung-specific promoter comprising a liver detargeting element is provided. In some embodiments, a synthetic lung-specific promoter comprising a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof is provided. In some embodiments, the synthetic lung-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic lung-specific promoter that includes a liver-detargeting element reduces expression in the liver by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a comparable synthetic lung-specific promoter that does not include the liver-detargeting element. In some embodiments, the addition of a liver-detargeting element to a synthetic lung-specific promoter does not affect its lung-specific expression.

[0064] In a ninth aspect of the present invention there is provided an expression cassette comprising a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention operably linked to a sequence encoding an expression product, preferably a gene, for example a transgene.

[0065] In some embodiments, the expression product is a therapeutic expression product. In some preferred embodiments, the therapeutic expression product is suitable for use in the treatment of a disease or condition associated with aberrant gene expression in muscle (i.e., a muscle disease), optionally in cardiac and / or skeletal muscle. In some preferred embodiments, therapeutic expression products include those useful in the treatment of muscle diseases.

[0066] The therapeutic expression product may be a therapeutic expression product useful in the treatment of any condition for which expression in muscle may be useful, for example, for the treatment of a muscular condition or for the treatment of a condition for which secretion of the therapeutic expression product from muscle may be desirable.

[0067] In a tenth aspect, there is provided an expression cassette comprising a synthetic muscle-specific promoter comprising CRE0145 or a functional variant thereof and DES_MT_enhancer_48bp or a functional variant thereof, wherein the synthetic promoter is operably linked to a sequence encoding an expression product and to a target sequence for miR122.

[0068] In some embodiments, the synthetic muscle-specific promoter further comprises SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0069] In some preferred embodiments, the expression cassette comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product and a target sequence for miR122.

[0070] In some preferred embodiments, the target sequence for miR122 comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 19, or a functional variant thereof. In some preferred embodiments, the target sequence for miR122 comprises or consists of a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 19.

[0071] In an eleventh aspect, there is provided a vector comprising a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention. In some embodiments, the vector is a gene therapy vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a gene therapy vector, preferably an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector. AAV vectors are of particular interest. The AAV vector may be selected from the group consisting of AAV2, AAV6, AAV8, AAV9, BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8, and AAV2G9, or derivatives thereof. AAV serotype 9 (AAV9) has been shown to achieve efficient transduction in cardiac and skeletal muscle; therefore, AAV9 and its derivatives represent one non-limiting example of a suitable AAV vector. In some embodiments, the rAAV vector is an AAV3b serotype, such as, but not limited to, an AAV3b265D virion, an AAV3b265D549A virion, an AAV3b549A virion, an AAV3bQ263Y virion, or an AAV3bSASTG virion (i.e., a virion comprising an AAV3b capsid containing the Q263A / T265 mutation). In some embodiments, the virion can be a rational haploid, or chimeric, or any mutant, such as a capsid tailored to increase uptake in a desired location, such as the heart. Other capsids can include capsids derived from any of the known AAV serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV7, and AAV10. In some preferred embodiments, the AAV vector is AAV2i8.

[0072] A vector according to the present invention may be an AAV vector comprising a nucleic acid encoding a therapeutic expression product for the treatment of heart failure, wherein the nucleic acid is operably linked to a muscle-specific promoter.

[0073] In a twelfth aspect of the present invention, a gene therapy vector is provided comprising a synthetic promoter comprising SEQ ID NO: 15 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 14 or a functional variant thereof. In some embodiments, a gene therapy vector comprises a synthetic promoter comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, a gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 17 or a functional variant thereof. In some embodiments, a gene therapy vector comprises a synthetic promoter comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

[0074] In some preferred embodiments, the gene therapy vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some preferred embodiments, the vector is an AAV vector. Suitable AAV vectors are described above.

[0075] In a thirteenth aspect, there is provided a gene therapy AAV vector comprising an expression cassette, the expression cassette comprising a synthetic promoter operably linked to a sequence encoding an expression product and a target sequence for miR122. In some embodiments, the synthetic promoter is a muscle-specific promoter. In some embodiments, the synthetic promoter is a muscle-specific promoter according to any of the first to fifth aspects of the invention.

[0076] In some embodiments, the gene therapy AAV vector is AAV2. AAV2 has been shown to provide efficient transduction in the liver and muscle. In some embodiments, the gene therapy AAV vector is AAV9. AAV9 has been shown to provide efficient transduction in muscle and the CNS and is currently used in clinical settings. In some embodiments, the gene therapy AAV vector is AAV5. AAV5 has been shown to provide efficient transduction in the lung. In some embodiments, the gene therapy AAV vector is AAV8. AAV8 has been shown to provide efficient transduction in muscle and is currently used in clinical settings.

[0077] In some embodiments, the targeting sequence for miR122 is expected to reduce hepatic expression of the gene therapy AAV vector. In some embodiments, the targeting sequence for miR122 comprises or consists of Mir122 miRNA target sequence 1. In some embodiments, the targeting sequence for miR122 comprises or consists of SEQ ID NO: 16 or a functional variant thereof. In some embodiments, the targeting sequence for miR122 comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the targeting sequence for miR122 comprises or consists of Mir122 miRNA target sequence 2. In some embodiments, the targeting sequence for miR122 comprises or consists of SEQ ID NO: 19. In some embodiments, the target sequence for miR122 comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19.

[0078] In some embodiments, a gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest) and Mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, a gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest) and Mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and adjacent to each other.

[0079] In some embodiments, a gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, an additional expression product (e.g., a second gene of interest), and mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, a gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, an additional expression product (e.g., a second gene of interest), and mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0080] In some embodiments, a gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest) and mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, a gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest) and mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0081] In some embodiments, a gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, an additional expression product (e.g., a second gene of interest), and mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, a gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, an additional expression product (e.g., a second gene of interest), and mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0082] In a fourteenth aspect, there is provided a virion (virus particle) comprising a vector according to the invention, preferably a viral vector. In some embodiments, the virion is an AAV virion. Suitable virions are described above.

[0083] In a fifteenth aspect, there is provided a pharmaceutical composition comprising a synthetic muscle-specific promoter, expression cassette, vector, or virion according to the invention.

[0084] In a sixteenth aspect, there is provided a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to the invention for use in therapy, i.e., the prevention or treatment of a medical condition or disease. In some embodiments, the synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to the invention is for use in therapy of a subject in need thereof. Suitably, the condition or disease is associated with aberrant gene expression, optionally in muscle cells (myocytes) or muscle tissue. Suitably, the condition or disease is associated with aberrant gene expression in cardiomyocytes or cardiac tissue. Suitably, the condition or disease is associated with aberrant gene expression in skeletal muscle or skeletal tissue. Suitably, there is provided a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to the invention for use in expressing a therapeutic expression product in skeletal muscle and / or cardiac muscle.

[0085] In one embodiment, the disease can be a cardiovascular disease or heart disease or disorder. In one embodiment, the disease can be heart failure, such as congestive heart failure. In one embodiment, the disease can be ischemia, arrhythmia, myocardial infarction (MI), abnormal cardiac contractility, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormal Ca. 2+ In some embodiments, the disease may be selected from the group consisting of congestive heart failure, myocardial injury, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, dysfunctional conduction system, dysfunctional coronary artery, and pulmonary heart hypertension. In some embodiments, the disease may be selected from congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, myocardial injury, idiopathic cardiomyopathy, cardiac arrhythmia, muscular dystrophy, muscle mass abnormalities, muscle degeneration, infectious myocarditis, drug- or toxin-induced myopathy, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease.

[0086] Preferably, the use is for gene therapy, preferably for use in the treatment of diseases involving aberrant gene expression. Preferably, the gene therapy involves expression of a therapeutic expression product in muscle cells or muscle tissue, preferably in cardiomyocytes or cardiac tissue, preferably in skeletal muscle cells or skeletal muscle tissue, or preferably in heart and bone cells and tissue.

[0087] Preferably, the subject in need of therapy will exhibit symptoms characteristic of a cardiovascular disease, such as the heart disease or heart failure described above, or a skeletal muscle condition. Medical applications typically involve ameliorating the symptoms exhibited by the subject in need thereof by expressing a therapeutic amount of a therapeutic product. In some embodiments, the expression cassette comprises a gene encoding a protein phosphate 1 (PP1) inhibitor operably linked to a cardiac-specific or cardiac-selective promoter. The therapy preferably comprises expressing a therapeutic amount of the PP1 inhibitor in cardiac tissue of the subject. Preferably, expression of a therapeutic amount of the PP1 inhibitor in cardiac tissue alleviates the subject's symptoms of heart failure or heart disease. Preferably, expression of a therapeutic amount of the PP1 inhibitor in cardiac tissue may attenuate cardiac remodeling, improve exercise capacity, or improve cardiac contractility. Preferably, expression of a therapeutic amount of the PP1 inhibitor in cardiac tissue may result in myocyte shortening, a decrease in the time constant for relaxation, and an accelerated decay of the calcium signal, an improvement in the end-systolic pressure dimension relationship, and combinations thereof.

[0088] In a seventeenth aspect, there is provided a cell comprising a synthetic muscle-specific promoter, expression cassette, vector, or virion of the invention. In some embodiments, the cell is a eukaryotic cell, optionally a mammalian cell, optionally a human cell. Suitably, the cell may be a muscle cell, optionally the cell is a human muscle cell. Suitably, the cell may be a human skeletal muscle cell or a human cardiac muscle cell. The synthetic muscle-specific promoter, expression cassette, vector, or virion of the invention may be episomal or may be present in the genome of the cell.

[0089] In an eighteenth aspect, there is provided a synthetic muscle-specific CRM, synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition as described herein for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

[0090] In a nineteenth aspect, there is provided a method of producing an expression product, the method comprising providing a synthetic muscle-specific expression cassette of the invention in a muscle cell and expressing an expression product, preferably a gene, present in the synthetic muscle-specific expression cassette. The method can be performed in vitro or ex vivo, or can be performed in vivo. In some embodiments, the method is a bioprocessing method. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell.

[0091] In a twentieth aspect, there is provided a method of expressing an expression product in a muscle cell, the method comprising introducing into the muscle cell a synthetic muscle-specific expression cassette, vector, or virion described herein. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. The method can be performed in vitro or ex vivo, or can be performed in vivo.

[0092] In a twenty-first aspect, there is provided a method of treating a subject, preferably a human, in need thereof, comprising: - administering to a subject an expression cassette, vector, virion, or pharmaceutical composition described herein comprising a sequence encoding a therapeutic product operably linked to a synthetic muscle-specific promoter according to the invention; and - expressing a therapeutic amount of a therapeutic product in the muscle of said subject The method may be performed ex vivo or in vivo. The subject may be a mammal other than a human.

[0093] In one embodiment, the muscle cells are cardiac muscle cells. In one embodiment, the muscle cells are skeletal muscle cells. Suitably, the method of treating a subject comprises expression of a therapeutic amount of a therapeutic product in cardiac muscle and / or skeletal muscle.

[0094] In some embodiments, the method comprises: - introducing into the muscle of a subject an expression cassette, vector, virion, or pharmaceutical composition described herein, comprising a gene encoding an expression product, preferably a therapeutic product; and - expressing a therapeutic amount of a therapeutic product in the muscle of said subject Includes.

[0095] In one embodiment, the muscle cells are cardiomyocytes. In one embodiment, the muscle cells are skeletal muscle cells. Suitably, the method comprises expression of a therapeutic amount of a therapeutic product in the cardiac muscle and / or skeletal muscle of said subject.

[0096] Suitably, the method comprises administering to a subject a vector, virion, or pharmaceutical composition described herein. In some preferred embodiments, the vector is a viral gene therapy vector, preferably an AAV vector.

[0097] In one embodiment, any of the aspects herein relating to muscle-specific promoters may also relate to CNS-, kidney-, or lung-specific promoters as defined above.

[0098] Further features and embodiments of the invention will be described below in the following sections. Any feature or embodiment of any section may be combined with any other feature or embodiment, or with any aspect of the invention, in any workable combination. [Brief explanation of the drawings]

[0099] [Figure 1]Figure 1 shows the in vivo activity of the control promoter CK8 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. CK8 has high activity in heart, gastrocnemius, and tibialis anterior, and activity in diaphragm and soleus is also shown. CK8 has lower activity in liver (about 13-fold lower in liver than in the lowest-expressing muscle tissue (diaphragm); about 570-fold lower in liver than in the highest-expressing muscle tissue (heart)). [Figure 2] Figure 1 shows the in vivo activity of the control promoter CK7 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as in non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of protein extracted. CK7 has high activity in gastrocnemius and tibialis anterior, and also shows activity in diaphragm, heart, soleus, and liver (liver is approximately 2-fold lower than the lowest-expressing muscle tissue (diaphragm); liver is approximately 100-fold lower than the highest-expressing muscle tissue (gastrocnemius)). [Figure 3] This figure shows the in vivo activity of the synthetic promoter SP0525 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as in non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0525 has high activity in the heart and also shows activity in skeletal muscle (gastrocnemius, soleus, tibialis anterior, diaphragm). SP0525 has lower activity in the liver (about 2-fold lower in the liver than in the lowest-expressing muscle tissue (diaphragm) and about 100-fold lower in the liver than in the highest-expressing muscle tissue (heart)). [Figure 4]Figure 1 shows the in vivo activity of the synthetic promoter SP0526 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as in non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0526 has high activity in heart and gastrocnemius, and also shows activity in diaphragm and tibialis anterior. SP0526 exhibits lower activity in soleus. SP0526 exhibits lower activity in liver (about 4-fold lower in liver than in the lowest-expressing muscle tissue (soleus); about 980-fold lower in liver than in the highest-expressing muscle tissue (heart)). [Figure 5] This figure shows the in vivo activity of the synthetic promoter SP0527 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0527 has high activity in heart and gastrocnemius, and also shows activity in diaphragm and tibialis anterior. SP0527 shows lower activity in soleus. SP0527 also shows lower activity in liver (about 4-fold lower in liver than the lowest-expressing muscle tissue (soleus); about 570-fold lower in liver than the highest-expressing muscle tissue (gastrocnemius)). [Figure 6] Figure 1 shows the in vivo activity of the synthetic promoter SP0528 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0528 has high activity in heart, tibialis anterior, and gastrocnemius, and also shows activity in the diaphragm. SP0528 exhibits lower activity in soleus. SP0528 exhibits lower activity in liver (about 2-fold lower in liver than the lowest-expressing muscle tissue (soleus); about 175-fold lower in liver than the highest-expressing muscle tissue (heart)). [Figure 7]Figure 1 shows the in vivo activity of synthetic expression cassette 529 (SP0524 + expression product + mir122 target sequence) in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as in non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. Expression cassette 529 exhibits high activity in cardiac muscle, as well as activity in skeletal muscle (diaphragm, gastrocnemius, soleus, and tibialis anterior). Expression cassette 529 exhibits lower activity in liver (about 13-fold lower in liver than in the lowest-expressing muscle tissue (soleus); about 7520-fold lower in liver than in the highest-expressing muscle tissue (heart)). [Figure 8] Figure 1 shows the in vivo activity of the synthetic promoter SP0530 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0530 exhibits high activity in cardiac muscle, as well as activity in skeletal muscle (diaphragm, gastrocnemius, soleus, and tibialis anterior) and liver (about 2-fold lower activity in liver than the least active muscle tissue (soleus); about 1430-fold lower activity in liver than the most active muscle tissue (heart)). [Figure 9] Figure 1 shows the in vivo activity of the synthetic promoter SP0531 in various muscle tissues, such as diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as in non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of protein extracted. SP0531 exhibits high activity in the diaphragm, as well as activity in the heart, gastrocnemius, soleus, tibialis anterior, and liver (about 137-fold lower activity in the liver compared to the most active muscle tissue, gastrocnemius). [Figure 10]Figure 1 shows saline control (background) in various muscle tissues, e.g., diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars represent standard deviation. Y-axis is luciferase activity (relative light units) per mg of protein extracted. [Figure 11] Figure 1 shows the vector copy number per diploid genome for tested synthetic promoters and control promoters in the heart. The y-axis is the vector copy number per diploid genome. Error bars are the standard deviation. [Figure 12] Figure 1 shows the vector copy number per diploid gene for tested synthetic promoters and control promoters in the liver. The y-axis is the vector copy number per diploid genome. Error bars are the standard deviation. [Figure 13] This figure shows lifetime imaging using the IVIS Spectrum in vivo imaging system to detect bioluminescence. Luminescence is represented by color on the image, with blue representing areas of low luminescence and red representing areas of high luminescence on a scale of 0.2-2.1 p / sec / cm / sr (photon emission or radiance from the subject). Luminescence detected in the lower half of the mouse primarily corresponds to skeletal muscle (including the soleus, tibialis anterior, and gastrocnemius muscles) and occasionally to the intestine. Luminescence detected in the upper half of the mouse corresponds to various tissues, such as the liver and heart. [Figure 14] Figure 1 shows the in vivo activity of the control promoter CK7 (low dose) in various muscle tissues, such as the heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus muscles, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. CK7 shows high activity in the quadriceps, gastrocnemius, tibialis anterior, and psoas muscles, as well as activity in the diaphragm, soleus, and heart. CK7 shows lower activity in the liver (approximately 5-fold lower than the lowest-expressing muscle tissue). [Figure 15]Figure 1 shows the in vivo activity of the control promoter CK7 (medium dose) in various muscle tissues, such as the heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus muscles, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. CK7 shows high activity in the quadriceps, gastrocnemius, tibialis anterior, and psoas muscles, as well as activity in the diaphragm, soleus, and heart. CK7 shows lower activity in the liver (approximately 80-fold lower than the lowest-expressing muscle tissue). [Figure 16] Figure 1 shows the in vivo activity of the synthetic promoter SP0527 (low dose) in various muscle tissues, such as heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of protein extracted. SP0527 exhibits high activity in the heart, as well as activity in the diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus muscles. SP0527 exhibits lower activity in the liver (approximately 10-fold lower than the lowest-expressing muscle tissue). [Figure 17] Figure 1 shows the in vivo activity of the synthetic promoter SP0527 (medium dose) in various muscle tissues, such as heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0527 exhibits high activity in the heart as well as diaphragm, quadriceps, gastrocnemius, and tibialis anterior. SP0527 also exhibits activity in psoas and soleus. SP0527 exhibits lower activity in the liver (approximately 40-fold lower than the lowest-expressing muscle tissue). [Figure 18]Figure 1 shows the in vivo activity of the synthetic promoter SP0527 (high dose) in various muscle tissues, such as heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0527 exhibits high activity in heart, as well as diaphragm, quadriceps, gastrocnemius, and tibialis anterior. SP0527 also exhibits activity in psoas and soleus. SP0527 exhibits even lower activity in liver (approximately 950-fold lower than the lowest-expressing muscle tissue). [Figure 19] Figure 1 shows saline control (background) in various muscle tissues, e.g., heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissue (liver). Error bars represent standard deviation. The Y-axis is luciferase activity (relative light units) per mg of protein extracted. [Figure 20] Figure 1 shows the vector copy number per diploid genome in non-muscle tissue (liver) for control promoter CK7 (low and medium doses), SP0527 (low, medium, and high doses), and saline background. The y-axis is the vector copy number per diploid genome. Error bars are standard deviation. [Figure 21]This figure shows the in vivo activity of the synthetic promoter SP0527 (high dose) in various muscle tissues, such as the heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissues, such as the liver, brain, lung, colon, spleen, testis, and kidney. Error bars represent standard deviation. The Y-axis represents luciferase activity (relative light units) per mg of extracted protein. SP0527 exhibits high activity in the heart, as well as the diaphragm, quadriceps, gastrocnemius, and tibialis anterior muscles. SP0527 also exhibits activity in the psoas and soleus muscles. SP0527 exhibits low activity in the brain, lung, and kidney (expression in the brain, lung, and kidney is approximately 30-165 fold lower than that in muscle tissue, which has the lowest expression). SP0527 shows even lower activity in liver, colon, and testis (expression in liver, colon, and testis is approximately 400-960 fold lower compared to muscle tissue, which has the lowest expression). SP0527 shows the lowest expression in spleen (expression in spleen is approximately 2100 fold lower compared to muscle tissue, which has the lowest expression). [Figure 22] Figure 1 shows saline control (background) in various muscle tissues, such as heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, and non-muscle tissues, such as liver, brain, lung, colon, spleen, testis, and kidney. Error bars represent standard deviation. The Y-axis is luciferase activity (relative light units) per mg of protein extracted. DETAILED DESCRIPTION OF THE INVENTION

[0100] Muscle-specific muscle type selectivity and muscle specificity The CREs, CRMs, promoter elements, and synthetic promoters of the present invention may be active in various muscle tissues, but may be particularly, but not exclusively, active in skeletal muscle and / or cardiac muscle. CREs, CRMs, promoter elements, and synthetic promoters of the present invention that are active in at least one muscle tissue type or at least one muscle cell type may be referred to as "muscle-specific." For ease of use, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention may be further classified into subtypes depending on whether they are primarily active in skeletal muscle or cardiac muscle.

[0101] In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are primarily active in skeletal muscle and less active or not active in cardiac muscle. These CREs, CRMs, promoter elements, and synthetic promoters of the present invention are referred to as "skeletal muscle-specific" or "skeleton-selective." In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are skeletal muscle-specific or skeletal-selective.

[0102] In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are primarily active in cardiac muscle and less active or not active in skeletal muscle. These CREs, CRMs, promoter elements, and synthetic promoters of the present invention are referred to as "cardiac muscle-specific" or "cardiac selective." In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are cardiac muscle-specific or cardiac selective. Examples of muscle-specific promoters that are primarily active in cardiac muscle include SP0525 (SEQ ID NO: 1) and SP0530 (SEQ ID NO: 27). An example of a muscle-specific expression cassette that is primarily active in cardiac muscle includes expression cassette 529.

[0103] In some embodiments, muscle-specific CREs, CRMs, promoter elements, and synthetic promoters that are active in both skeletal and cardiac muscle are preferred. If promoter activity is required in both skeletal muscle and the heart (in cardiac muscle), these CREs, CRMs, promoter elements, and synthetic promoters may be preferred. Examples of muscle-specific promoters that are active in both skeletal and cardiac muscle include SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0531 (SEQ ID NO: 28), and SP0524 (SEQ ID NO: 7).

[0104] In some embodiments, skeletal muscle-specific or skeletal muscle-selective CREs, CRMs, promoter elements, and synthetic promoters are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is required in skeletal muscle with little or no activity in the heart (myocardium).

[0105] Skeletal muscle-specific or skeletal-selective promoters may be active in fast and / or slow muscles. In some embodiments, skeletal muscle-specific or skeletal-selective CREs, CRMs, promoter elements, and synthetic promoters that are active in fast muscles may be preferred. In some embodiments, skeletal muscle-specific or skeletal-selective CREs, CRMs, promoter elements, and synthetic promoters that are active in slow muscles may be preferred. In some embodiments, skeletal muscle-specific or skeletal-selective CREs, CRMs, promoter elements, and synthetic promoters that are active in both slow and fast muscles may be preferred.

[0106] In some embodiments, cardiac-specific or cardiac-selective CREs, CRMs, promoter elements, and synthetic promoters are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is required in the heart (myocardium) with little or no activity in skeletal muscle.

[0107] The myocardium-specific or cardiac-selective CRE, CRM, promoter element, and synthetic promoter of the present invention may be active in various cells of the heart. The main cell types in the heart are ventricular cardiomyocytes, atrial cardiomyocytes, cardiac fibroblasts, or cardiac endothelial cells (EC), as well as perivascular cells and pacemaker cells. Furthermore, the myocardium-specific or cardiac-selective CRE, CRM, promoter element, and synthetic promoter of the present invention may be active in various regions of the heart, such as any or all of the following cardiac regions: aortic arch (AA); aorta; cardiomyocytes (CM); endothelial or endocardial cells (EC); inferior vena cava (ICV); interventricular septum (IVS); left atrium (LA); left superior vena cava (LSCV); left ventricle (LV); outflow tract (OT); pulmonary artery (PO); epicardial precursor tissue (PEO); pulmonary veins (PV); right atrium (RA); right superior vena cava (RSCV); right ventricle (RV); superior vena cava (SCV); and cardiac smooth muscle cells (SM).

[0108] In some embodiments, skeletal muscle and cardiac muscle-specific or cardiac-selective CREs, CRMs, promoter elements, and synthetic promoters are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is required in skeletal muscle and the heart (cardiac muscle). This may be particularly beneficial in diseases such as DMD.

[0109] Cis-regulatory elements and their functional variants Disclosed herein are various CREs that can be used in constructing muscle-specific promoters. These CREs are generally derived from genomic promoter and enhancer sequences, but they are used herein in a context quite different from their natural genomic environment. Generally, CREs constitute a small portion of a much larger genomic regulatory domain, which normally controls the expression of associated genes. Surprisingly, many of these CREs are very small and can be isolated from their normal environment and retain muscle-specific regulatory activity when used to construct various synthetic promoters. This is surprising because removal of regulatory sequences from the complex and "three-dimensional" natural context in the genome often results in a significant reduction in activity, and therefore a given CRE cannot be expected to maintain the observed level of activity after being removed from its natural environment. Combinations of these CREs have been tested and found to be highly effective in enhancing the activity of muscle-specific promoters when combined with minimal and proximal promoters. It should be noted that the sequences of the CREs of the present invention can be altered without resulting in a substantial reduction in activity. Functional variants of CREs can be prepared by modifying the sequence of the CRE, provided that modifications that are significantly detrimental to the activity of the CRE are avoided. Given the information provided in this disclosure, modifying a CRE to provide a functional variant is straightforward. Furthermore, this disclosure provides a methodology for easily evaluating the functionality of any given CRE variant. Functional variants for each CRE are described below.

[0110] The relatively small size of certain CREs according to the present invention is advantageous because it allows CREs, and more particularly promoters containing them, to be provided in vectors while minimizing the vector payload, which is particularly important when CREs are used in vectors with limited CRE capacity, such as AAV-based vectors.

[0111] The CREs disclosed herein contain certain muscle-specific transcription factor binding sites (TFBSs). It is generally desirable for these muscle-specific TFBSs to maintain functionality in functional variants of CREs. Those skilled in the art are well aware that TFBS sequences can be altered while maintaining functionality. Taking this into consideration, sequences for TFBSs are typically represented by consensus sequences, which usually have some degree of variation. Further information about the variations that occur in TFBSs can be displayed using a positional weight matrix (PWM), which represents the frequency with which a given nucleotide is typically found at a given position in the consensus sequence. Details of TF consensus sequences and associated positional weight matrices can be found, for example, in the Jaspar or Transfac databases (http: / / jaspar.genereg.net / and http: / / gene-regulation.com / pub / databases.html). This information allows those skilled in the art to modify the sequence of any given TFBS of a CRE in a manner that maintains and, in some cases, increases the functionality of the CRE. With this in mind, those skilled in the art have ample guidance on how to vary the TFBS for any given transcription factor (TF) while maintaining the ability to bind to the desired TF. The Jaspar system, for example, will score putative TFBSs based on their similarity to a given PWM. Furthermore, all TFBSs can be identified / analyzed by scanning the CRE against all PWMs from the JASPAR database. Those skilled in the art can, of course, find additional guidance in the literature and can further use routine experimentation to confirm the TFs that bind to the putative TFBS in any variant CRE. It will be apparent that significant sequence modifications can be made in CREs while maintaining function, even within the TFBS in the CRE.

[0112] A functional variant of a CRE can contain substitutions, deletions, and / or insertions compared to a reference CRE, provided that they do not render the CRE substantially non-functional.

[0113] CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) are muscle-specific cis-regulatory elements (CREs) that have been shown to provide significant muscle-specific enhancer activity when combined with suitable promoter elements and / or added to suitable synthetic promoters.

[0114] Promoter elements and their functional variants The CRMs of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters, collectively referred to as promoter elements.

[0115] A functional variant of a promoter element comprises a sequence that differs from the reference promoter element but substantially maintains activity as a muscle-specific promoter element. It will be understood by those skilled in the art that the sequence of a promoter element can be altered while maintaining its ability to promote expression. A functional variant of a promoter element can include substitutions, deletions, and / or insertions compared to the reference promoter element, provided that they do not render the promoter element substantially non-functional.

[0116] A functional variant of a promoter element can be considered a promoter element that, when substituted for a reference promoter element in a synthetic promoter, substantially maintains its activity. For example, a muscle-specific synthetic promoter comprising a functional variant of a given promoter element preferably maintains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and even more preferably at least 100% of its activity (compared to the reference promoter comprising the unmodified promoter element).

[0117] Preferably, functional variants of a promoter element maintain a high level of sequence identity to the reference promoter element. Preferably, functional variants comprise a sequence that is at least 70% identical to the reference promoter element, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference promoter element.

[0118] Maintenance of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an identical promoter, but containing the replaced promoter element, under equivalent conditions. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, e.g., in the Examples.

[0119] SCP1 (SEQ ID NO: 12) is a promoter element that has been shown to provide significant muscle-specific activity when combined with the cis-regulatory elements CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) as shown in particular on page 9, lines 7-11, and Figures 5, 6, and 18 of International Application PCT / GB2022 / 051611, which is incorporated herein by reference.

[0120] CRE0053 (SEQ ID NO: 26) is a promoter element, preferably a minimal promoter, which has been shown to provide significant muscle-specific activity, preferably significant cardiac muscle-specific or cardiac-selective activity, when combined with the cis-regulatory elements CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) as shown in particular on page 9, lines 1-6, and Figures 5, 6, and 17 of International Application PCT / GB2022 / 051611, the entire disclosure of which is incorporated herein by reference.

[0121] Detargeting Elements and Liver Detargeting Elements Detargeting elements can be added to the synthetic promoter. In some embodiments, the addition of detargeting elements reduces expression from the synthetic promoter in specific tissues or cells. In some embodiments, the addition of detargeting elements alters the expression profile of the synthetic muscle-specific promoter compared to a reference promoter that does not include the detargeting element. In some embodiments, the addition of detargeting elements alters the expression profile of the synthetic muscle-specific promoter in various muscle tissues compared to a reference promoter that does not include the detargeting element. In some embodiments, the detargeting element is a binding site for a protein that is highly expressed in a specific tissue or cell. In some embodiments, the detargeting element is a liver detargeting element. In some embodiments, the addition of a liver detargeting element alters the expression profile of the synthetic muscle-specific promoter in various muscle tissues compared to a reference promoter that does not include the liver detargeting element.

[0122] In one embodiment, a liver detargeting element reduces liver expression of a synthetic promoter containing the element by at least 10% compared to an appropriate control. In one embodiment, a liver detargeting element reduces liver expression of a synthetic promoter containing the element by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more compared to an appropriate control. As used herein, "appropriate control" refers to the liver expression level of a synthetic promoter that does not contain a detargeting element in an otherwise identical sample. One skilled in the art can assess the expression level of a given synthetic promoter (with or without a detargeting element) in the liver using standard techniques, e.g., PCR-based assays, for measuring mRNA levels in liver cell and / or tissue samples.

[0123] The alpha-fetoprotein (AFP) gene is activated in fetal liver but repressed postnatally. ZBTB20 is a zinc finger protein that functions as a transcriptional repressor of AFP promoter-mediated activity in the liver (Xie Z, Zhang H, Tsai W, Zhang Y, Du Y, Zhong J, Szpirer C, Zhu M, Cao X, Barton MC, Grusby MJ, Zhang WJ. Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10859-64. Doi: 10.1073 / pnas.0800647105. Epub 2008 Jul 31. PMID: 18669658; PMCID: PMC2504784). Various regions to which ZBTB20 binds have been identified in the literature (Zhang, H., Cao, D., Zhou, L et al. ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene. Sci Rep 5, 11979 (2015). https: / / doi.org / 10.1038 / srep11979). In particular, Zhang et al. (2015) teach not to reduce the size of the -151 / -53 region of AFP (which they identified as the region to which ZBTB20 binds) because it reduces promoter activity.

[0124] SEQ ID NO: 15 shows the minimal ZBTB20 binding site. Liver-detargeted sequence 1 (SEQ ID NO: 13), liver-detargeted sequence 2 (SEQ ID NO: 14), and liver-detargeted sequence 3 (SEQ ID NO: 17) contain minimal ZBTB20 binding sites.

[0125] In some embodiments, the detargeting element is a binding site for an miRNA that is highly expressed in a particular tissue or cell, hi some embodiments, the detargeting element is a liver detargeting element.

[0126] miR122 is highly expressed in the liver, and miR122 target sequences are effective in reducing hepatic expression (Qiao C, Yuan Z, Li J, He B, Zheng H, Mayer C, Li J, Xiao X. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibit transgene expression in the liver. Gene Ther. 2011 Apr;18(4):403-10. Doi: 10.1038 / gt.2010.157. Epub 2010 Dec 9. PMID: 21150938; PMCID: PMC3686499).

[0127] SEQ ID NO: 19 shows the minimal Mir122 miRNA target sequence. Mir122 miRNA target sequence 1 (SEQ ID NO: 16) contains three times the minimal Mir122 miRNA target sequence.

[0128] A functional variant of a detargeting element or liver detargeting element comprises a sequence that differs from the reference detargeting element or liver detargeting element but substantially maintains the activity of the detargeting element or liver detargeting element. It will be understood by those skilled in the art that it is possible to alter the sequence of a detargeting element while maintaining its ability to reduce expression from a synthetic promoter in a particular tissue or cell. It will be understood by those skilled in the art that it is possible to alter the sequence of a liver detargeting element while maintaining its ability to reduce expression from a synthetic promoter in liver tissue or cells. A functional variant of a detargeting element or liver detargeting element can include substitutions, deletions, and / or insertions compared to the reference detargeting element or liver detargeting element, provided that they do not render the detargeting element or liver detargeting element substantially non-functional.

[0129] A functional variant of a detargeting element or liver-detargeting element can be considered a detargeting element or liver-detargeting element that substantially maintains its activity when substituted for a reference detargeting element or liver-detargeting element in a synthetic promoter. For example, a muscle-specific synthetic promoter comprising a given detargeting element or functional variant of a liver-detargeting element preferably maintains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and even more preferably 100% of its activity (compared to a reference promoter comprising an unmodified detargeting element or liver-detargeting element).

[0130] Preferably, a functional variant of a detargeting element or liver-detargeting element maintains a high level of sequence identity to the reference detargeting element or liver-detargeting element. Preferably, the functional variant comprises a sequence that is at least 70% identical to the reference detargeting element or liver-detargeting element, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference detargeting element or liver-detargeting element.

[0131] Maintenance of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an identical promoter but containing the replaced detargeting element or liver detargeting element under equivalent conditions.

[0132] Suitable methods for assessing muscle-specific promoter activity are disclosed herein, for example in the Examples.

[0133] Synthetic muscle-specific CRM and its functional variants Disclosed herein are various synthetic muscle-specific CRMs that can be used in constructing synthetic muscle-specific promoters. The CRMs of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters.

[0134] A functional variant of a CRE comprises a sequence that differs from the CRM element of the reference but substantially maintains activity as a muscle-specific CRM. It will be understood by those skilled in the art that the sequence of a CRM can be altered while maintaining its ability to recruit suitable muscle-specific transcription factors (TFs), thereby enhancing expression. A functional variant of a CRE can include substitutions, deletions, and / or insertions compared to the reference CRM, provided that they do not render the CRM substantially non-functional.

[0135] In some embodiments, a functional variant of a CRE can be considered a CRM that substantially maintains its activity when substituted for a reference CRM in a promoter. For example, a muscle-specific promoter comprising a functional variant of a given CRM preferably maintains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and even more preferably 100% of its activity (compared to a reference promoter comprising an unmodified CRM).

[0136] Preferably, functional variants of a CRE maintain a high level of sequence identity to the reference CRM. Preferably, functional variants comprise a sequence that is at least 70% identical to the reference CRM, more preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference CRM.

[0137] Maintenance of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an identical promoter, but containing the replaced CRM, under equivalent conditions. Suitable methods for assessing the activity of muscle-specific promoters are disclosed herein, e.g., in the Examples.

[0138] A functional variant of a given CRM can, in some embodiments, include one or more functional variants of the CREs present in the reference CRM, for example, a functional variant of a given CRM can include one, two, three, four, five, or six functional variants of the CREs present in the reference CRM.

[0139] A functional variant of a given CRM may, in some embodiments, contain the same combination of CREs as the reference CRM, but the CREs may be present in a different order than in the reference CRM. It is usually preferred that the CREs be present in the same order as in the reference CRM (thus, a functional variant of a CRM preferably contains the same permutations of the CREs as listed in the reference CRM).

[0140] In some embodiments, a functional variant of a given CRM may include one or more additional CREs relative to the CRE present in the reference CRM. The additional CREs may be provided upstream of the CRE present in the reference CRM, downstream of the CRE present in the reference CRM, and / or between the CREs present in the reference CRM. The additional CREs may be CREs disclosed herein or may be other CREs. Generally, it is preferred that a functional variant of a given CRM includes the same CRE (or a functional variant thereof) and does not include additional CREs.

[0141] A functional variant of a given CRM may contain one or more additional regulatory elements compared to a reference CRM, such as inducible or repressible elements, intronic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, segments of terminal repeats, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, and splicing elements, so long as they do not render the CRM substantially non-functional.

[0142] A functional variant of a given CRM may include additional spacers between adjacent CREs, or, if one or more spacers are present in the reference CRM, said one or more spacers may be longer or shorter (or may be absent) than those present in the reference CRM.

[0143] The CRMs disclosed herein, or functional variants thereof, can be combined with any suitable promoter elements to provide a synthetic muscle-specific promoter according to the present invention.

[0144] In some embodiments, a synthetic muscle-specific CRM according to the present invention is operably linked to a promoter element to form a synthetic muscle-specific promoter. The promoter element can be a minimal or proximal promoter. The proximal promoter is preferably a muscle-specific proximal promoter. In some embodiments, a synthetic muscle-specific CRM according to the present invention is operably linked to promoter element SCP1 (SEQ ID NO: 12). In some embodiments, a synthetic muscle-specific CRM according to the present invention is operably linked to promoter element CRE0053 (SEQ ID NO: 26).

[0145] In a CRM, the regulatory elements are preferably present in the order listed and are preferably contiguous with each other (i.e., without intervening regulatory elements). The regulatory elements may be contiguous or non-contiguous (i.e., they can be located immediately adjacent to each other, or they can be separated by a spacer or other sequence). In some embodiments, some or all of the regulatory elements may be CREs. The CREs are preferably present in the order listed and are preferably contiguous with each other. The CREs may be contiguous or non-contiguous (i.e., they can be located immediately adjacent to each other, or they can be separated by a spacer or other sequence). In some preferred embodiments, the regulatory elements or functional variants thereof are provided in the order listed and are contiguous with each other. For example, a synthetic skeletal muscle-specific or skeletal-selective CRM may include CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 1, etc. In some embodiments, it is preferred that some or all of the CREs or regulatory elements are contiguous.

[0146] In some embodiments, the synthetic muscle-specific CRM comprises one or more regulatory elements in addition to the regulatory elements listed above. In some embodiments, the one or more additional regulatory elements may be one or more other regulatory elements according to the present invention or other regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more CREs according to the present invention or other CREs. In some embodiments, the one or more additional regulatory elements may be one or more promoter elements. In some embodiments, the one or more additional regulatory elements may be one or more detargeting elements or liver-detargeting elements. In some embodiments, the one or more additional regulatory elements may be one or more introns.

[0147] In some embodiments, the synthetic muscle-specific CRM comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 9, 20-22.

[0148] In some embodiments, the synthetic muscle-specific CRM is active in both skeletal and cardiac muscle. In some embodiments, the synthetic muscle-specific CRM is active primarily in skeletal muscle. In some embodiments, the synthetic muscle-specific CRM is active primarily in cardiac muscle.

[0149] CRMs including CRE0145 and DES_MT_enhancer_48bp have been shown to provide significant muscle-specific enhancer activity in both skeletal and cardiac muscle when combined with suitable promoter elements as shown in International Application No. PCT / GB2022 / 051611, particularly at page 9, lines 1-11, and Figures 5, 6, 17, and 18, which is incorporated herein by reference.

[0150] Shorter promoter sequences are often preferred, particularly for use in situations where the capacity of a vector (e.g., a viral vector such as AAV) is limited. Thus, in some embodiments, the synthetic muscle-specific CRM has a length of 300 nucleotides or less, e.g., 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 60, 50 nucleotides or less. In some embodiments, the synthetic muscle-specific CRM has a length of 270 nucleotides or less, preferably 220 nucleotides or less, more preferably 190 nucleotides or less, and most preferably 170 nucleotides or less.

[0151] Synthetic muscle-specific promoters and their functional variants A variety of synthetic muscle-specific promoters are disclosed herein. A functional variant of a reference synthetic muscle-specific promoter is a promoter that contains a sequence that differs from the reference synthetic muscle-specific promoter but substantially maintains muscle-specific promoter activity. It will be understood by those skilled in the art that the sequence of a synthetic muscle-specific promoter can be altered while maintaining its ability to recruit appropriate muscle-specific transcription factors (TFs) and recruit RNA polymerase II to provide muscle-specific expression of an operably linked sequence (e.g., an open reading frame). A functional variant of a synthetic muscle-specific promoter can include substitutions, deletions, and / or insertions compared to the reference promoter, provided that such substitutions, deletions, and / or insertions do not render the synthetic muscle-specific promoter substantially non-functional compared to the reference promoter.

[0152] Thus, in some embodiments, a functional variant of a synthetic muscle-specific promoter can be considered a variant that substantially maintains the muscle-specific promoter activity of a reference promoter. For example, a functional variant of a synthetic muscle-specific promoter preferably maintains at least 70% of the activity of the reference promoter, more preferably at least 80% of that activity, more preferably at least 90%, 91%, 92%, 93%, 94% of that activity, more preferably at least 95%, 96%, 97%, 98%, and even more preferably 100% of that activity. In some embodiments, a functional variant of a synthetic muscle-specific promoter maintains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter.

[0153] Functional variants of synthetic muscle-specific promoters often maintain a high level of sequence similarity to the reference synthetic muscle-specific promoter. In some embodiments, functional variants comprise a sequence that is at least 70% identical to the reference synthetic muscle-specific promoter, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference synthetic muscle-specific promoter.

[0154] In one embodiment, a synthetic muscle-specific promoter described herein increases expression in muscle, e.g., of a gene product driven by the promoter, by at least 10% compared to a reference level. In one embodiment, a synthetic muscle-specific promoter described herein increases expression in muscle, such as of a gene product driven by the promoter, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a reference level, or by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold or more compared to a reference level. As used herein, "reference level" refers to the expression level of a product (e.g., a promoter or a gene product driven by a promoter) in muscle driven by a non-synthetic muscle-specific promoter or a control muscle-specific promoter in an otherwise identical sample. One skilled in the art can assess the expression level of a given product in muscle using standard techniques, such as, for example, PCR-based assays or Western blotting, to measure mRNA or protein levels, respectively.

[0155] The activity of functional variants can be assessed by comparing the expression of a suitable reporter under the control of a reference synthetic muscle-specific promoter with that of the putative functional variant under equivalent conditions. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, e.g., in the Examples.

[0156] A functional variant of a given synthetic muscle-specific promoter may include a functional variant of one or more CREs present in the reference synthetic muscle-specific promoter. Functional variants of CREs are described above.

[0157] A functional variant of a given synthetic muscle-specific promoter can include a functional variant of a CRM present in the reference synthetic muscle-specific promoter. Functional variants of CRMs are described above.

[0158] A functional variant of a given synthetic muscle-specific promoter may contain functional variants or different promoter elements when compared to a reference synthetic muscle-specific promoter. Functional variants of promoter elements are described above.

[0159] A functional variant of a given synthetic muscle-specific promoter may contain the same CREs as the reference synthetic muscle-specific promoter, but the CREs may be present in a different order than in the reference synthetic muscle-specific promoter.

[0160] A functional variant of a given synthetic muscle-specific promoter may contain one or more additional CREs in addition to the CRE present in the reference synthetic muscle-specific promoter. The additional CREs may be provided upstream of the CRE present in the reference synthetic muscle-specific promoter, downstream of the CRE present in the reference synthetic muscle-specific promoter, and / or between the CREs present in the reference synthetic muscle-specific promoter. The additional CREs may be CREs disclosed herein or may be other CREs.

[0161] A functional variant of a given synthetic muscle-specific promoter may include additional spacers between the adjacent CRE and promoter elements, or if one or more spacers are present in the reference synthetic muscle-specific promoter, said one or more spacers may be longer or shorter (or may be absent) than those present in the reference synthetic muscle-specific promoter.

[0162] It will be apparent that the synthetic muscle-specific promoters of the invention can comprise a CRM of the invention and additional regulatory elements, such as one or more additional CRMs, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, segments of terminal repeats, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, introns, splicing elements, and the like, provided they do not render the promoter substantially non-functional.

[0163] Preferred synthetic muscle-specific promoters of the present invention exhibit muscle-specific promoter activity in muscle cells that is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity exhibited by the CBA, CK8, CK7, or RSV promoter. While higher levels of promoter activity are often preferred, this is not always the case; thus, in some cases, more moderate levels of expression may be preferred. In some cases, it may be desirable to have available a variety of promoters with different activity levels that allow expression levels to be tailored to requirements; the present disclosure provides promoters with such a wide range of activity. The activity of a given synthetic muscle-specific promoter of the invention relative to CBA, CK8, CK7, or RSV can be assessed by comparing the muscle-specific expression of a reporter gene under the control of the synthetic muscle-specific promoter with the expression of the same reporter under the control of the CBA, CK8, CK7, or RSV promoter when the two promoters are provided in otherwise equivalent expression constructs and under equivalent conditions. Suitable methods for testing the activity of muscle-specific promoters can be found, for example, in the Examples.

[0164] Preferably, the synthetic muscle-specific promoters of the invention may be capable of increasing expression of a gene (e.g., a therapeutic gene or gene of interest) in a muscle or muscle cell of a subject by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, at least 500%, at least 1000%, or more compared to a known muscle-specific promoter, preferably the SPc5-12 promoter (Gene Ther. 2008 Nov;15(22):1489-99).

[0165] Suitably, the synthetic muscle-specific promoters of the invention exhibit activity in non-muscle cells (e.g., Huh7 and HEK293 cells) that is 50% or less compared to CMV-IE, preferably 25% or less of CMV-IE, more preferably 10% or less of CMV-IE, and in some cases 5% or less of CMV-IE, or 1% or less of CMV-IE.

[0166] In many cases, shorter promoter sequences are preferred, particularly for use in situations where the capacity of a vector (e.g., a viral vector such as AAV) is limited. Thus, in some embodiments, a synthetic muscle-specific promoter has a length of 400 nucleotides or less, e.g., 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250 nucleotides or less. In some embodiments, a synthetic muscle-specific promoter has a length of 390 nucleotides or less, more preferably 380 nucleotides or less, even more preferably 350 nucleotides or less, and most preferably 330 nucleotides or less. In some embodiments, a synthetic muscle-specific promoter has a length of 320 nucleotides or less, preferably 310 nucleotides or less, more preferably 280 nucleotides or less, and most preferably 250 nucleotides or less.

[0167] Particularly preferred synthetic muscle-specific promoters are those that are short and exhibit high levels of activity.

[0168] In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, 27-28.

[0169] It is generally preferred that a promoter according to the invention that is a functional variant of any one of SEQ ID NOs: 1-4, 27-28 maintains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter. Preferably, said activity is assessed using one of the examples described herein, although other methods may also be used.

[0170] In some embodiments, the synthetic muscle-specific promoter is active in both skeletal and cardiac muscle. In some embodiments, the synthetic muscle-specific promoter is active primarily in skeletal muscle. In some embodiments, the synthetic muscle-specific promoter is active primarily in cardiac muscle.

[0171] A synthetic promoter comprising CRE0145 and DES_MT_enhancer_48bp operably linked to SCP1 has been found to provide significant muscle-specific enhancer activity in both skeletal and cardiac muscle, as shown in particular on page 9, lines 7-11, and Figures 5, 6, and 18 of International Application No. PCT / GB2022 / 051611, which is incorporated herein by reference.

[0172] A synthetic promoter comprising CRE0145 and DES_MT_enhancer_48bp operably linked to CRE0053 has been found to provide significant myocardium-specific or cardiac-selective activity, as shown in particular on page 9, lines 1-6, and Figures 5, 6, and 17 of International Application No. PCT / GB2022 / 051611, which is incorporated herein by reference.

[0173] In a synthetic muscle-specific promoter, the regulatory elements are preferably present in the order listed and are preferably contiguous with each other (i.e., without intervening regulatory elements). The regulatory elements may be contiguous or non-contiguous (i.e., they can be located directly adjacent to each other, or they can be separated by a spacer or other sequence). In some embodiments, some or all of the regulatory elements may be CREs. The CREs are preferably present in the order listed and are preferably contiguous with each other. The CREs may be contiguous or non-contiguous (i.e., they can be located directly adjacent to each other, or they can be separated by a spacer or other sequence). In some preferred embodiments, the regulatory elements or functional variants thereof are present in the order listed and are contiguous with each other. Promoter elements are located downstream of other regulatory elements and are typically adjacent to proximal regulatory elements. Promoter elements may be contiguous with adjacent regulatory elements or may be separated by a spacer. In some embodiments, promoter elements are located downstream of CREs and are typically adjacent to proximal CREs. The promoter element may be contiguous with the adjacent CRE or may be separated by a spacer.

[0174] In some embodiments, a promoter element is located downstream of other regulatory elements and is typically adjacent to proximal regulatory elements. A promoter element can be contiguous with an adjacent regulatory element or can be separated by a spacer.

[0175] In some embodiments, the synthetic muscle-specific promoter comprises one or more regulatory elements in addition to the regulatory elements listed above. In some embodiments, the one or more additional regulatory elements may be one or more other regulatory elements according to the present invention or other regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more other CREs according to the present invention or other CREs. In some embodiments, the one or more additional regulatory elements may be one or more promoter elements. In some embodiments, the one or more additional regulatory elements may be one or more detargeting elements or liver-detargeting elements. In some embodiments, the one or more additional regulatory elements may be one or more introns.

[0176] Synthetic muscle-specific expression cassette Also disclosed herein are synthetic muscle-specific expression cassettes, which may comprise a synthetic muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding an expression product, preferably a gene (e.g., a transgene, such as a therapeutic transgene).

[0177] In some embodiments, the expression product is a therapeutic expression product. In some preferred embodiments, the therapeutic expression product is suitable for use in the treatment of a disease or condition. In some embodiments, the disease or condition is associated with abnormal gene expression, optionally in muscle (i.e., a muscle disease), optionally in cardiac and / or skeletal muscle. In some preferred embodiments, therapeutic expression products include those useful in the treatment of diseases, preferably muscle diseases.

[0178] The term "muscle disease" is generally understood by a skilled artisan. The term relates to a disease suitable for treatment and / or prevention by administration of an active compound to muscle, particularly muscle cells. In some embodiments, the muscle disease is a skeletal muscle disease. In some embodiments, the muscle disease is a cardiac muscle disease. In some embodiments, the muscle disease is a skeletal and cardiac muscle disease.

[0179] Suitable diseases and expression products are detailed below.

[0180] Preferably, the synthetic muscle-specific expression cassette comprises sequences providing or encoding one or more, preferably all, of a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Preferably, the expression cassette comprises nucleic acid encoding a post-transcriptional regulatory element. Preferably, the expression cassette comprises nucleic acid encoding a polyA element.

[0181] Activity of muscle-specific promoters and expression cassettes heart In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention exhibits an expression level in cardiac cells or tissue that is at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 110%, of the control promoter CK7. 0%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 10,000%, 15,000%, 20,000%, preferably 30,000%, 31,000%, 32,000%, 33,000%, more preferably at least 35,000% of the activity of the control promoter CK7 in cardiac cells or tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in cardiac cells or tissue than the control promoter CK7. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 50, 100, 150, 200-fold, preferably at least 210, 220, 230, 240, 250, 260, 270, 280, 290, 300-fold, more preferably at least 310, 320, 330, 340, or 350-fold greater activity in cardiac cells or tissue than the control promoter CK7.

[0182] As shown in Figures 2 and 3, SP0525 (SEQ ID NO: 1) exhibits approximately 1500% of the activity of CK7 in the heart (15-fold higher activity than CK7 in the heart). As shown in Figures 2 and 4, SP0526 (SEQ ID NO: 2) exhibits approximately 800% of the activity of CK7 in the heart (8-fold higher activity than CK7 in the heart). As shown in Figures 2 and 5, SP0527 (SEQ ID NO: 3) exhibits approximately 3600% of the activity of CK7 in the heart (36-fold higher activity than CK7 in the heart). As shown in Figures 2 and 6, SP0528 (SEQ ID NO: 4) exhibits approximately 1400% of the activity of CK7 in the heart (14-fold higher activity than CK7 in the heart). As shown in Figures 2 and 7, expression cassette 529 exhibits approximately 35000% of the activity of CK7 in the heart (350-fold higher activity than CK7 in the heart). As shown in Figures 2 and 8, SP0530 (SEQ ID NO: 27) exhibits approximately 23,000% of the activity of CK7 in the heart (230-fold higher activity than CK7 in the heart). As shown in Figures 2 and 9, SP0531 (SEQ ID NO: 28) exhibits approximately 300% of the activity of CK7 in the heart (3-fold higher activity than CK7 in the heart). In some preferred embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has at least 300% of the activity of the control promoter CK7 in cardiac cells or cardiac tissue. In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has at least 3-fold higher activity than the control promoter CK7 in cardiac cells or cardiac tissue.

[0183] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention exhibits an activity in cardiac cells or tissue that is at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 110%, of the control promoter CK8. 0%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in cardiac cells or tissue than the control promoter CK8.

[0184] As shown in Figures 1 and 7, expression cassette 529 exhibits approximately 250% of the activity of CK8 in the heart (two-fold higher activity than CK8 in the heart). As shown in Figures 1 and 8, SP0530 (SEQ ID NO: 27) exhibits approximately 160% of the activity of CK8 in the heart.

[0185] In some preferred embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200% of the activity of the control promoter CK8 in cardiac cells or tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2-fold higher activity in cardiac cells or tissue than the control promoter CK8.

[0186] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 110%, 120%, 130%, 140%, 150%, 160%, preferably at least 170%, 180%, 190%, 200%, and most preferably at least 210%, 220%, 230%, 240%, 250% of the activity of SP0524 in cardiac cells or tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least two-fold greater activity than SP0524 in cardiac cells or tissue.

[0187] As shown in Figure 8 and International Application PCT / GB2022 / 051611 (particularly page 9, lines 7-11 and Figures 5, 6, and 18), SP0530 exhibits approximately 230% of the activity of SP0524 in the heart (two-fold higher activity than SP0524 in the heart). Thus, the addition of the tMCK SA / SD intron to SP0524 results in a promoter (SP0530) with higher activity in the heart.

[0188] diaphragm In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention is at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 11% of the control promoter CK7 in diaphragm cells or tissue. 00%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in diaphragm cells or tissue than the control promoter CK7.

[0189] As shown in Figures 2 and 4, SP0526 (SEQ ID NO: 2) exhibits approximately 1500% of the activity of CK7 in the diaphragm (15-fold higher activity than CK7 in the diaphragm). As shown in Figures 2 and 5, SP0527 (SEQ ID NO: 3) exhibits approximately 1100% of the activity of CK7 in the diaphragm (11-fold higher activity than CK7 in the diaphragm). As shown in Figures 2 and 6, SP0528 (SEQ ID NO: 4) exhibits approximately 300% of the activity of CK7 in the diaphragm (3-fold higher activity than CK7 in the diaphragm). As shown in Figures 2 and 7, expression cassette 529 exhibits approximately 1800% of the activity of CK7 in the diaphragm (18-fold higher activity than CK7 in the diaphragm). As shown in Figures 2 and 8, SP0530 (SEQ ID NO: 27) exhibits approximately 2400% of the activity of CK7 in the diaphragm (24-fold higher activity than CK7 in the diaphragm). As shown in Figures 2 and 9, SP0531 (SEQ ID NO: 28) exhibits approximately 2800% of the activity of CK7 in the diaphragm (28-fold higher activity than CK7 in the diaphragm).

[0190] In some preferred embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 300% of the activity of the control promoter CK7 in diaphragm cells or tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 3-fold higher activity in diaphragm cells or tissue than the control promoter CK7.

[0191] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an expression level in diaphragm cells or tissue that is at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 11% of the control promoter CK8. 00%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in diaphragm cells or tissue than the control promoter CK8.

[0192] As shown in Figures 1 and 4, SP0526 (SEQ ID NO: 2) exhibits approximately 180% of the activity of CK8 in the diaphragm. As shown in Figures 1 and 5, SP0527 (SEQ ID NO: 3) exhibits approximately 130% of the activity of CK8 in the diaphragm. As shown in Figures 1 and 7, expression cassette 529 exhibits approximately 200% of the activity of CK8 in the diaphragm (two-fold higher activity than CK8 in the diaphragm). As shown in Figures 1 and 8, SP0530 (SEQ ID NO: 27) exhibits approximately 280% of the activity of CK8 in the diaphragm (two-fold higher activity than CK8 in the diaphragm). As shown in Figures 1 and 9, SP0531 (SEQ ID NO: 28) exhibits approximately 300% of the activity of CK8 in the diaphragm (three-fold higher activity than CK8 in the diaphragm).

[0193] gastrocnemius In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention exhibits at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 11% of the control promoter CK7 in gastrocnemius muscle cells or gastrocnemius muscle tissue. 00%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in gastrocnemius muscle cells or tissue than the control promoter CK7.

[0194] As shown in Figures 2 and 4, SP0526 (SEQ ID NO: 2) exhibits approximately 400% of the activity of CK7 in the gastrocnemius muscle (4-fold higher activity than CK7 in the gastrocnemius muscle). As shown in Figures 2 and 5, SP0527 (SEQ ID NO: 3) exhibits approximately 260% of the activity of CK7 in the gastrocnemius muscle (2-fold higher activity than CK7 in the gastrocnemius muscle). As shown in Figures 2 and 9, SP0531 (SEQ ID NO: 28) exhibits approximately 200% of the activity of CK7 in the gastrocnemius muscle (2-fold higher activity than CK7 in the gastrocnemius muscle).

[0195] In some preferred embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200% of the activity of the control promoter CK7 in gastrocnemius muscle cells or gastrocnemius muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2-fold higher activity in gastrocnemius muscle cells or gastrocnemius muscle tissue than the control promoter CK7.

[0196] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has about 100% or less than 100% of the activity of the control promoter CK8 in gastrocnemius muscle cells or gastrocnemius muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has similar or less activity in gastrocnemius muscle cells or gastrocnemius muscle tissue than the control promoter CK8.

[0197] In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has at least 110%, preferably 120%, most preferably 130% of the activity of SP0524 in gastrocnemius muscle cells or tissue. In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has at least 150%, 160%, 170%, 180%, 190%, 200%, preferably 210%, 250%, 300%, 350%, 400%, 450%, most preferably 500% or 550% of the activity of SP0524 in gastrocnemius muscle cells or tissue.

[0198] As shown in Figure 8 and International Application PCT / GB2022 / 051611 (particularly page 9, lines 7-11, and Figures 5, 6, and 18), SP0530 exhibits approximately 130% of the activity of SP0524 in gastrocnemius muscle. Thus, the addition of the tMCK SA / SD intron to SP0524 results in a promoter (SP0530) with higher activity in gastrocnemius muscle.

[0199] As shown in Figure 9 and International Application PCT / GB2022 / 051611 (particularly page 9, lines 7-11, and Figures 5, 6, and 18), SP0531 exhibits approximately 550% of the activity of SP0524 in gastrocnemius muscle. Thus, the addition of an MVM truncated intron to SP0524 results in a promoter (SP0531) with higher activity in gastrocnemius muscle. Furthermore, the addition of an MVM truncated intron to SP0524 results in a promoter (SP0531) with lower activity in diaphragm, heart, soleus, and tibialis anterior muscles.

[0200] soleus muscle In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less than 100% of the activity of the control promoter CK7 in soleus muscle cells or soleus muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less activity than the control promoter CK7 in soleus muscle cells or soleus muscle tissue.

[0201] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less than 100% of the activity of the control promoter CK8 in soleus muscle cells or soleus muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less activity than the control promoter CK8 in soleus muscle cells or soleus muscle tissue.

[0202] tibialis anterior In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention exhibits an expression level that is at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, of the control promoter CK7 in tibialis anterior muscle cells or tissue. %, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36-fold greater activity in tibialis anterior muscle cells or tissue than the control promoter CK7.

[0203] As shown in Figures 2 and 4, SP0526 (SEQ ID NO: 2) exhibits approximately 240% of the activity of CK7 in the tibialis anterior muscle (two-fold higher activity than CK7 in the tibialis anterior muscle), and as shown in Figures 2 and 5, SP0527 (SEQ ID NO: 3) exhibits approximately 140% of the activity of CK7 in the tibialis anterior muscle.

[0204] liver In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less than 100% of the activity of the control promoter CK7 or CK8 in liver cells or liver tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less activity than the control promoter CK8 or CK7 in liver cells or liver tissue.

[0205] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less than 100% of the activity of SP0524 in liver cells or liver tissue. In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has less activity than SP0524 in liver cells or liver tissue.

[0206] In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has a nucleotide sequence of 1.2e 12 In some embodiments, the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity of 1.2 e 12At doses higher than vg / 200 μl, it has less activity than SP0524 in liver cells or liver tissue.

[0207] In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has activity in the liver that is about 2-, 3-, 4-, 5-, preferably 6-, 7-, 8-, 9-, 10-, and most preferably 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold less than the muscle tissue with the lowest activity. In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has activity in the liver that is 2-, 3-, 4-, 5-, preferably 6-, 7-, 8-, 9-, 10-, and most preferably 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold less than the muscle tissue with the lowest activity. The muscle cells with the lowest activity are those muscle tissues in which the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention is least active (i.e. the lowest average activity).

[0208] In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has activity in the liver that is about 100-, 200-, 300-, 400-, 500-, preferably about 600-, 700-, 800-, 900-, and most preferably about 1000-, 1100-, 1200-, 1300-, 1400-, or 1500-fold less than the muscle tissue with the highest activity. In some embodiments, a synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or an expression cassette according to the ninth or tenth aspect of the invention has activity in the liver that is at least 100-, 200-, 300-, 400-, 500-, preferably 600-, 700-, 800-, 900-, and most preferably 1000-, 1100-, 1200-, 1300-, 1400-, or 1500-fold less than the muscle tissue with the highest activity. The muscle tissue with the highest activity is the muscle tissue in which the synthetic muscle-specific promoter according to any of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention is most active (i.e. the highest average activity).

[0209] Vectors and viral particles Disclosed herein are various vectors comprising a synthetic muscle-specific promoter according to any aspect of the present invention or an expression cassette according to the present invention.

[0210] In some embodiments of the invention, the vector is a plasmid. Such a plasmid may contain a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments of the invention, the vector is a viral vector.

[0211] In some embodiments of the present invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic cell expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other vectors are well known and commercially available. In the case of mammalian adenovirus vectors, the pSV and pCMV series of vectors are particularly well-known, non-limiting examples. For example, there are many well-known yeast expression vectors, including, but not limited to, the Yeast Integrating Plasmid (Yip) and the Yeast Replicating Plasmid (Yrp). For plants, the Ti plasmid of Agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0212] In some preferred embodiments, the vector is a gene therapy vector. Various gene therapy vectors are known in the art, and mention may be made of AAV vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors. When the vector is a gene therapy vector, the vector preferably comprises a nucleic acid sequence operably linked to the synthetic muscle-specific promoter of the present invention encoding a therapeutic product, preferably a therapeutic protein. The therapeutic protein may be a secretable protein. Non-limiting examples of secretable therapeutic proteins include clotting factors, such as factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, toxic proteins, etc.

[0213] In some embodiments of the present invention, the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector. In some preferred embodiments, the vector is an AAV vector. In some preferred embodiments, the AAV has a serotype suitable for muscle transduction. In some embodiments, the AAV is selected from the group consisting of AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8, and AAV2G9, or derivatives thereof. The AAV vector is preferably used as a self-complementary double-stranded AAV vector (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., conversion of single-stranded AAV to double-stranded AAV), although the use of a single-stranded AAV vector (ssAAV) is also encompassed herein. In some embodiments of the present invention, the AAV vector is chimeric, meaning that it contains components from at least two AAV serotypes, such as the ITRs of AAV2 and the capsid protein of AAV5. AAV9 is known to transduce skeletal muscle and cardiac muscle particularly effectively, and therefore AAV9 and its derivatives are particularly interesting for targeting skeletal muscle and cardiac muscle. AAV1, AAV6, AAV7, and AAV8 are also known to target skeletal muscle, and therefore these AAV serotypes and their derivatives are also particularly interesting for targeting skeletal muscle. AAV1 and AAV8 are also known to target cardiac muscle, and therefore these AAV serotypes and their derivatives are also particularly interesting for targeting cardiac muscle. In some embodiments, the rAAV vector is an AAV3b serotype, such as, but not limited to, an AAV3b265D virion, an AAV3b265D549A virion, an AAV3b549A virion, an AAV3bQ263Y virion, or an AAV3bSASTG virion (i.e., a virion comprising an AAV3b capsid containing the Q263A / T265 mutation).In some embodiments, the virions can be rational haploid, chimeric, or any variant, such as a capsid tailored to increase uptake in a desired location, such as the heart. Other capsids can include capsids derived from any of the known AAV serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV7, and AAV10. In some embodiments, the vector is AAV9. In some embodiments, the vector is AAVMYO. AAVMYO capsids have been shown to have high transduction potential in muscle, as described in Weinmann, J., Weis, S., Sippel, J., et al., "Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants." Nat Commun 11, 5432 (2020) (https: / / doi.org / 10.1038 / s41467-020-19230-w), which is incorporated herein by reference. In some embodiments, the vector is a MyoAAV vector.MyoAAV capsids have been shown to have high transduction potential in muscle, Mohammadsharif Tabebordbar, Kim A. Lagerborg, Alexandra Stanton, Emily M. King, Simon Ye, Liana Tellez, Allison Krunnfusz, Sahar Tavakoli, Jeffrey J. Widrick, Kathleen A. Messemer, Emily C. Troiano, Behzad Moghadaszadeh, Bryan L. Peacker, Krystynne A. Leacock, Naftali Horwitz, Alan H. Beggs, Amy J. Wagers, Pardis C. Sabeti, Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species, Cell, Volume 184, Issue 19, 2021, pp. 4919-4938.e22, ISSN 0222-2245. 0092-8674 (https: / / doi.org / 10.1016 / j.cell.2021.08.028), which is incorporated herein by reference.

[0214] The present invention further provides a recombinant virion (virus particle) comprising the vector described above.

[0215] Pharmaceutical Composition The synthetic muscle-specific promoter, expression cassette, vector, or virion of the invention may be formulated in a pharmaceutical composition together with one or more pharmaceutically acceptable excipients, i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition may be provided in the form of a kit. Suitable pharmaceutical compositions and delivery systems for AAV vectors, as well as methods and uses thereof, are known in the art.

[0216] Therapeutic and Other Methods and Uses The synthetic muscle-specific promoters, expression cassettes, vectors, virions, or pharmaceutical compositions according to various aspects of the invention may be for use in the treatment of diseases, preferably diseases associated with aberrant gene expression in muscle (e.g., muscle diseases), as appropriate.

[0217] In one embodiment, the invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the invention for use in treating skeletal muscle disease. In one embodiment, the invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the invention for use in treating myocardial disease. In one embodiment, the invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the invention for use in treating skeletal and myocardial muscle disease.

[0218] Associated diseases and expression products are described below.

[0219] The synthetic muscle-specific promoters, expression cassettes, vectors, or virions according to various aspects of the invention may be for use in the manufacture of a pharmaceutical composition for the treatment of any condition or disease mentioned herein.

[0220] cell The present invention further provides a cell comprising a synthetic muscle-specific promoter, expression cassette, vector, or virion according to various aspects of the invention. Preferably, the cell is a eukaryotic cell. The eukaryotic cell may preferably be a fungal cell (e.g., a yeast cell), an animal (metazoan) cell (e.g., a mammalian cell), or a plant cell. Alternatively, the cell may be a prokaryotic cell. In some embodiments of the invention, the cell is ex vivo, e.g., in cell culture. In other embodiments of the invention, the cell may be part of a tissue or a multicellular organism.

[0221] In a preferred embodiment, the cells are muscle cells (myocytes), which may be ex vivo or in vivo. In a preferred embodiment, the cells are cardiomyocytes, which may be ex vivo or in vivo. In an alternative preferred embodiment, the cells are skeletal muscle cells, which may be ex vivo or in vivo. The muscle cells may be primary muscle cells or cells of a muscle-derived cell line, e.g., an immortalized cell line. The cell may be present within a muscle tissue environment (e.g., within the muscle of an animal) or may be isolated from muscle tissue, e.g., it may be present in cell culture. Preferably, the cell is a human cell. Preferably, the cell is a non-human mammalian cell.

[0222] The skeletal muscle cells may be derived from fast or slow muscle fibers. The skeletal muscle cells may be selected from intermediate muscle cells, myocytes, myotubes, myoblasts, and satellite cells.

[0223] The cardiomyocytes may be selected from ventricular cardiomyocytes, atrial cardiomyocytes, pericytes, cardiac smooth muscle cells, cardiac fibroblasts, or cardiac endothelial cells (ECs), as well as perivascular cells and pacemaker cells.

[0224] A synthetic muscle-specific promoter, expression cassette, or vector according to the invention may be inserted into the genome of the cell, or it may be episomal (e.g., present on an episomal vector).

[0225] Method for producing expression product Also disclosed herein is a method for producing an expression product, comprising the steps of providing a synthetic muscle-specific expression cassette according to the present invention in a cell, preferably a muscle cell (preferably in a vector as set out above), and expressing the expression product, preferably a gene, present in the synthetic muscle-specific expression cassette. The method preferably comprises maintaining said muscle cell under conditions suitable for expression of the expression product, preferably a gene. When culturing, this may comprise incubating the cell, or tissue comprising the cell, under suitable culture conditions. Preferably, culture conditions may be at 37°C, 5% CO2. Expression may, of course, be in vivo, for example in one or more cells in a muscle of a subject. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. Types of skeletal muscle cells and cardiomyocytes are described above.

[0226] Preferably, the method comprises introducing a synthetic muscle-specific expression cassette into muscle cells. Various methods for transfecting muscle cells are well known in the art. A preferred method for transfecting muscle cells is to transduce the cells with a viral vector, e.g., an AAV vector, comprising the synthetic muscle-specific expression cassette.

[0227] Various methods of transfecting cells are well known in the art, such as viral-mediated transfection, e.g., transfection using viral vectors; chemical-based transfection, e.g., lipofection, calcium phosphate transfection, cationic polymers of Fu gene reagents, etc.; non-chemical-based transfection, e.g., electroporation, etc.; microinjection; Agrobacterium-mediated transfer; gene guns; impale infection; hydrostatic pressure; direct DNA uptake; whisker-mediated transformation; and particle bombardment.

[0228] Methods for expressing expression products Also disclosed herein is a method for expressing an expression product, preferably a therapeutic transgene, in a muscle cell, comprising introducing an expression cassette or vector according to the present invention into the muscle cell. Preferably, the step of introducing the expression cassette or vector may comprise transfecting the muscle cell with the expression cassette or vector. Various methods for transfecting muscle cells are well known in the art. A preferred method for transfecting muscle cells is transducing the cells with a viral vector, e.g., an AAV vector, comprising a synthetic muscle-specific expression cassette. The muscle cell may be in vitro or ex vivo. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. Types of skeletal muscle cells and cardiomyocytes are described above.

[0229] Gene therapy methods The synthetic muscle-specific promoters, expression cassettes, vectors, pharmaceutical compositions, or virions according to various aspects of the invention may be used for gene therapy, and the use of such nucleic acid constructs in gene therapy therefore forms part of the present invention.

[0230] The expression cassette, vector, pharmaceutical composition or virion according to the invention may be for use in gene therapy in a subject, preferably gene therapy by muscle-specific expression of an expression product, preferably a therapeutic gene. Suitably, the expression cassette, vector, pharmaceutical composition or virion according to the invention may be for use in gene therapy by cardiac muscle-specific (or heart-selective) expression and / or skeletal muscle-specific (or skeleton-selective) expression of an expression product, preferably a therapeutic gene. The therapy may involve treatment of disease by secretion of an expression product (suitably a gene, therapeutic transgene or therapeutic product) from muscle cells, preferably in diseases involving abnormal gene expression in muscle. Suitable diseases are described below.

[0231] 1. A method of gene therapy in a subject, preferably a human, in need thereof, comprising: - administering to a subject (preferably introducing into the muscle of a subject) a synthetic muscle-specific expression cassette, vector, virion, or pharmaceutical composition of the invention comprising a gene encoding an expression product, preferably a therapeutic expression product. Also disclosed herein are methods comprising: Suitable expression products are detailed below.

[0232] In one embodiment, the muscle is cardiac muscle. In one embodiment, the muscle is skeletal muscle.

[0233] The method preferably comprises expressing in the muscle of said subject a therapeutic amount of an expression product (preferably a therapeutic expression product) from a gene. Various conditions and diseases that can be treated and suitable diseases are described below.

[0234] In some embodiments, the method comprises administering to a subject a vector or virion according to the invention. Suitably, the vector is a viral gene therapy vector, for example an AAV vector.

[0235] In some embodiments, the method includes systemically administering the viral gene therapy vector. Systemic administration can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection). Preferred routes of injection include intravenous, intramuscular, subcutaneous, intraarterial, intraarticular, intrathecal, and intradermal injection.

[0236] In some embodiments, viral gene therapy vectors may be administered simultaneously or sequentially with one or more additional therapeutic agents or one or more saturating agents designed to prevent removal of the vector by the reticuloendothelial system.

[0237] If the vector is an AAV vector, the dosage of the vector is 1 x 10 10 gc / kg to 1 × 10 15 gc / kg or more, preferably 1 x 10 12 gc / kg to 1×10 14gc / kg, preferably 5×10 12 gc / kg to 5 × 10 13 In some preferred embodiments, the dose of AAV is 1e 14 gc / kg or approximately 1e 14 gc / kg.

[0238] Generally, the subject in need of such treatment will be a mammal, preferably a primate, more preferably a human. In some embodiments, the subject is not human. Typically, the subject in need of such treatment will exhibit symptoms characteristic of a disease. The method typically includes the step of ameliorating the symptoms exhibited by the subject in need thereof by expressing a therapeutic amount of an expression product, preferably a therapeutic expression product.

[0239] Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well known in the art and will not be described in detail here. Briefly, they include intramuscular injection, intrainterstitial injection, airway infusion, endothelial application, intrahepatic parenchyma, and intravenous or intraarterial administration (e.g., intrahepatic artery, intrahepatic vein) of plasmid DNA vectors or viral vectors. Various devices have been developed to increase the availability of DNA to target cells. A simple approach is to physically contact the target cells with a catheter or implant material containing the relevant vector, while more complex approaches may use jet propellants. Gene transfer into mammalian muscle cells has been performed using both ex vivo and in vivo techniques. Ex vivo approaches typically require the harvesting of muscle cells, in vitro transduction with a suitable expression vector, and subsequent reintroduction of the transduced myocytes into the muscle. In vivo gene transfer has been achieved by intramuscular injection of DNA or viral vectors. In some preferred embodiments, the preferred route of administration is intravenous administration. Intravenous delivery is particularly preferred for gene therapy targeted to muscle. In some preferred embodiments, the preferred route of administration is intracoronary administration. Intracoronary delivery is particularly preferred for gene therapy targeted to myocardium.

[0240] According to some preferred embodiments, the above methods may be used for the treatment of subjects with diseases described below, such as muscular dystrophy or congestive heart failure.

[0241] disease The disease may be any disease. Preferably, the condition or disease is associated with abnormal gene expression, optionally in muscle cells (myocytes) or muscle tissue. Preferably, the condition or disease is associated with abnormal gene expression, optionally in cardiac muscle cells or skeletal muscle cells. In a preferred embodiment, the disease is one that can be alleviated by muscle-specific expression of a suitable expression product.

[0242] In some embodiments, the disease is vascular disease, muscular dystrophy, cardiomyopathy, hypertonia, muscle atrophy, myoclonus-dystonia (gene affected: SGCE), mitochondrial myopathy, rhabdomyolysis, fibromyalgia, and / or myofascial pain syndrome.

[0243] In one embodiment, the disease can be a cardiovascular disease, heart disease, or heart disorder. In one embodiment, the disease can be heart failure, such as congestive heart failure. In one embodiment, the disease can be ischemia, arrhythmia, myocardial infarction (MI), abnormal cardiac contractions, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormal Ca. 2+ In some embodiments, the disease may be selected from the group consisting of congestive heart failure, myocardial injury, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, dysfunctional conduction system, dysfunctional coronary artery, and pulmonary heart hypertension. In some embodiments, the disease may be selected from congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, myocardial injury, idiopathic cardiomyopathy, cardiac arrhythmia, muscular dystrophy, muscle mass abnormalities, muscle degeneration, infectious myocarditis, drug- or toxin-induced myopathy, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease.

[0244] In some embodiments, the disease is a myocardial disorder. In some embodiments, the myocardial disorder is hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy, restrictive cardiomyopathy, left ventricular noncompaction, takotsubo cardiomyopathy, myocarditis, eosinophilic myocarditis, or ischia cardiomyopathy. Preferably, the arrhythmogenic right ventricular dysplasia is ARVD1 (gene: TGFB3), ARVD2 (gene: RYR2), ARVD3, ARVD4, ARVD5 (gene: TMEM43), ARVD6, ARVD7 (gene: DES), ARVD8 (gene: DSP), ARVD9 (gene: PKP2), ARVD10 (gene: DSG2), ARVD11 (gene: DSC2), and / or ARVD12 (gene: JUP). In some embodiments, the disease is hypertrophic cardiomyopathy. Preferably, the hypertrophic cardiomyopathy is CMH1 (gene: MYH7), CMH2 (gene: TNNT2), CMH3 (gene: TPM1), CMH4 (gene: MYBPC3), CMH5, CMH6 (gene: PRKAG2), CMH7 (gene: TNNI3), CMH8 (gene: MYL3), CMH9 (gene: TTN), CMH10 (gene: MYL2), CMH11 (gene: ACTC1), or CMH12 (gene: CSRP3).

[0245] In some embodiments, the disease is a vascular disease. The vascular disease can be coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery stenosis, or aortic aneurysm.

[0246] In some embodiments, the disease may be a myocardial disorder, which may be hypertensive heart disease, heart failure (e.g., congestive heart failure, etc.), pulmonary heart disease, arrhythmia, inflammatory heart disease (e.g., endocarditis, inflammatory cardiac hypertrophy, myocarditis, etc.), valvular heart disease, congenital heart disease, or rheumatic heart disease.

[0247] In some embodiments, the disease is muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (gene affected: DMD), Becker muscular dystrophy (gene affected: DMD), limb-girdle muscular dystrophy (subtypes and genes affected: LGMD1A (gene: TTID), LGMD1B (gene: LMNA), LGMD1C (gene: CAV3), LGMD1D (gene: DNAJB6), LGMD1E (gene: DES), LGMD1F (gene: TNP03), LGMD1G (gene: HNRPDL). , LGMD1H, LGMD2A (gene: CAPN3), LGMD2B (gene: DYSF), LGMD2C (gene: SGCG), LGMD2D (gene: SGCA), LGMD2E (gene: SGCB), LGMD2F (gene: SGCD), LGMD2G (gene: TCAP), LGMD2H (gene: TRIM32), LGMD2I (gene: FKRP), LGMD2J (gene: TTN), LGMD2K (gene: POMT1), LGMD2L (gene: AN05), LGMD2M (gene: FKTN), LGMD2 N (gene: POMT2), LGMD20 (gene: POMGNT1), LGMD2Q (gene: PLEC1)), congenital muscular dystrophy, distal muscular dystrophy (subtypes and affected genes: Miyoshi myopathy (gene: DYSF), pretibial onset distal myopathy (gene: DYSF), Welander distal myopathy (gene: TIA1), Gowers-Leyne distal myopathy (gene: MYH7), Nonaka distal myopathy, hereditary inclusion body myositis type 1, distal myopathy with vocal cord and pharyngeal weakness, ZASP In some preferred embodiments, the disease is limb-girdle muscular dystrophy type 2i (LGMD2I; gene affected: FKRP).

[0248] In some embodiments, the disease is myotonia, hi some embodiments, the myotonia is myotonia congenita (gene affected: CLCN1; subtypes: Thomsen, Becker) and / or paramyotonia congenita (gene affected: SCN4A).

[0249] In some embodiments, the disease is Duchenne muscular dystrophy (gene: DMD), myotubular myopathy (gene: MTM1), spinal muscular atrophy (gene: SMA), glycogen storage disease type II (Pompe disease, gene: GAA), or cardiomyopathy. In some preferred embodiments, the disease is Duchenne muscular dystrophy (gene affected: DMD).

[0250] Further exemplary diseases include, but are not limited to, acid maltase deficiency (AMD), alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis (ALS), Andersen-Tawil syndrome, Becker muscular dystrophy (BMD), Becker myotonia congenita, Bethlem myopathy, carnitine deficiency, carnitine palmityltransferase deficiency (CPT deficiency), central core myopathy (CCD), centronuclear myopathy, Charcot-Marie-Tooth disease (CMT), congenital myasthenic syndrome (CMS), congenital myotonic dystrophy, Cori's disease (Debranchor's enzyme deficiency), Debranchor's enzyme deficiency, Dejerine-Sottas disease (DSD), dermatomyositis (DM), endocrine myopathy, Eulenberg's disease (congenital paramyotonia), Forbes' disease (Debranchor's enzyme deficiency), Friedreich's ataxia (FA), glycogen storage disease type 10, glycogen storage disease type 11, glycogen storage disease type 2, glycogen storage disease type 3, glycogen storage disease type 5, glycogen storage disease type 7, glycogen storage disease type 9, Gowers-Leyne distal myopathy, Hauptmann-Tannhäuser MD (Emery-Dreyfus muscular dystrophy), hereditary inclusion body myositis, hereditary motor and sensory neuropathy (Schizophrenia) Charcot-Marie-Tooth disease), hyperthyroid myopathy, hypothyroid myopathy, inclusion body myositis (IBM), hereditary myopathy, integrin-deficient congenital muscular dystrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome (LEMS), McArdle disease (phosphorylase deficiency), metabolic disorders of muscle, mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease, myo-oculocerebral disease, myasthenia gravis (MG), myoadenylate deaminase deficiency, myofibrillar myopathy, myophosphorylase deficiency, myotonia congenita ( MC), myotonic muscular dystrophy (MMD), myotubular myopathy (MTM or MM), nemaline myopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy (OPMD), congenital paramyotonia, Pearson syndrome, periodic paralysis, peroneal muscular atrophy (Charcot-Marie-Tooth disease), phosphofructokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, phosphorylase deficiency, polymyositis (PM), Pompe disease (acid maltase deficiency), progressive external ophthalmoplegia (PEO),These include rod body disease (nemaline myopathy), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), Steinert disease (myotonic muscular dystrophy), Tarui disease (phosphofructokinase deficiency), Thomsen disease (myotonia congenita), Ullrich congenital muscular dystrophy, Walker-Warburg syndrome (congenital muscular dystrophy), Welander distal myopathy, and ZASP-related myopathy.

[0251] In some preferred embodiments, the disease is a myocardial disease. In some preferred embodiments, the disease is congestive heart failure. In some preferred embodiments, the disease is a congenital heart defect.

[0252] In some preferred embodiments, the disease is a cardiac or skeletal muscle disease, hi some embodiments, the disease is Duchenne muscular dystrophy (genetic: DMD).

[0253] In some preferred embodiments, the disease is Danon disease, an X-linked dominant disorder associated with hypertrophic cardiomyopathy, skeletal muscle weakness, and intellectual disability.

[0254] Due to its small size (less than 390 bp), the promoter according to the present invention is particularly useful in diseases requiring the expression of a large expression product (e.g., a large transgene). The promoter according to the present invention is particularly useful in diseases requiring the reduction of expression of the expression product in non-muscle tissues and cells (e.g., the liver). The promoter according to the present invention is particularly useful in diseases requiring the expression of a large expression product and requiring the reduction of hepatic expression of the expression product.

[0255] Expression product The expression product can be any product whose expression is desired. Expression can be desired in skeletal muscle and / or cardiac muscle. The expression product can be a gene. The expression product can be a gene encoding a desired gene expression product (e.g., a product of interest), such as a polypeptide (protein) or RNA. The expression product can be a transgene. The expression product can be a protein or polypeptide. The expression product can be a nucleic acid sequence. The expression product can be a therapeutic expression product.

[0256] In some preferred embodiments, the expression product is lysosome-associated membrane protein type 2 (LAMP2). Mutations in LAMP2 cause Danon disease, and providing a non-diseased copy of LAMP2 may be desirable in treating Danon disease.

[0257] In some preferred embodiments, the expression product is fukutin-related protein (FKRP). Mutations in FKRP cause limb-girdle muscular dystrophy-dystroglycanopathy (LGMD), and providing a replacement, non-disease copy of FKRP may be desirable in treating LGMD. In some embodiments, the expression product, preferably a gene, is a non-disease-mediated variant, such as DMD, GALGT2, SMA, GAA, MTM1, TTID, LMNA, CAV3, DNAJB6, DES, TNP03, HNRPDL, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, FKRP, TTN, POMT1, AN05, FKTN, POMT2, PFEC1, DYSF, TIA1, M The gene encodes a wild-type variant of at least one human gene selected from the group consisting of YH7, DUX4, SMCHD, PABPN1, DMPK, ZNF9, CFCN1, SCN4A, MYH7, TNNT2, TPM1, MYBPC3, PRKAG2, TNNI3, MYF3, TTN, MYF2, ACTC1, CSRP3, TGFB3, RYR2, TMEM43, DES, DSP, PKP2, DSG2, DSC2, JUP, and HYPP. In some preferred embodiments, the gene is DMD.

[0258] In some embodiments, the expression product is dystrophin (including microdystrophin), β1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthetase I, α-1 antitrypsin, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, porphobilinase, or a combination of these. These include acetylcholine deaminase, cystathione β-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, and cystic fibrosis transmembrane conductance regulator (CFTR).

[0259] In some embodiments, the expression product may be a dystrophin protein. Mutations in the DMD gene that impair the function of the dystrophin protein cause Becker muscular dystrophy or Duchenne muscular dystrophy, X-linked recessive muscular dystrophy disorders characterized by muscle weakness. Providing a non-disease copy of dystrophin may be desirable in the treatment of Becker muscular dystrophy and / or Duchenne muscular dystrophy. The DMD gene is the largest gene known in humans (approximately 2.4 million base pairs). Therefore, the full-length DMD gene is too large to be packaged into some capacity-limited viral vectors, such as AAV vectors. A truncated version of the DMD gene (called mini-dystrophin) has been used to address this issue. Nevertheless, even mini-dystrophin is quite large (e.g., 3.5-4 kB), still making it difficult to package with AAV. Thus, synthetic muscle-specific promoters of short length (e.g., less than 400 nucleotides, less than 350 nucleotides, preferably less than 300 nucleotides, even more preferably less than 290 nucleotides, and most preferably less than 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 70, 68 nucleotides) may be particularly preferred in expression cassettes in which the sequence encoding the expression product is the DMD gene or a miniature version of the DMD gene (mini-dystrophin). In some preferred embodiments, the expression product is dystrophin, preferably mini-dystrophin.

[0260] In some preferred embodiments, the expression product is a smaller version of utrophin (e.g., a mini-utrophin or a micro-utrophin). In some preferred embodiments, the expression product is a smaller version of dystrophin (e.g., a mini-dystrophin or a micro-dystrophin). In some embodiments, the expression product is Song Y, Morales L, Malik AS, Mead AF, Greer CD, Mitchell MA, Petrov MT, Su LT, Choi ME, Rosenblum ST, Lu Stedman HH. Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models. Nat Med. 2019 Oct;25(10):1505~1511. doi: 10.1038 / s41591-019-0594-0. Epub 2019 Oct 7. PMID: 31591596; PMCID: PMC7274039 and particularly Extended Data Figure 1, which is incorporated herein by reference. In some embodiments, the expression product may be micro-dystrophin, as disclosed in Duan D. Micro-Dystrophin Gene Therapy Goes Systemic in Duchenne Muscular Dystrophy Patients. Hum Gene Ther. 2018 Jul;29(7):733-736. doi: 10.1089 / hum.2018.012. Epub 2018 Apr 5. PMID: 29463117; PMCID: PMC6066190, which is incorporated herein by reference.In some embodiments, the expression product may be micro-utrophin, as disclosed in Duan D. Micro-utrophin Therapy for Duchenne Muscular Dystrophy. Mol Ther. 2019 Nov 6;27(11):1872-1874. doi: 10.1016 / j.ymthe.2019.10.011. Epub 2019 Oct 22. PMID: 31653398; PMCID: PMC6838911 and particularly in FIG. 1 , which is incorporated herein by reference. In some embodiments, the expression product may be microutrophin, as disclosed in Starikova, AV, Skopenkova, VV, Polikarpova, AV et al. Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression. Sci Rep 12, 848 (2022) (https: / / doi.org / 10.1038 / s41598-022-04892-x) and particularly in FIG. 1, which is incorporated herein by reference. In some embodiments, the expression product may be micro-utrophin, as disclosed in Kennedy TL, Guiraud S, Edwards B, Squire S, Moir L, Babbs A, Odom G, Golebiowski D, Schneider J, Chamberlain JS, Davies KE. Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2 / mdx Mice. Mol Ther Methods Clin Dev. 2018 Oct 16;11:92-105. doi: 10.1016 / j.omtm.2018.10.005. PMID: 30417024; PMCID: PMC6216100, which is incorporated herein by reference.In some embodiments, the expression product may be microutrophin, as disclosed in Banks GB, Chamberlain JS, Odom GL. Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects. PLoS Genet. 2020 Nov 11;16(11):e1009179. doi: 10.1371 / journal.pgen.1009179. PMID: 33175853; PMCID: PMC7682874 and particularly in FIG. 1 , which is incorporated herein by reference. In some embodiments, the expression product may be micro-dystrophin as disclosed in Howard ZM, Dorn LE, Lowe J, Gertzen MD, Ciccone P, Rastogi N, Odom GL, Accornero F, Chamberlain JS, Rafael-Fortney JA. Micro-dystrophin gene therapy prevents heart failure in an improved Duchenne muscular dystrophy cardiomyopathy mouse model. JCI Insight. 2021 Apr 8;6(7):e146511. doi: 10.1172 / jci.insight.146511. PMID: 33651713; PMCID: PMC8119181, and particularly the methods therein, which are incorporated herein by reference.In some embodiments, the expression product can be micro-dystrophin as disclosed in Mendell JR, Sahenk Z, Lehman K, et al., Assessment of Systemic Delivery of rAAVrh74.MHCK7.micro-dystrophin in Children With Duchenne Muscular Dystrophy: A Nonrandomized Controlled Trial. JAMA Neurol. 2020;77(9):1122-1131. doi:10.1001 / jamaneurol.2020.1484, and particularly in the introduction, which is incorporated herein by reference. In some embodiments, the expression product can be micro-dystrophin as disclosed at https: / / www.clinicaltrials.gov / ct2 / show / NCT03368742, which is incorporated herein by reference.

[0261] Still other expression products include enzymes useful in enzyme replacement therapy, which are useful in a variety of conditions resulting from deficient enzyme activity. For example, enzymes containing mannose-6-phosphate may be utilized in therapy for lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)).

[0262] In some embodiments, exemplary polypeptide expression products include neuroprotective and anti-angiogenic polypeptides. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), urturing, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-β), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotrophin 6 (NT-6), epidermal growth factor (EGF), pigment epithelium-derived factor (PEDF), Wnt polypeptides, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibodies, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and members of the hedgehog family (such as sonic hedgehog, Indian hedgehog, and desert hedgehog).

[0263] In some embodiments, the expression product is a hormone and growth and differentiation factor, such as, but not limited to, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), and the like. F-II), any one of the transforming growth factor α superfamily, for example, TGFα, activin, inhibin, or any one of the bone morphogenetic proteins (BMP) BMP1 to 15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase, etc.

[0264] In some embodiments, expression products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons (α, β, and γ), stem cell factor, flk-2 / flt3 ligand, and the like. Gene products produced by the immune system are also useful in the present invention. In some embodiments, expression products include immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. In some embodiments, the expression products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

[0265] In some embodiments, the expression product comprises any one of a receptor for a hormone, growth factor, cytokine, lymphokine, regulatory protein, and immune system protein. Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and / or lipid modulation, such as low-density lipoprotein (LDL) receptor, high-density lipoprotein (HDL) receptor, very-low-density lipoprotein (VLDL) receptor, and scavenger receptors. The present invention also encompasses the use of gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptor and estrogen receptor, vitamin D receptor, and other nuclear receptors. Further useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MyoD, and myogenin, ETS box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding proteins, STATs, GATA box-binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0266] In some embodiments, expression products include those used to treat hemophilia, e.g., hemophilia B (including factor IX) and hemophilia A (factor VIII and its variants, e.g., including heterodimeric light and heavy chains and the B deleted domain; U.S. Patent Nos. 6,200,560 and 6,221,349).

[0267] In some embodiments, the expression product can be a modulator of phosphatase activity, e.g., type 1 phosphatase activity. The modulator can be a protein that inhibits phosphatase activity, e.g., type 1 phosphatase activity. The modulator can be a nucleic acid that increases expression of an endogenous nucleic acid encoding a protein that inhibits phosphatase activity, such as a transcription factor. The modulator can be a regulatory sequence integrated in or near the endogenous nucleic acid encoding the protein that inhibits phosphatase activity. The modulator can be a nucleic acid that can provide a nucleic acid modulator of gene expression, such as an siRNA.

[0268] In some embodiments, the expression product can be an inhibitor of protein phosphate 1 (PP1), e.g., an I-1 polypeptide. Phosphatase inhibitor 1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatase. Increasing I-1 levels or activity can restore beta-adrenergic responsiveness in dysfunctional human cardiomyocytes. Suitably, the I-1 protein can be constitutively active, such as an I-1 protein in which threonine 35 is replaced with glutamic acid instead of aspartic acid. The expression product can be one or more of the following inhibitors selected from: phosphatase inhibitor 2 (PP2); okadaic acid or calyculin; and nippl, an endogenous nuclear inhibitor of protein phosphatase 1.

[0269] In some embodiments, the expression product can be any protein that regulates cardiac activity, such as a phosphatase type 1 inhibitor, such as I-1, or a sarcoplasmic reticulum Ca2+ ATPase (SERCA), such as SERCA1 (e.g., 1a or 1b), SERCA2 (e.g., 2a or 2b), or SERCA3.

[0270] In some embodiments, the expression product can be a nucleic acid sequence encoding a mutant form of phosphatase inhibitor 1 protein, wherein the mutant form contains at least one amino acid at a position that is a PKC-α phosphorylation site in the wild-type, and the at least one amino acid is constitutively unphosphorylated or mimics the unphosphorylated state in the mutant form. The expression product can be adenylyl cyclase 6 (AC6, also known as adenylyl cyclase VI), S100A1, β-adrenergic receptor kinase-ct (βARKct), sarcoplasmic / endoplasmic reticulum (SR) Ca-ATPase (SERCA2a), IL-18, VEGF, VEGF activator, urocortin, and B-cell lymphoma 2 (Bcl2)-associated anthanogen 3 (BAG3).

[0271] In some embodiments, the expression product can be an inhibitor of a cytokine, such as an IL-18 inhibitor. The expression product can encode a β-adrenergic signaling protein (β-ASP) (including a β-adrenergic receptor (β-Ar), a G protein receptor kinase inhibitor (GRK inhibitor), and adenylyl cyclase (Ac)) to enhance cardiac function.

[0272] In some embodiments, the expression product may be an angiogenic protein. Angiogenic proteins promote the development and differentiation of blood vessels. Examples of angiogenic proteins include members of the fibroblast growth factor (FGF) family, such as aFGF (FGF-1), bFGF (FGF-2), FGF-4 (also known as "hst / KS3"), FGF-5, and FGF-6, the vascular endothelial growth factor (VEGF) family, the platelet-derived growth factor (PDGF) family, the insulin-like growth factor (IGF) family, and the like.

[0273] In some embodiments, the expression product comprises a non-naturally occurring polypeptide, such as a chimeric or hybrid polypeptide having a non-naturally occurring amino acid sequence containing insertions, deletions, or amino acid substitutions.

[0274] Further suitable expression products include microRNA (miRNA), interfering RNA, antisense RNA, ribozymes, and aptamers.

[0275] In some preferred embodiments, the expression product may be an inhibitor of protein phosphate 1 (PP1).

[0276] In some embodiments of the present invention, the expression product is useful for gene editing, such as a gene encoding a site-specific nuclease, e.g., a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats system (CRISPR-Ca). Preferably, the site-specific nuclease is adapted to edit a desired target genomic locus, making a cut (typically a site-specific double-strand break) followed by repair via non-homologous end joining (NHEJ) or homology-dependent repair (HDR), resulting in the desired edit. The edit can be the partial or complete repair of a dysfunctional gene, or the knockdown or knockout of a functional gene. Alternatively, the edit can be via base editing or prime editing, using a suitable system known in the art.

[0277] The expression product can be a gene. The gene typically encodes a desired gene expression product, such as a polypeptide (protein) or RNA. The gene can be a full-length cDNA or genomic DNA sequence, or any fragment, subunit, or variant thereof that has at least some desired biological activity.

[0278] If the gene encodes a protein, it can be essentially any type of protein. By way of non-limiting example, the protein can be an enzyme, an antibody or antibody fragment (e.g., a monoclonal antibody), a viral protein (e.g., REP-CAP, REV, VSV-G, or RD114), a therapeutic protein, or a toxic protein (e.g., caspase 3, 8, or 9).

[0279] In some preferred embodiments, the gene encodes a therapeutic expression product, preferably a therapeutic polypeptide suitable for use in the treatment of a disease or condition associated with aberrant gene expression in muscle, optionally cardiac and / or skeletal muscle.

[0280] The expression product may be a therapeutic expression product. The therapeutic expression product may be useful in the treatment of cardiovascular or cardiac diseases and disorders, such as heart attack or CHF. In some preferred embodiments, the therapeutic expression product may be useful in the treatment of DMD. In some preferred embodiments, the therapeutic expression product may be useful in the treatment of Danon disease. The expression product may be a LAMP2 protein. The therapeutic expression product may be any protein that regulates cardiac activity, such as a phosphatase type 1 inhibitor, such as I-1, or a sarcoplasmic reticulum Ca2+ ATPase (SERCA), such as SERCA1 (e.g., 1a or 1b), SERCA2 (e.g., 2a or 2b), or SERCA3.

[0281] The therapeutic expression product can be a modulator of phosphatase activity, such as type 1 phosphatase activity. The modulator can be a protein that inhibits phosphatase activity, such as type 1 phosphatase activity. The modulator can be a nucleic acid that increases expression of an endogenous nucleic acid encoding a protein that inhibits phosphatase activity, such as a transcription factor. The modulator can be a regulatory sequence integrated in or near the endogenous nucleic acid encoding the protein that inhibits phosphatase activity. The modulator can be a nucleic acid that can provide a nucleic acid modulator of gene expression, such as an siRNA.

[0282] The therapeutic expression product can be an inhibitor of protein phosphate 1 (PP1), e.g., an I-1 polypeptide. Phosphatase inhibitor 1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatase. Increasing I-1 levels or activity can restore beta-adrenergic responsiveness in dysfunctional human cardiomyocytes. Suitably, the I-1 protein can be constitutively active, such as an I-1 protein in which threonine 35 is replaced with glutamic acid instead of aspartic acid. The therapeutic expression product can be any one or more of the following inhibitors selected from: phosphatase inhibitor 2 (PP2); okadaic acid or calyculin; and nippl, an endogenous nuclear inhibitor of protein phosphatase 1. In some preferred embodiments, the expression cassette comprises a muscle-specific promoter operably linked to an inhibitor of protein phosphate 1 (PP1). Type 1 phosphatases include, but are not limited to, PP1cα, PP1cβ, PP1cδ, and PP1cγ.

[0283] The therapeutic expression product can be a nucleic acid sequence encoding a mutant form of phosphatase inhibitor 1 protein, where the mutant form contains at least one amino acid at a position that is a PKC-α phosphorylation site in the wild-type, and the at least one amino acid is constitutively unphosphorylated or mimics the unphosphorylated state in the mutant form. The therapeutic expression product can be adenylyl cyclase 6 (AC6, also called adenylyl cyclase VI), S100A1, β-adrenergic receptor kinase-ct (βARKct), sarcoplasmic / endoplasmic reticulum (SR) Ca-ATPase (SERCA2a), IL-18, VEGF, VEGF activator, urocortin, and B-cell lymphoma 2 (Bcl2)-associated anthanogen 3 (BAG3).

[0284] The therapeutic expression product can be an inhibitor of a cytokine, such as an IL-18 inhibitor. The therapeutic expression product can encode a β-adrenergic signaling protein (β-ASP), such as a β-adrenergic receptor (β-Ar), a G protein receptor kinase inhibitor (GRK inhibitor), and adenylyl cyclase (Ac), to enhance cardiac function.

[0285] The therapeutic expression product can be an angiogenic protein. Angiogenic proteins promote the development and differentiation of blood vessels. Examples of angiogenic proteins include members of the fibroblast growth factor (FGF) family, e.g., aFGF (FGF-1), bFGF (FGF-2), FGF-4 (also known as "hst / KS3"), FGF-5, and FGF-6, the vascular endothelial growth factor (VEGF) family, the platelet-derived growth factor (PDGF) family, the insulin-like growth factor (IGF) family, and the like.

[0286] Definitions and General Aspects While the making and use of various embodiments of the invention are described in more detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a variety of specific contexts. The specific embodiments described herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0287] The discussion of the background to the invention herein is included to explain the context of the invention and is not an admission that any of the material mentioned was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0288] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of documents specifically mentioned herein are incorporated by reference.

[0289] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell physiology, cell culture, molecular biology, transgenic biology, bacteriology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and Sons Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd Edition, (Sambrook et al. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J. Gait ed., 1984); U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins ed., 1984); Transcription and Translation (Hames and Higgins ed., 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Series, Methods in Enzymology (Abelson and Simon ed. -in-chief, Academic Press, Inc., New York), in particular, see volumes 154 and 155 (eds. Wu et al.) and 185, "Gene Expression Technology" (ed. Goeddel); Gene Transfer Vectors For Mammalian Cells (eds. Miller and Calos, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987); Handbook of Experimental Immunology, volumes I-IV (eds. Weir and Blackwell, 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0290] To facilitate understanding of the present invention, several terms are defined or explained below. Terms used herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "the" do not refer only to a singular entity, but encompass general classes of which specific examples of the class may be used for purposes of illustration. While terms herein are used to describe particular embodiments of the present invention, their use does not limit the present invention except as outlined in the claims.

[0291] The term "muscle" is well understood by those skilled in the art. Preferably, the muscle is skeletal muscle (including the diaphragm) or cardiac muscle. Preferably, the muscle is muscle of a vertebrate, more preferably a mammal, even more preferably a human subject. Preferably, the muscle is striated muscle. The synthetic promoters of the present invention may be active in skeletal muscle and / or cardiac muscle.

[0292] The term "muscle cell" or "myocyte" refers to a cell found in muscle (muscle tissue) or a cell derived from muscle tissue. Muscle cells can be primary cells or cell lines (e.g., C2C12 or H2K cells (skeletal muscle cell lines) or H9C2 cells (cardiac cell line)). Muscle cells can be in vivo (e.g., muscle tissue) or in vitro (e.g., cell culture). Myocytes found in muscle tissue are typically long, tubular cells that develop from myoblasts to form muscle in a process known as myogenesis. As used herein, the term muscle cell or myocyte includes myocytes derived from skeletal muscle and myocytes derived from cardiac muscle (cardiomyocytes). The synthetic promoters of the present invention can be active in skeletal muscle cells and / or cardiac muscle cells.

[0293] The term "cis-regulatory element" or "CRE" is well known to those skilled in the art and refers to nucleic acid sequences such as enhancers, promoters, insulators, and silencers that can regulate or modulate the transcription of adjacent genes (i.e., in cis). CREs are found near the genes they control. CREs typically regulate gene transcription by binding to TFs, i.e., they contain TFBSs. A single TF can bind to many CREs and thus control the expression of many genes (pleiotropy). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene they regulate. "Enhancers" in this context are CREs that enhance (i.e., upregulate) the transcription of genes with which they are operatively associated and can be found upstream, downstream, or even within introns of the genes they regulate. Multiple enhancers can function in concert to control the transcription of a single gene. "Silencers" in this context refer to CREs that bind to TFs called repressors, which function to prevent or downregulate gene transcription. The term "silencer" can also refer to a region in the 3' untranslated region of a messenger RNA that binds to a protein that represses translation of that mRNA molecule, although this usage differs from its use in describing a CRE. Generally, a CRE of the present invention is a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) enhancer element (often referred to as a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) CRE, or a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) CRE enhancer, etc.). In some embodiments, CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) are muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) enhancer elements.In this context, it is preferred that the CRE is located within 2500 nucleotides of the transcription start site (TSS), more preferably within 2000 nucleotides of the TSS, more preferably within 1500 nucleotides of the TSS, and suitably within 1000, 750, 500, 250, 200, 150, or 100 nucleotides of the TSS. CREs of the present invention are preferably relatively short, preferably 500 nucleotides or less in length; for example, they may be 400, 300, 200, 175, 150, 90, 80, 70, 60, or 50 nucleotides or less in length. Typically, the CREs of the present invention are provided in combination with an operably linked promoter element, which may be a minimal promoter or a proximal promoter. The CREs of the present invention enhance the muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) activity of the promoter element. In any combination of CREs or functional variants thereof disclosed herein, some or all of the listed CREs and promoter elements may be suitably located adjacent to each other in the promoter (i.e., without any intervening CREs or other regulatory elements). The CREs may be contiguous or non-contiguous (i.e., they may be located directly adjacent to each other, or they may be separated by a spacer or other sequence). The CREs may be in any order. In some preferred embodiments, the CREs or functional variants thereof are provided in the listed order and adjacent to each other. For example, a synthetic muscle-specific synthetic promoter may include CRE0145 immediately upstream of DES_MT_enhancer_48bp, etc. In some embodiments, it is preferred that some or all of the CREs are contiguous.

[0294] The term "cis-regulatory module" or "CRM" refers to a functional regulatory nucleic acid module that typically includes two or more CREs or other regulatory elements; in the present invention, a CRE is typically a muscle-specific, cardiac-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) enhancer, and thus, a CRM is a synthetic muscle-specific, cardiac-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) regulatory nucleic acid sequence. Thus, in the present application, a CRM typically includes multiple muscle-specific, cardiac-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) CREs and, where appropriate, other regulatory elements. Typically, multiple CREs within a CRM function together (e.g., additively or synergistically) to enhance the transcription of a gene with which a synthetic promoter comprising the CRM is operably associated. Within a CRM, there is considerable room for shuffling (i.e., rearranging), inverting (i.e., reverse orientation), and altering the spacing of CREs. Thus, functional variants of CRMs of the present invention include, inter alia, variants of the referenced CRMs in which the CREs therein have been shuffled and / or inverted and / or the spacing between the CREs has been altered. CRMs may contain other regulatory elements, such as inducible or repressible elements, intronic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, segments of terminal repeats, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, and splicing elements, provided that they do not render the CRM substantially non-functional.

[0295] As used herein, the term "promoter" refers to a region of DNA that is generally located upstream of a nucleic acid sequence to be transcribed and is necessary for transcription to occur, i.e., initiates transcription. A promoter allows for the appropriate activation or repression of transcription of the coding sequence under its control. A promoter typically contains specific sequences that are recognized and bound by multiple TFs. TFs bind to the promoter sequence, resulting in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene. A variety of promoters are known in the art.

[0296] In some cases, the term "promoter" or "composite promoter" is used herein to refer to a combination of a promoter and one or more additional regulatory elements. In some cases, a promoter has a desired expression profile, and the addition of one or more additional regulatory elements modulates the expression profile. Modulation means increasing, decreasing, or maintaining the level of activity in any particular cell type or tissue. In some cases, additional regulatory elements may be located immediately downstream of the transcription start site (TSS), such as introns. Such sequences downstream of the TSS may contribute to regulating expression at the transcription and / or translation levels. In some cases, additional regulatory elements may be located between the CRE and promoter elements, such as detargeting elements or liver detargeting elements. Such sequences located between the CRE and promoter elements may contribute to lower expression in certain tissues or cell types, for example, in the liver or hepatocytes.

[0297] As used herein, the term "synthetic promoter" refers to a promoter that does not exist in nature. In the context of the present invention, a synthetic promoter typically comprises a CRE and / or CRM of the present invention operably linked to a promoter element, such as a minimal (or core) promoter or a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) proximal promoter. The CRE and / or CRM of the present invention serves to enhance muscle-specific, cardiac muscle-specific (or cardiac muscle-selective), or skeletal muscle-specific (or skeleton-selective) transcription of a gene operably linked to the synthetic promoter. While a portion of a synthetic promoter may exist in nature (e.g., a minimal promoter or one or more CREs within a promoter), a synthetic promoter as an entity does not exist in nature.

[0298] As used herein, a "minimal promoter" (also referred to as a "core promoter") refers to a typically short DNA segment that is inactive or nearly inactive by itself but can mediate transcription when combined with other transcriptional regulatory elements. Minimal promoter sequences can be derived from a variety of different sources, such as prokaryotic and eukaryotic genes. Examples of minimal promoters include the desmin minimal promoter, the dopamine β-hydroxylase gene minimal promoter, the cytomegalovirus (CMV) immediate-early gene minimal promoter (CMV-MP), and the herpes thymidine kinase minimal promoter (MinTK). Minimal promoters typically contain a transcription start site (TSS) and its immediate upstream elements, a binding site for RNA polymerase II, and a general transcription factor binding site (often a TATA box). Minimal promoters may also contain some elements downstream of the TSS, but these typically function poorly in the absence of additional regulatory elements.

[0299] As used herein, the term "proximal promoter" refers to the proximal sequence upstream of a gene, which tends to include a minimal promoter and at least some additional regulatory sequences, typically key regulatory elements. Proximal promoters often extend up to about 250 base pairs upstream of the TSS and include specific TFBSs. Proximal promoters may also include one or more regulatory elements downstream of the TSS, such as UTRs or introns. In this case, the proximal promoter may be a naturally occurring muscle-specific, cardiac muscle-specific (or cardiac muscle-selective), or skeletal muscle-specific (or skeletal-selective) proximal promoter that can be combined with one or more CREs or CRMs of the present invention. However, proximal promoters can also be synthetic.

[0300] As used herein, "promoter element" refers to either a minimal promoter or a proximal promoter as defined above. In the context of the present invention, a promoter element is typically combined with a CRM, or one or more CREs, and optionally one or more additional regulatory elements, to provide a synthetic muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) promoter of the present invention.

[0301] A "functional variant" of a CRE, CRM, promoter element, promoter, or other regulatory nucleic acid in the context of the present invention is a variant of the reference sequence that maintains the ability to function similarly to a reference sequence, e.g., a muscle-specific, cardiac-specific (or cardiac-selective), or skeletal-specific (or skeletal-selective) CRE, a muscle-specific, cardiac-specific (or cardiac-selective), or skeletal-selective CRM, a muscle-specific, cardiac-specific (or cardiac-selective), or skeletal-selective promoter element, or a muscle-specific, cardiac-specific (or cardiac-selective), or skeletal-selective promoter. A "functional variant" of an additional regulatory element, detargeting element, liver-detargeting element, or intron is a variant of the reference sequence that maintains the ability to function similarly to a reference sequence, e.g., an additional regulatory element, detargeting element, liver-detargeting element, or intron. Alternative terms for such functional variants include "biological equivalent" or "equivalent."

[0302] It will be understood that the ability of a given CRE, CRM, promoter, or other regulatory sequence to function as a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) enhancer is largely determined by the sequence's ability to bind to the same muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) TFs that bind to the reference sequence. Thus, in most cases, a functional variant of a CRE, CRM, promoter, or other regulatory sequence will contain TFBSs for most or all of the same TFs as the reference CRE, CRM, promoter, or other regulatory sequence. It is preferred, but not required, that the TFBSs of the functional variant be in the same relative position (i.e., order and overall location) as the reference CRE, CRM, promoter, or other regulatory sequence. It is also preferred, but not required, that the TFBSs of the functional variant be in the same orientation as the reference sequence (it will be noted that in some cases, the TFBSs may be present in the opposite orientation, e.g., as the reverse complement of a sequence in the reference sequence). It is also preferred, but not required, that the TFBSs of the functional variant be on the same strand as the reference sequence. Thus, in a preferred embodiment, the functional variant contains TFBSs for the same TFs in the same order, at the same positions, in the same orientation, and on the same strand as the reference sequence. It will also be understood that sequences between TFBSs (sometimes referred to as spacer sequences, etc.) are not critical to the function of the CRE, CRM, promoter, or other regulatory sequence. Such sequences can typically vary significantly, and their length can also vary. However, in a preferred embodiment, the spacing (i.e., the distance between adjacent TFBSs) is substantially the same in the functional variant as in the reference sequence (e.g., it does not vary by more than 20%, preferably does not vary by 10% or less, and more preferably is approximately the same).It will be apparent that in some cases, a functional variant of a CRE, CRM, promoter, or other regulatory sequence may exist in the opposite orientation, e.g., it may be the reverse complement of the CRE, CRM, promoter, or other regulatory sequence described above, or a variant thereof.

[0303] The level of sequence identity between a functional variant and a reference sequence can also be an indicator of maintained functionality. A high level of sequence identity in a CRE, CRM, or promoter TFBS is generally more important than sequence identity in a spacer sequence (where there is little or no need for sequence conservation). However, given that the sequence of a functional TFBS need not exactly match the consensus sequence, it will be appreciated that a considerable degree of sequence variation can be tolerated even within a TFBS.

[0304] The ability of one or more TFs to bind to TFBSs in a given functional variant can be determined by any relevant means known in the art, including, but not limited to, electromobility shift assays (EMSA), binding assays, chromatin immunoprecipitation (ChIP), and ChIP-sequencing (ChIP-seq). In a preferred embodiment, the ability of one or more TFs to bind to a given functional variant is determined by EMSA. Methods for performing EMSA are well known in the art. A suitable approach is described by Sambrook et al., cited above. Many relevant articles describing this procedure are available, for example, Hellman and Fried, Nat Protoc. 2007; 2(8): 1849-1861.

[0305] "Muscle-specific" or "muscle-specific expression" refers to the ability of a cis-regulatory element, cis-regulatory module, promoter element, or promoter to enhance or drive expression of a gene preferentially or predominantly in muscle cells (or muscle-derived cells) compared to other tissues (e.g., liver, kidney, spleen, heart, lung, brain). The expression of the gene can be in the form of mRNA or protein. In preferred embodiments, muscle-specific expression is negligible in other (i.e., non-muscle) tissues or cells, i.e., expression is highly muscle-specific. For example, expression in muscle cells is at least 75%, 80%, 85%, 90%, or 95% as opposed to other cells. "Myocardial-specific," "cardiac-selective," or "myocardial-specific expression" refers to the ability of a cis-regulatory element, cis-regulatory module, promoter element, or promoter to enhance or drive expression of a gene preferentially or predominantly in cardiac muscle compared to other tissues (e.g., liver, kidney, spleen, heart, lung, brain) or skeletal muscle tissue. "Skeletal muscle-specific," "skeletal-selective," or "skeletal muscle-specific expression" refers to the ability of a cis-regulatory element, cis-regulatory module, promoter element, or promoter to enhance or drive expression of a gene preferentially or predominantly in skeletal muscle compared to other tissues (e.g., liver, kidney, spleen, heart, lung, brain) or cardiac muscle tissue. There may be instances where a lesser degree of specificity is desired, and this is also part of the invention.

[0306] "CNS-specific" or "CNS-specific expression" refers to the ability of a promoter to enhance or drive expression of a gene preferentially or predominantly in central nervous system (CNS) cells (or CNS-derived cells) compared to other tissues (e.g., liver, kidney, spleen, heart, lung, muscle, and brain). The expression of the gene can be in the form of mRNA or protein. In preferred embodiments, CNS-specific expression is negligible expression in other (i.e., non-CNS) tissues or cells, i.e., expression is highly CNS-specific. For example, expression in CNS cells as opposed to other cells is at least 75%, 80%, 85%, 90%, or 95%. A lesser degree of specificity may be desired, and is also part of the invention.

[0307] "Kidney-specific" or "kidney-specific expression" refers to the ability of a promoter to enhance or drive expression of a gene preferentially or predominantly in kidney cells (or kidney-derived cells) compared to other tissues (e.g., liver, CNS, spleen, heart, lung, muscle, and brain). The expression of the gene can be in the form of mRNA or protein. In preferred embodiments, kidney-specific expression is negligible expression in other (i.e., non-kidney) tissues or cells, i.e., expression is highly kidney-specific. For example, expression in kidney cells as opposed to other cells is at least 75%, 80%, 85%, 90%, or 95%. A lower degree of specificity may be desired and is also part of the invention.

[0308] "Lung-specific" or "lung-specific expression" refers to the ability of a promoter to enhance or drive expression of a gene preferentially or predominantly in lung cells (or lung-derived cells) compared to other tissues (e.g., liver, CNS, spleen, heart, muscle, and brain). The expression of the gene can be in the form of mRNA or protein. In preferred embodiments, lung-specific expression is negligible in other (i.e., non-lung) tissues or cells, i.e., expression is highly lung-specific. For example, expression in lung cells as opposed to other cells is at least 75%, 80%, 85%, 90%, or 95%. A lower degree of specificity may be desired and is also part of the present invention.

[0309] The ability of a CRE, CRM, or promoter to function as a muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) CRE, CRM, or promoter can be easily evaluated by one skilled in the art. Thus, one skilled in the art can easily determine whether a particular CRE, CRM, or promoter listed above remains functional (i.e., whether it is a functional variant as defined above). For example, any given CRE or CRM to be evaluated can be operably linked to a minimal promoter (e.g., located upstream of a CMV-MP), and the ability of the CRE or CRM to drive muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) expression of a gene (typically a reporter gene) can be measured. Alternatively, a variant of a CRE or CRM can be substituted for the reference CRE or CRM with a synthetic muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) promoter, and the effect on muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) expression driven by the modified promoter can be determined and compared to the unmodified form. Similarly, the ability of a promoter to drive muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeleton-selective) expression can be easily evaluated by one skilled in the art (e.g., as described in the Examples below). The expression level of a gene driven by a variant of the reference promoter can be compared to the expression level driven by the reference promoter. In some embodiments, a variant remains functional if the muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) expression level driven by the variant promoter is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the expression level driven by the reference promoter.Suitable nucleic acid constructs and reporter assays for assessing enhanced muscle-specific, cardiac muscle-specific (or heart-selective), or skeletal muscle-specific (or skeletal-selective) expression can be readily constructed, and the examples provided below demonstrate suitable methodologies.

[0310] Muscle-specificity, cardiac muscle-specificity (or cardiac selectivity), or skeletal muscle-specificity (or skeletal selectivity) can be identified when expression of a gene (e.g., a therapeutic gene or reporter gene) occurs preferentially or predominantly in muscle-derived cells (or muscle tissue), cardiac muscle-derived cells (or cardiac tissue), or skeletal muscle-derived cells (or skeletal tissue). Preferential or predominant expression can be defined, for example, when expression levels are higher, preferably significantly higher, in muscle-derived, cardiac muscle-derived, or skeletal muscle-derived cells than in other types of cells (i.e., non-muscle-derived, non-cardiac muscle-derived, or non-skeletal muscle-derived cells). For example, expression in muscle-derived, cardiac muscle-derived, or skeletal muscle-derived cells is suitably at least 1.2-fold, 1.5-fold, 2-fold, 4-fold, or 5-fold higher than in non-muscle, non-cardiac muscle, or non-skeletal muscle cells, and preferably at least 10-fold higher, and in some cases may be 50-fold or higher, than in non-muscle, non-cardiac muscle, or non-skeletal muscle cells. Conveniently, muscle-specific expression may be suitably demonstrated by comparing expression levels in a muscle cell line (e.g., a muscle-derived cell line such as C2C12 or H2K cells (skeletal muscle) or H9C2 cells (cardiac muscle)) compared to expression levels in a liver-derived cell line (e.g., Huh7 or HepG2), a kidney-derived cell line (e.g., HEK-293), a cervical tissue-derived cell line (e.g., HeLa), and / or a lung-derived cell line (e.g., A549). Cardiac muscle-specific or cardiac selective expression may suitably be demonstrated by comparison of expression levels in a cardiac muscle cell line (e.g., a cardiac muscle-derived cell line, such as, for example, H9C2) or primary cardiac muscle cells compared to expression levels in a liver-derived cell line (e.g., Huh7 or HepG2), a kidney-derived cell line (e.g., HEK-293), a cervical tissue-derived cell line (e.g., HeLa), a lung-derived cell line (e.g., A549), and / or a skeletal muscle-derived cell (e.g., C2C12 or H2K).Skeletal muscle-specific or skeletal-selective expression may suitably be demonstrated by comparison of expression levels in skeletal muscle-derived cells (e.g., C2C12 or H2K) or primary skeletal muscle cells compared to expression levels in liver-derived cell lines (e.g., Huh7 or HepG2), kidney-derived cell lines (e.g., HEK-293), cervical tissue-derived cell lines (e.g., HeLa), lung-derived cell lines (e.g., A549), and / or cardiac muscle-derived cell lines (e.g., H9C2).

[0311] The synthetic muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) promoters of the present invention preferably exhibit reduced expression in non-muscle-derived cells, preferably Huh7, HEK-293, HeLa, and / or A549 cells, when compared to non-tissue-specific promoters such as CMV-IE. The synthetic muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) promoters of the present invention preferably have 50% or less, preferably 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less activity than the CMV-IE promoter in non-muscle-derived cells (preferably Huh7, HEK-293, HeLa, and / or A549 cells). Generally, it is preferred to minimize expression in non-muscle-derived cells, although in some cases this may not be necessary. Even if a synthetic promoter of the invention has higher expression in, for example, one or two non-muscle cells, it can still be a muscle-specific promoter as long as it generally exhibits higher overall expression in various muscle cells versus non-muscle cells. In some embodiments, the muscle-specific promoter expresses a gene at least 25%, or at least 35%, or at least 45%, or at least 55%, or at least 65%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or any integer between 25% and 95% in muscle cells compared to non-muscle cells.

[0312] The synthetic muscle-specific promoters of the invention are preferably suitable for promoting expression in the muscle of a subject, e.g., for driving muscle-specific expression of a transgene, preferably a therapeutic transgene. The synthetic cardiac muscle-specific or cardiac-selective promoters of the invention are preferably suitable for promoting expression in the heart of a subject, e.g., for driving cardiac muscle-specific or cardiac-selective expression of a transgene, preferably a therapeutic transgene. The synthetic skeletal muscle-specific or skeletal-selective promoters of the invention are preferably suitable for promoting expression in the skeletal muscle of a subject, e.g., for driving skeletal muscle-specific or skeletal-selective expression of a transgene, preferably a therapeutic transgene. Preferred synthetic muscle-specific promoters of the invention are suitable for driving muscle-specific transgene expression and have an activity in muscle cells that is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, 120%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity of the CBA, CK7, or CK8 promoter. In some embodiments, the synthetic muscle-specific promoters of the invention are suitable for driving muscle-specific transgene expression at a level that is at least 100%, preferably 150%, 200%, 300%, or 500% of the activity of the CBA, spc5-12, CK7, or CK8 promoter. In some embodiments, the synthetic myocardium-specific or heart-selective promoters of the present invention are suitable for promoting myocardium-specific or heart-selective transgene expression at a level that is at least 100% of the activity of the Tnnt2 or Myl2 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the Tnnt2 or Myl2 promoter.In some embodiments, the synthetic skeletal muscle-specific or skeletal-preferred promoters of the invention are suitable for driving skeletal muscle-specific or skeletal-preferred transgene expression at a level that is at least 100% of the activity of the Tnnt2 or Myl2 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the spc5-12 promoter. Such muscle-specific expression is preferably specified in muscle-derived cells, such as C2C12 or H2K cells (skeletal muscle) or H9C2 cells (cardiac), or primary muscle cells (preferably primary human cardiomyocytes). The CK8 promoter is disclosed in Himeda, CL, Chen, X., Hauschka, SD (2011). Design and Testing of Regulatory Cassettes for Optimal Activity in Skeletal and Cardiac Muscles. In: Duan, D. (ed.) Muscle Gene Therapy. Methods in Molecular Biology, vol. 709. Humana Press. (https: / / doi.org / 10.1007 / 978-1-61737-982-6_1).

[0313] The synthetic muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) promoters of the invention may also be capable of promoting muscle-specific, cardiac muscle-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) expression of a gene at a level of at least 50%, 100%, 150%, or 200% compared to CMV-IE in muscle-derived cells (e.g., C2C12 or H2K cells (skeletal muscle) or H9C2 cells (cardiac muscle)).

[0314] The term "nucleic acid," as used herein, typically refers to an oligomer or polymer (preferably a linear polymer) of any length essentially composed of nucleotides. A nucleotide unit generally comprises a heterocyclic base, a sugar group, and at least one, e.g., one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include, inter alia, purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), which are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (methylated) non-natural bases or derivatized bases. Sugar groups may include, inter alia, pentose (pentofuranos) groups, such as ribose and / or 2-deoxyribose, commonly found in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids contemplated herein may include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may contain modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications of the phosphate group or sugar may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA-RNA hybrids. A nucleic acid can be naturally occurring, e.g., found in or isolated from nature; or it can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partially or wholly chemically or biochemically synthesized. A "nucleic acid" can be double-stranded, partially double-stranded, or single-stranded. If single-stranded, the nucleic acid can be the sense strand or the antisense strand. Furthermore, a nucleic acid can be circular or linear.

[0315] The term "isolated," when referring to a nucleic acid, means a nucleic acid molecule that lacks all or part of sequences that are normally associated with that nucleic acid molecule in nature; or a sequence that is present in nature but has heterologous sequences associated with it; or a molecule that has been separated from a chromosome.

[0316] The terms "identity" or "identical" and the like refer to sequence similarity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules. Sequence alignment and sequence identity determination can be performed, for example, using the Basic Local Alignment Search Tool (BLAST), originally described by Altschul et al., 1990 (J Mol Biol 215: 403-10), or the "Blast 2 sequences" algorithm, described by Tatusova and Madden, 1999 (FEMS Microbiol Lett 174: 247-250).

[0317] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed discussion of sequence alignment methods and homology calculation methods can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0318] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, such as the National Center for Biotechnology Information (Bethesda, MD), as well as on the Internet when used in conjunction with several sequence analysis programs. Instructions on how to determine sequence identity using this program are available on the Internet in the "Help" section of BLAST™. For comparison of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program can be used with default parameters. Nucleic acid sequences that are more similar to a reference sequence will exhibit a higher percentage of identity when assessed by this method. Typically, the percentage of sequence identity is calculated over the entire length of the sequence.

[0319] For example, the optimal global alignment is preferably found by the Needleman-Wunsch algorithm using the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2. The percentage of identity of the resulting optimal global alignment is preferably calculated by multiplying the ratio of the number of aligned bases to the total length of the alignment (the alignment length includes both matches and mismatches) by 100.

[0320] The term "hybridize" refers to the annealing of two at least partially complementary nucleotide sequences in a hybridization process. To allow hybridization to occur, complementary nucleic acid molecules are generally thermally or chemically denatured to dissolve the duplex into two single strands and / or to remove hairpins or other secondary structures from single-stranded nucleic acids. Hybridization strategies are influenced by conditions such as temperature, salt concentration, and the composition of the hybridization buffer. Conventional hybridization conditions are described, for example, in Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, CSH, New York. However, those skilled in the art will understand that numerous different hybridization conditions can be designed depending on the known or predicted homology and / or length of the nucleic acid sequences. High stringency conditions for hybridization include elevated temperatures and / or low sodium / salt concentrations (salts include, for example, NaCl and sodium citrate) and / or the inclusion of formamide in the hybridization buffer and / or reduced concentrations of compounds such as SDS (sodium dodecyl sulfate detergent) in the hybridization buffer and / or the elimination of compounds such as dextran sulfate or polyethylene glycol from the hybridization buffer. As a non-limiting example, typical salt and temperature conditions for stringent hybridization are 1x SSC, 0.5% SDS at 65°C. The abbreviation SSC refers to the buffer used in nucleic acid hybridization solutions. One liter of 20x (20x concentrated) stock SSC buffer solution (pH 7.0) contains 175.3 g of sodium chloride and 88.2 g of sodium citrate. A typical time to achieve hybridization is 12 hours.

[0321] The term "transcription factor binding site" (TFBS) is well known in the art. It will be apparent to one of skill in the art that alternative TFBS sequences can be used as long as they are subject to binding by the intended TF. Consensus sequences for various TFBSs are known in the art, and one of skill in the art can readily use this information to determine alternative TFBSs. Furthermore, the ability of a TF to bind to a given putative sequence can be readily determined experimentally by one of skill in the art (e.g., by EMSA and other techniques well known in the art and described herein).

[0322] The meaning of "consensus sequence" is well known in the art. In this application, the following notation is used for consensus sequences unless the context dictates otherwise: The following exemplary DNA sequence: A[CT]N{A}YR If you consider A means that A is always found at that position; [CT] represents either C or T at that position; N means that any base can be at that position; {A} means that any base other than A can be at that position. Y represents any pyrimidine and R represents any purine.

[0323] As used herein, "synthetic" refers to a nucleic acid molecule that does not exist in nature. Synthetic nucleic acids of the invention are typically produced artificially by recombinant technology or de novo synthesis. Such synthetic nucleic acids may contain naturally occurring sequences (e.g., promoters, enhancers, introns, and other regulatory sequences), but these are present in a context that does not occur in nature. For example, a synthetic gene (or portion of a gene) typically contains one or more nucleic acid sequences that are not contiguous in nature (chimeric sequences) and / or may include substitutions, insertions, and deletions, as well as combinations thereof.

[0324] "Complementary" or "complementarity," as used herein, refers to Watson-Crick base pairing of two nucleic acid sequences. For example, in the sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'. Complementarity between two nucleic acid sequences can be "partial," where only a portion of the bases bind to their complements, or complete, where all bases in the sequence bind to their complementary bases. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0325] As used herein, "transfection" broadly refers to any process by which nucleic acid is intentionally introduced into a cell, encompassing the introduction of viral and non-viral vectors, and including terms and processes such as transformation, transduction, and the like, or equivalents thereof. Examples include, but are not limited to, transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; whisker-mediated transformation; and particle bombardment (Klein et al. (1987) Nature 327:70).

[0326] As used herein, the term "transgene" refers to an exogenous nucleic acid sequence. In one embodiment, the transgene is a gene encoding an industrially or pharmaceutically useful compound or a gene encoding a desirable property. In yet another embodiment, the transgene encodes a useful nucleic acid, such as an antisense nucleic acid sequence, where expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. The transgene preferably encodes a therapeutic product, such as a protein.

[0327] The term "vector" is well known in the art and, as used herein, refers to a nucleic acid molecule, e.g., double-stranded DNA, into which a nucleic acid sequence according to the present invention may be inserted. A vector is preferably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all the necessary elements that allow the inserted nucleic acid molecule to be transcribed, and preferably the transcription product to be translated into a polypeptide. A vector typically contains all the necessary elements so that, after the vector enters a host cell, the vector can replicate independently of or simultaneously with the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule can be generated. The vectors of the present invention can be episomal vectors (i.e., not integrated into the genome of the host cell) or vectors that integrate into the host cell genome. This definition encompasses both non-viral and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, Bluescript vectors (pBS), and pBR322, or their derivatives lacking bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors, such as artificial chromosomes (bacterial (BAC), yeast (YAC), or human (HAC)), can be used to accommodate larger inserts. Viral vectors are derived from viruses and include, but are not limited to, retroviral, lentiviral, adeno-associated viral, adenoviral, herpesviral, and hepatitis viral vectors. Typically, but not necessarily, viral vectors are replication-deficient if they have lost the ability to propagate in a given cell because viral genes essential for replication have been removed from the viral vector. However, some viral vectors can also be adapted to replicate specifically in certain cells, such as cancer cells, and are typically used to cause (cancer) cell-specific lysis (oncolysis).Virosomes are a non-limiting example of vectors that contain both viral and non-viral elements; in particular, they combine liposomes with inactivated HIV or influenza viruses (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0328] The terms "operably linked," "operably connected," or equivalent expressions, as used herein, refer to the arrangement of various nucleic acid elements relative to one another such that the elements are functionally connected and can interact with one another in the intended manner. Such elements may include, but are not limited to, a promoter, a CRE (e.g., an enhancer or other regulatory element), a detargeting element (e.g., a liver detargeting element), a promoter element, a polyadenylation sequence, one or more introns and / or exons, one or more UTRs, and the coding sequence of the gene of interest to be expressed. When nucleic acid sequence elements are appropriately oriented or operably linked, they can act together to modulate each other's activity, ultimately affecting the level of expression of the expression product. Modulate means to increase, decrease, or maintain the level of activity of a particular element. The position of each element relative to other elements can be expressed in terms of the 5' and 3' ends of each element or their position upstream or downstream of another element or position (e.g., a TSS or promoter element), and the distance between any particular elements can be referenced by the number of intervening nucleotides or base pairs between the elements. As will be understood by those skilled in the art, operably linked refers to functional activity and does not necessarily refer to linkage at the natural position. Indeed, when used in a nucleic acid expression cassette, a CRE is typically positioned immediately upstream of the promoter element (although this is generally the case and should not be construed as a limitation or exclusion of the position within the nucleic acid expression cassette), but this is not necessarily the case in vivo; for example, a naturally occurring regulatory element sequence downstream of a gene whose transcription is affected can function in the same way as if it were located upstream of the promoter. Thus, in certain embodiments, the regulatory or enhancing effect of a regulatory element can be position-independent.

[0329] As used herein, a "spacer sequence" or "spacer" is a nucleic acid sequence that separates two functional nucleic acid sequences (e.g., TFBS, CRE, CRM, promoter element, etc.). A spacer can have essentially any sequence, so long as it does not prevent the functional nucleic acid sequence (e.g., a cis-regulatory element) from functioning as desired (e.g., this may occur if the spacer contains a silencer sequence, preventing the binding of a desired transcription factor, etc.). A spacer is typically non-functional, as it exists only to separate adjacent functional nucleic acid sequences from each other. In some embodiments, a spacer can have a length of 75, 50, 40, 30, 20, or 10 nucleotides or less.

[0330] The term "pharmaceutically acceptable," as used herein, is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0331] Phrases such as "therapeutically effective amount" refer to a dosage or plasma concentration in a subject that provides the desired specific pharmacological effect, e.g., to express a therapeutic gene in muscle. A therapeutically effective amount may not always be effective in treating the conditions described herein, even if such a dosage is considered therapeutically effective by those skilled in the art. A therapeutically effective amount may vary based on the route and form of administration, the age and weight of the subject, and / or the disease or condition being treated.

[0332] The term " AAV vector " used herein is well known in the art, and generally refers to the AAV vector nucleic acid sequence that comprises various nucleic acid sequences.The AAV vector used herein typically comprises the heterologous nucleic acid sequence that is not of AAV origin as part of the vector.This heterologous nucleic acid sequence typically comprises the promoter disclosed herein and other sequences that are intended for the genetic transformation of cells.Generally, the heterologous nucleic acid sequence is flanked by at least one, generally two AAV inverted terminal repeats (ITR).

[0333] "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" means a viral particle composed of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an encapsidated polynucleotide AAV vector. When a particle contains heterologous nucleic acid (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it may be referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV virion or AAV particle necessarily includes production of an AAV vector, and as such, the vector is contained within the AAV virion or AAV particle.

[0334] "Small interfering RNA" or "short interfering RNA" or siRNA is an RNA duplex of nucleotides that targets a gene of interest (a "target gene"). "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. An siRNA is "targeted" to a gene, and the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides long. In some embodiments, the duplex length is 19-25 nucleotides long. The RNA duplex portion of an siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can include a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also include a 3' or 5' overhang portion. In some embodiments, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0335] The terms "treatment" or "treating" mean reducing, ameliorating, or eliminating one or more signs, symptoms, or effects of a disease or condition. Thus, "treatment," as used herein, includes any treatment of a disease in a mammal, particularly a human, and includes the following: (a) preventing the disease from occurring in a subject susceptible to or at risk of having the disease but not yet diagnosed with it; (b) inhibiting the disease, i.e., halting the progression of the disease; and (c) palliating the disease, i.e., causing regression of the disease.

[0336] "Administration" of an agent to a subject includes any route by which the agent is introduced or delivered to the subject to perform its intended function. Administration can be by any suitable route, such as oral, intranasal, intraocular, ophthalmic, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another. Intramuscular administration is of particular interest in the present invention.

[0337] The terms "individual," "subject," and "patient" are used interchangeably and refer to any individual subject having a disease or condition in need of treatment. For purposes of the present invention, a subject may be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or cow. In some embodiments, the subject is not a human.

[0338] As used herein, the term "gene therapy vector" refers to a vector, as defined above, suitable for or intended for gene therapy. Gene therapy vectors typically contain a promoter operably linked to a therapeutically useful expression product (e.g., a transgene useful in the treatment of a disease or condition).

[0339] As used herein, the term "detargeting element" refers to a nucleic acid sequence that, when added to a CRM, synthetic promoter, expression cassette, or vector, has the purpose or function to reduce expression of the CRM, synthetic promoter, expression cassette, or vector in a particular tissue or cell. Examples of detargeting elements include Kopp F, Schnoedt M, Haase R, Wagner E, Roidl A, Ogris M. De-targeting by miR-143 decreases unwanted transgene expression in non-tumorigenic cells. Gene Ther. 2013 Nov;20(11):1104~9. doi: 10.1038 / gt.2013.37. Epub 2013 Jun 27. PMID: 23804075, Dhungel B, Ramlogan-Steel CA, Layton CJ, Steel JC. MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma. Mol Ther Nucleic Acids. 2018 Dec 7;13:78~88. doi: 10.1016 / j.omtn.2018.08.016.Epub 2018 Aug 24. PMID: 30245470; PMCID: PMC6148835 (especially Figure 2 and Materials and Methods), and Dhungel, B., Ramlogan-Steel, CA & Steel, JC Synergistic and independent action of endogenous microRNAs 122a and 199a for post-transcriptional liver detargeting of gene vectors. Sci Rep 8, 15539 (2018). (https: / / doi.org / 10.1038 / s41598-018-33801-4) (especially Figure 2), Juliette Hordeaux, Elizabeth L.Buza, Brianne Jeffrey, Chunjuan Song, Tahsin Jahan and Yuan Yuan, Yanqing Zhu, Peter Bell, Mingyao Li, Jessica A. Chichester, Roberto Calcedo and James M. Wilson, "MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates," Science Translational Medicine, Volume 12, 569, 2020, doi 10.1126 / scitranslmed.aba9188, which is incorporated herein by reference. The detargeting element may be a binding site for a protein or miRNA that is highly expressed in a particular cell or tissue. In some embodiments, the detargeting element may be a liver detargeting element. Addition of a liver detargeting element to a CRM, synthetic promoter, expression cassette, or vector may reduce expression of the CRM, synthetic promoter, expression cassette, or vector in liver tissue or cells.

[0340] As used herein, the term "additional regulatory elements" refers to nucleic acid sequences that can be added to a CRM or synthetic promoter. The addition of additional regulatory elements is expected to adjust the expression profile of the CRM or synthetic promoter (i.e., increase, decrease, or maintain the level of activity in any particular cell type or tissue) without rendering the CRM or synthetic promoter substantially non-functional. Addition of additional regulatory elements may be desirable to increase or decrease the level of activity in a desired tissue. For example, the addition of the tMCK SA / SD intron (SEQ ID NO: 29) to SP0524 (synthetic muscle-specific promoter SP0527) increases activity in the heart. Additional regulatory elements can be selected from CREs, CRMs, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, segments of terminal repeats, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, introns, UTRs, splicing elements, and the like. The term "aberrant gene expression" refers to gene expression associated with a disease or pathology. Aberrant gene expression can be caused by mutations in the gene or mRNA.

[0341] The invention will now be described with reference to the following non-limiting examples. [Example]

[0342] In vivo data The strength of synthetic muscle-specific promoters according to embodiments of the present invention was tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase.

[0343] Materials and Methods A selection of synthetic muscle-specific promoters was tested in vivo.

[0344] In vivo experiments AAV containing a synthetic promoter (e.g., SP0527) operably linked to luciferase was diluted in 0.9% saline and injected into 8-week-old male Balb / c mice at a dose of 1 e per mouse. 11 Vg / 200 μl (6 mice per group) was administered by tail vein injection. Mice were sacrificed 6 weeks after injection according to Schedule 1. AAV9 was used.

[0345] Diaphragm (skeletal muscle), heart (cardiac muscle), gastrocnemius (skeletal muscle), soleus (skeletal muscle), tibialis anterior (TA) (skeletal muscle), and liver were harvested from each mouse 6 weeks post-injection. The following tissues were harvested for IHC: heart, TA, soleus, liver, and gastrocnemius. For luciferase (Luc) expression and vector copy number (VCN) analysis, all samples were snap-frozen in liquid nitrogen immediately after dissection, then transferred to dry ice and stored at -80°C. Tissues for IHC were mounted on corks, slowly frozen in isopentane over liquid nitrogen, and then transferred to dry ice. Mouse gastrocnemius muscles contain fast-twitch fibers (approximately 80%) and slow-twitch fibers (approximately 20%).

[0346] Live imaging All six mice in each group were imaged four weeks after injection to confirm luciferase expression. An IVIS® Spectrum Lumina III series in vivo imaging system was used for imaging. The IVIS device detects bioluminescence by imaging the light emitted by enzyme-catalyzed reactions, reporting activity at the molecular level. Bioluminescent reporters require small chemical substrates for noninvasive imaging in cell biology and small animal research. This protocol specifically requires IP injection of D-luciferin and anesthesia induction with isophane. The imaging procedure was as follows: - A stock solution of D-luciferin potassium salt (as substrate) was prepared using luciferin (cat# MB000102, Syd Labs, USA) lot# Ro405-017 and luciferin (cat# LUCK-1G, GoldBio, USA) lot# 016067LUCK. For reconstitution, PBS (Gibco Cat# 14190-094, lot# 2241142) was used. - Luciferin stock was prepared at 15 mg / ml (W / V) (1500 mg / 100 ml PBS), aliquoted, labeled, wrapped in foil to protect from light and stored in a -20 / -80°C freezer. - D-luciferin substrate was administered intraperitoneally to mice in 300 μl / mouse of luciferin stock. - Fill the anesthetic mixer with isoflurane / isoflurane. - The mouse was moved to the anesthesia induction chamber. - The oxygen supply was set to 2.5 L / min, and the mice were observed to sleep. After the mice were anesthetized, during a 5-minute countdown, the mice were moved from the induction chamber to the IVIS chamber of the bioimaging device in the order corresponding to the animal experiment number (group). - The image was taken. - Luciferase expression was compared between the upper half of the animal (including the heart, diaphragm, and liver) and the lower half (including the soleus, tibialis anterior, and gastrocnemius muscles).

[0347] Tissue homogenization and lysis Tissues stored at -80°C were thawed at room temperature. Reporter lysis buffer (Promega, Catalog No. E4030) was pipetted into each tube containing sample tissue. Qiagen Carbide Beads (QIAGEN, Catalog No. 69997) were added at 1-2 per tube. The tubes were placed in a Qiagen Tissue Lyser II (QIAGEN, Catalog No. 85220) and homogenized at 25.0 Hz for 75 seconds. After 75 seconds, the adapter was rotated, and the sample was homogenized for an additional 75 seconds. The samples were then flash-frozen at -80°C for 10-15 minutes, followed by thawing at 37°C for 10 minutes using a dry heating bath system. The tubes were centrifuged at 10,000 × g for 3 minutes. The supernatant was transferred to a fresh 1.5 ml Eppendorf tube, taking care not to disturb the pellet. The above steps were repeated once more by adding additional reporter lysis buffer to the pelleted tissue. The supernatant from the repeated process was added to the same 1.5 ml Eppendorf tube. The tube was vortexed thoroughly and stored at -80°C.

[0348] Measurement of luciferase activity - Luciferase activity was measured using Promega's Luciferase Assay System (Promega, Cat. No. #E4550). - Luciferase (LAR) reagent working solution was thawed at room temperature 30 minutes before use, while sample tissue lysates were thawed on ice before use. - The sample of interest was diluted 1:10 in a fresh 1.5 ml Eppendorf tube. - 10 μl of diluted samples of interest and negative control (reporter lysis buffer) were manually pipetted (in triplicate) into a 96-well flat-bottom solid white plate. Luminescence (RLU) was measured in a BMG Fluostar plate reader by injecting 50 μl of LAR into each well. Luciferase expression (RLU per μg / μL) was calculated by normalizing relative light units to protein concentration. Data were displayed as mean ± standard deviation using GraphPad Prism 9.

[0349] Protein quantification Protein was extracted from the collected tissues and quantified using the BCA Pierce Protein Assay Kit (ThermoFisher 23225). The manufacturer's instructions were as follows: Bovine serum albumin (BSA) standards were prepared by diluting albumin to a working range of 20-20,000 μg / ml. An appropriate amount of BCA working reagent (WR) was prepared by mixing 50 parts BCA Reagent A with 1 part BCA Reagent B (50:1, Reagent A:B). The microplate procedure (1:8 sample to WR ratio) was performed as follows: - Samples were diluted 1:10 in reporter lysis buffer (1x) - 10 μL of each standard or unknown sample replicate was pipetted into wells of a 96-well plate in duplicate. - 200 μL of WR was added to each well and the plate was placed on a plate shaker and mixed for 30 seconds. - The plate was covered and incubated at 37°C for 30 minutes. - The plate was cooled to room temperature and the absorbance was measured at 562 nm in a BMG Fluostar plate reader. The average 562 nm absorbance reading of the blank standard replicates was subtracted from the 562 nm absorbance readings of all other BSA standard replicates and sample replicates. A standard curve was generated by plotting the blank-corrected average 562 nm readings for each BSA standard against its concentration (μg / ml). The standard curve was used to determine the protein concentration of each sample.

[0350] Vector copy number The copy number of the vector was determined by dual Taqman qPCR. DNA was extracted using the DNeasy® Blood & Tissue Kit (250) (QIAGEN, Cat. No. 69506). Taqman qPCR was performed on each sample using luciferase- and GAPDH-specific primer and probe sets.

[0351] [Table 1]

[0352] For analytical purposes, standard curves for Luc and GAPDH were used. The following final concentrations of reagents and DNA were used in the multiplex qPCR protocol: Luc2 FW primer (350 nM), Luc2 RV primer (350 nM), mGapdH FW primer (350 nM), mGapdH RV primer (350 nM), Luc2 probe (250 nM), mGapdH probe (250 nM), and DNA (10 ng / µL). The PCR cycle protocol was as follows: 95°C for 20 seconds, PCR: 40 cycles, 95°C for 1 second, 60°C for 20 seconds. The ΔΔCt VCN (quantity) per genome was calculated by subtracting the average VCN (quantity) per genome (threshold) of the saline sample.

[0353] result SP0525 drives high expression in cardiac muscle (heart) as well as skeletal muscle (e.g., gastrocnemius, soleus, tibialis anterior). SP0525 drives lower expression in liver. This is shown in Figure 3.

[0354] SP0526 drives high expression in cardiac muscle (heart) as well as skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0526 drives lower expression in liver. This is shown in Figure 4.

[0355] SP0527 drives high expression in cardiac muscle (heart) as well as skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0527 drives lower expression in liver. This is shown in Figure 5.

[0356] SP0528 drives high expression in cardiac muscle (heart) as well as skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0528 drives lower expression in liver. This is shown in Figure 6.

[0357] As shown in Figure 7, expression cassette 529 drives high expression in cardiac muscle (heart). Expression cassette 529 also drives expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm, and soleus), which is lower than expression in cardiac muscle (heart). Expression cassette 529 drives low expression in liver. This is shown in Figure 7.

[0358] As shown in Figure 8, the synthetic muscle-specific promoter SP0530 drives high expression in cardiac muscle (heart). The synthetic muscle-specific promoter SP0530 also drives expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm, and soleus), which is lower than expression in cardiac muscle (heart). The synthetic muscle-specific promoter SP0530 drives low expression in liver. Notably, the addition of the tMCK SA / SD intron (SEQ ID NO: 29) appears to result in increased expression in the heart compared to the original design SP0524 (SP0530 is SP0524 + tMCK SA / SD intron).

[0359] As shown in Figure 9, the synthetic muscle-specific promoter SP0531 drives high expression in cardiac muscle (heart) and skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm, and soleus). The synthetic muscle-specific promoter SP0531 drives low expression in the liver.

[0360] Background (saline) expression is shown in Figure 10. Figures 11 and 12 show that vector copy numbers are similar between the different promoters and expression cassette 529 tested in the heart and liver, respectively.

[0361] Live imaging of a mouse injected with an AAV containing a synthetic promoter (e.g., SP0525) operably linked to luciferase is shown in Figure 13. [Example]

[0362] Further in vivo studies The strength of synthetic muscle-specific promoters according to embodiments of the present invention was tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase.

[0363] Materials and Methods A selection of synthetic muscle-specific promoters were tested in vivo at multiple doses.

[0364] In vivo experiments AAV containing a synthetic promoter (e.g., SP0525) operably linked to luciferase was diluted in 0.9% saline and administered at three doses: 1e per mouse. 11 vg / 200 μl (6 mice per group), 6 e per mouse 11 vg / 200 μl (5 or 6 mice per group), 1.2 e per mouse 12 The vaccine was administered to 8-week-old male Balb / c mice via tail vein injection at 200 μg / 200 μl (6 mice per group). Mice were sacrificed 6 weeks after injection using Schedule 1. AAV9 was used.

[0365] Diaphragm (skeletal muscle), heart (cardiac muscle), gastrocnemius (skeletal muscle), soleus (skeletal muscle), tibialis anterior (TA) (skeletal muscle), quadriceps (skeletal muscle), psoas (skeletal muscle), and liver were harvested from each mouse at all doses and saline at 6 weeks post-injection. The following additional tissues were harvested from each mouse: brain, kidney, spleen, lymph node, gallbladder, colon, adrenal gland, lung, and testis from the high-dose group (1.2 e per mouse). 12 vg / 200 μl) and saline only. For IHC, the following tissues were collected: TA, soleus, heart, quadriceps, psoas, and gastrocnemius. For luciferase (Luc) expression and vector copy number (VCN) analysis, all samples were snap-frozen in liquid nitrogen immediately after dissection, then transferred to dry ice and stored at -80°C. Tissues for IHC were mounted on corks and slowly frozen in isopentane over liquid nitrogen, then transferred to dry ice. Mouse gastrocnemius contains fast-twitch fibers (approximately 80%) and slow-twitch fibers (approximately 20%).

[0366] Tissue homogenization and lysis Tissues stored at -80°C were thawed at room temperature. 1x Reporter Lysis Buffer (RLB) (Promega, Catalog #E4030) was pipetted into each tube containing sample tissue. One to two Qiagen Carbide Beads (QIAGEN, Catalog #69997) were added per tube. The tubes were placed in a Qiagen Tissue Lyser II (QIAGEN, Catalog #85220) and homogenized at 25.0 Hz for 75 seconds. After 75 seconds, the adapter was rotated, and the sample was homogenized for an additional 75 seconds. The samples were then flash-frozen at -80°C for 10-15 minutes, followed by thawing at 37°C for 10 minutes using a dry heating bath system. The tubes were centrifuged at 10,000 x g for 3 minutes. The supernatant was transferred to a fresh 1.5 ml Eppendorf tube, taking care not to disturb the pellet. The above steps were repeated once more by adding additional reporter lysis buffer to the pelleted tissue. The supernatant from the repeated process was added to the same 1.5 ml Eppendorf tube. The tube was vortexed thoroughly and stored at -80°C.

[0367] Measurement of luciferase activity - Luciferase activity was measured using the Promega Luciferase Assay System (Promega, Cat. No. E4550). - Luciferase (LAR) reagent working solution was thawed at room temperature 30 minutes before use, and simultaneously, sample tissue lysates were thawed on ice before use. - The sample of interest was diluted 1:10 in a fresh 1.5 ml Eppendorf tube - 10 μl of diluted samples of interest and negative control (reporter lysis buffer) were manually pipetted (in triplicate) into a 96-well flat-bottom solid white plate and luminescence (RLU) was measured in a BMG Fluostar plate reader by injecting 50 μl of LAR into each well. Luciferase expression (RLU per μg / μL) was calculated by normalizing relative light units to protein concentration. Data were displayed as mean ± standard deviation using GraphPad Prism 9.

[0368] Protein quantification Protein was extracted from the collected tissues and quantified using the BCA Pierce Protein Assay Kit (ThermoFisher #23225). The manufacturer's instructions are as follows: Bovine serum albumin (BSA) standards were prepared by diluting albumin to a working range of 20-20,000 μg / ml. The appropriate amount of BCA working reagent (WR) was prepared by mixing 50 parts BCA Reagent A with 1 part BCA Reagent B (50:1, Reagent A:B). The microplate procedure (1:8 sample to WR ratio) was performed as follows: - Samples were diluted 1:10 in reporter lysis buffer (1x) - 10 μL of each standard or unknown sample replicate was pipetted into wells of a 96-well plate in duplicate. - 200 μL of WR was added to each well and the plate was placed on a plate shaker and mixed for 30 seconds. - The plate was covered and incubated at 37°C for 30 minutes. - The plate was cooled to room temperature and the absorbance was measured at 562 nm in a BMG Fluostar plate reader.

[0369] The average 562 nm absorbance reading of the blank standard replicates was subtracted from the 562 nm absorbance readings of all other BSA standard replicates and sample replicates. A standard curve was generated by plotting the blank-corrected average 562 nm readings for each BSA standard against its concentration (μg / ml). The standard curve was used to determine the protein concentration of each sample.

[0370] Vector copy number Vector copy number was determined by dual Taqman qPCR. DNA was extracted using the automated Maxwell RSC Tissue DNA Kit (Promega #AS1610). Multiplexed Taqman qPCR was performed on each sample using TaqMan® Fast Advanced Master Mix (#4444557, ThermoFisher) with luciferase-specific primer and probe sets for the target and GAPDH or HPRT1-specific primer and probe sets for the housekeeping gene.

[0371] [Table 2]

[0372] For analytical purposes, standard curves for Luc and GAPDH were used. The following final concentrations of reagents and DNA were used in the multiplex qPCR protocol: Luc2 FW primer (350 nM), Luc2 RV primer (350 nM), mGapdH FW primer (350 nM), mGapdH RV primer (350 nM), Luc2 probe (250 nM), mGapdH probe (250 nM), and DNA (10 ng / µL). The PCR cycle protocol was as follows: 95°C for 20 seconds, PCR: 40 cycles, 95°C for 1 second, 60°C for 20 seconds. The ΔΔCt VCN (quantity) per genome was calculated by subtracting the average VCN (quantity) per genome (threshold) of the saline sample.

[0373] mRNA RNA was isolated using the Maxwell simplyRNA tissue kit (Promega #AS1340) according to the manufacturer's instructions. The isolated RNA was treated with DNase I and retrotranscribed to its complementary cDNA using Superscript III reverse transcriptase (Life Technologies #18418020). Amplification was performed in a Life Technologies QuantStudio 7 using TaqMan® Fast Advanced Master Mix (#4444557, ThermoFisher). The primers and probe sets used are detailed in the previous table.

[0374] result Expression of CK7 (low and medium doses) in various muscle and non-muscle tissues (liver) is shown in Figures 14 and 15. Expression of SP0527 (low, medium, and high doses) in various muscle and non-muscle tissues (liver) is shown in Figures 16-18. SP0527 drives high expression in cardiac muscle (heart) as well as skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0527 drives lower expression in the liver.

[0375] Background (saline) expression is shown in Figure 19. Figure 20 shows the vector copy number in the liver for CK7 and SP0527 at various doses, where a dose-dependent pattern is seen, with lower doses resulting in lower vector copy numbers and higher doses resulting in higher vector copy numbers.

[0376] Figure 21 shows the expression of SP0527 (high dose) in muscle tissue and various non-muscle tissues (e.g., brain, liver, lung, colon, spleen, testis, kidney, etc.). SP0527 drives high expression in muscle tissue and lower expression in non-muscle tissues (e.g., brain, liver, lung, colon, spleen, testis, kidney, etc.). SP0527 shows low expression in all non-muscle tissues tested. Background (saline) expression is shown in Figure 22. [Example]

[0377] Liver detargeting Synthetic promoters SP0525-SP0528 are tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase. Expression cassette 529 is also tested by replacing the expression product (e.g., a gene of interest) with the reporter gene luciferase.

[0378] In vivo experiments AAV containing a synthetic promoter (e.g., SP0525) operably linked to luciferase or expression cassette 529 was diluted in 0.9% saline and administered at various doses to 8-week-old male Balb / c mice via tail vein injection. The dose was 1 e per mouse. 11 vg / 200μl, 6e 11 vg / 200μl, 1e 12 vg / 200μl, 6e 12 vg / 200μl, 1e 13 vg / 200 μl, and 6 e 13 vg / 200 μl (6 mice per group). Mice were sacrificed 6 weeks after injection by the method of Schedule 1. AAV9 was used. Tissues were collected and processed as detailed in Example 1.

[0379] SP0525, SP0526, SP0527, SP0528, and expression cassette 529 demonstrate muscle-specific activity at all doses tested. The expression profile of SP0525 in various muscle tissues is similar to the expression profile shown in Figure 3 at all doses tested. The expression profile of SP0526 in various muscle tissues is similar to the expression profile shown in Figure 4 at all doses tested. The expression profile of SP0527 in various muscle tissues is similar to the expression profile shown in Figure 5 at all doses tested. The expression profile of SP0528 in various muscle tissues is similar to the expression profile shown in Figure 6 at all doses tested. The expression profile of expression cassette 5295 in various muscle tissues is similar to the expression profile shown in Figure 7 at all doses tested.

[0380] 6e 12 At a dose of vg / 200 μl, SP0525, SP0526, SP0527, SP0528, and expression cassette 529 show lower activity in the liver compared to CK7 and / or CK8. 13 At a dose of vg / 200 μl, SP0525, SP0526, SP0527, SP0528, and expression cassette 529 show lower activity in the liver compared to CK7 and / or CK8. 13 At a dose of vg / 200 μl, SP0525, SP0526, SP0527, SP0528, and expression cassette 529 show lower activity in the liver compared to CK7 and / or CK8.

[0381] Sequence information

[0382] [Table 3]

[0383] [Table 4]

[0384] [Table 5]

[0385] [Table 6]

[0386] [Table 7]

[0387] [Table 8]

[0388] [Table 9]

[0389] Expression cassette 529 is represented below:

[0390] [Table 10]

[0391] [Table 11]

[0392] Clause

[0393] 1. A synthetic muscle-specific cis-regulatory module (CRM) comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof, DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, and at least one additional regulatory element, wherein optionally the additional regulatory element is a detargeting element or a functional variant thereof.

[0394] 2. The synthetic muscle-specific CRM of clause 1, wherein said at least one additional regulatory element is a detargeting element or a functional variant thereof, optionally a liver-detargeting element or a functional variant thereof.

[0395] 3. The liver detargeting element or a functional variant thereof is selected from the group consisting of: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof 3. The synthetic muscle-specific CRM of clause 2, selected from the group consisting of:

[0396] 4. The following: - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 1 (SEQ ID NO: 13); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 2 (SEQ ID NO: 14); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15); CRE0145 (SEQ ID NO: 10), liver detargeting sequence 3 (SEQ ID NO: 17), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); and CRE0145 (SEQ ID NO: 10), ZBTB20 binding site (SEQ ID NO: 15), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15), 4. The synthetic muscle-specific CRM of any one of clauses 1-3, comprising a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0397] 5. The synthetic muscle-specific CRM of any one of clauses 1-4, comprising or consisting of SEQ ID NOs: 20, 21, 22, and 9 or a functional variant thereof, optionally comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 20, 21, 22, and 9.

[0398] 6. A synthetic muscle-specific promoter comprising a CRM of any one of clauses 1-5 operably linked to a promoter element, optionally wherein the promoter element is SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0399] 7. The synthetic muscle-specific promoter comprises a synthetic muscle-specific CRM comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof, DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof, and at least one additional regulatory element, optionally the additional regulatory element being - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; and - ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof 7. The synthetic muscle-specific promoter of clause 6, wherein the liver-detargeting element is selected from the group consisting of:

[0400] 8. The synthetic muscle-specific promoter is operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof. - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 1 (SEQ ID NO: 13); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 2 (SEQ ID NO: 14); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15); CRE0145 (SEQ ID NO: 10), liver detargeting sequence 3 (SEQ ID NO: 17), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); and - CRE0145 (SEQ ID NO: 10), ZBTB20 binding site (SEQ ID NO: 15), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15); and a synthetic muscle-specific CRM comprising a combination of regulatory elements or functional variants thereof selected from the group consisting of: Optionally, the regulatory elements are present in the CRM in the order listed and adjacent to each other: 8. The synthetic muscle-specific promoter of any one of clauses 6-7.

[0401] 9. The synthetic muscle-specific promoter of any one of clauses 6 to 8, comprising or consisting of SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), or SP0528 (SEQ ID NO: 4), or a functional variant of any of them.

[0402] 10. The synthetic muscle-specific promoter of any one of clauses 6-9, comprising or consisting of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4.

[0403] 11. The synthetic muscle-specific promoter according to any one of clauses 6 to 10, comprising or consisting of SP0527 (SEQ ID NO: 3) or any functional variant thereof, optionally comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

[0404] 12. Synthetic muscle-specific promoter containing liver detargeting elements.

[0405] 13. The liver detargeting element comprises: - SEQ ID NO: 15 or a functional variant thereof; - SEQ ID NO: 13 or a functional variant thereof; - SEQ ID NO: 17 or a functional variant thereof; or - SEQ ID NO: 14 or a functional variant thereof 13. The synthetic muscle-specific promoter of clause 12, selected from the group consisting of:

[0406] 14. The synthetic muscle-specific promoter of clause 12 or 13, wherein the liver detargeting element comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:14.

[0407] 15. The following: - CRE0145 (SEQ ID NO: 10) or a functional variant thereof, - DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, - SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof; and - at least one additional regulatory element A synthetic muscle-specific promoter comprising or consisting of:

[0408] 16. The at least one additional regulatory element is a detargeting element, optionally a liver detargeting element, wherein the liver detargeting element is: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof 16. The synthetic muscle-specific promoter of clause 15, selected from the group consisting of:

[0409] 17. The synthetic muscle-specific promoter of clause 15, wherein the at least one additional regulatory element is an intron, optionally wherein the intron is selected from the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncated intron (SEQ ID NO: 30).

[0410] 18. The additional regulatory element is selected from the group consisting of: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof a liver detargeting element selected from the group consisting of: An intron selected from the tMCK SA / SD intron (SEQ ID NO: 29) and the MVM cleavage intron (SEQ ID NO: 30) 16. The synthetic muscle-specific promoter of clause 15, wherein

[0411] 19. The synthetic muscle-specific promoter of any one of clauses 15-18, comprising or consisting of a sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0530 (SEQ ID NO: 27), or SP0531 (SEQ ID NO: 28), or any one of SEQ ID NOs: 1-4, 27-28.

[0412] 20. An expression cassette comprising the synthetic muscle-specific promoter of any one of clauses 6-19 operably linked to a sequence encoding an expression product, or an expression cassette comprising a synthetic muscle-specific promoter comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, wherein the synthetic muscle-specific promoter is operably linked to the expression product and to a target sequence for miR122.

[0413] 21. A vector comprising the synthetic muscle-specific promoter according to any one of paragraphs 6 to 19 or the expression cassette according to paragraph 20.

[0414] 22. The vector of clause 21, which is an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector.

[0415] 23. A gene therapy vector comprising a synthetic promoter comprising a liver detargeting element, wherein the liver detargeting element is one of the following: - SEQ ID NO: 15 or a functional variant thereof; - SEQ ID NO: 13 or a functional variant thereof; - SEQ ID NO: 17 or a functional variant thereof; or - SEQ ID NO: 14 or a functional variant thereof A gene therapy vector selected from the group consisting of:

[0416] 24. The gene therapy vector of clause 23, comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:13, or SEQ ID NO:14.

[0417] 25. The gene therapy vector of clause 23 or 24 which is an AAV vector.

[0418] 26. A gene therapy AAV vector comprising an expression cassette, the expression cassette comprising a synthetic promoter operably linked to a sequence encoding an expression product and a target sequence for miR122.

[0419] 27. The gene therapy AAV vector according to clause 26, wherein said target sequence for miR122 comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 19 or a functional variant thereof.

[0420] 28. The gene therapy AAV vector of clause 26 or 27, wherein the target sequence for miR122 comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 19.

[0421] 29. A virion comprising the vector of any one of clauses 21 to 28.

[0422] 30. A pharmaceutical composition comprising the synthetic muscle-specific promoter of any one of clauses 6 to 19, the expression cassette of clause 20, the vector of any one of clauses 21 to 28, or the virion of clause 29.

[0423] 31. The synthetic muscle-specific promoter of any one of clauses 6-19, the expression cassette of clause 20, the vector of any one of clauses 21-28, the virion of clause 29, or the pharmaceutical composition of clause 30 for use in therapy.

[0424] 32. A cell comprising the synthetic muscle-specific promoter of any one of clauses 6-19, the expression cassette of clause 20, the vector of any one of clauses 21-28, or the virion of clause 29.

[0425] 33. The synthetic muscle-specific promoter of any one of clauses 6-19, the expression cassette of clause 20, the vector of any one of clauses 21-28, the virion of clause 29, or the pharmaceutical composition of clause 30 for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

[0426] 34. A method for producing an expression product, the method comprising providing an expression cassette according to paragraph 20 in a muscle cell and expressing the expression product present in the expression cassette.

[0427] 35. A method for expressing a therapeutic transgene in a muscle cell, the method comprising introducing into the muscle cell an expression cassette according to clause 20, a vector according to any one of clauses 21-28, or a virion according to clause 29.

[0428] 36. A method for treating a subject, preferably a human, in need of treatment, comprising: administering to the subject an expression cassette according to clause 20, a vector according to any one of clauses 21-28, a virion according to clause 29, or a pharmaceutical composition according to clause 30, comprising a sequence encoding a therapeutic product operably linked to a promoter according to any one of clauses 6-19; and expressing a therapeutic amount of a therapeutic product in the muscle of said subject. Methods including:

[0429] 37. The method of treating a subject of clause 36, wherein a therapeutic amount of said therapeutic product is expressed in skeletal muscle and / or cardiac muscle.

Claims

1. A synthetic muscle-specific cis-regulatory module (CRM) comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof, DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, and at least one additional regulatory element, such as a detargeting element, optionally comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-11; Synthetic muscle-specific cis-regulatory modules (CRMs).

2. The at least one additional regulatory element may be a detargeting element, optionally one of the following: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof; - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof a liver detargeting element selected from the group consisting of: Optionally, the synthetic muscle-specific CRM comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-17 and 18; 10. The synthetic muscle-specific CRM of claim 1.

3. CRM includes: - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 1 (SEQ ID NO: 13); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 2 (SEQ ID NO: 14); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15); CRE0145 (SEQ ID NO: 10), liver detargeting sequence 3 (SEQ ID NO: 17), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); and CRE0145 (SEQ ID NO: 10), ZBTB20 binding site (SEQ ID NO: 15), DES_MT_enhancer_48bp (SEQ ID NO: 11), and ZBTB20 binding site (SEQ ID NO: 15), and optionally the regulatory elements are present in the CRM in the order listed and adjacent to each other; Optionally, the synthetic muscle-specific CRM comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-11, 13-15, and 17; 3. The synthetic muscle-specific CRM of claim 1 or 2.

4. 4. The synthetic muscle-specific CRM of any one of claims 1 to 3, comprising or consisting of SEQ ID NOs: 20, 21, 22 and 9 or a functional variant thereof, optionally comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 20, 21, 22 and 9,

5. 5. A synthetic muscle-specific promoter comprising the CRM of any one of claims 1 to 4 operably linked to a promoter element, optionally the promoter element is SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof; optionally the synthetic muscle-specific promoter comprises or consists of SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), or SP0528 (SEQ ID NO: 4), or a functional variant of any of them; optionally the synthetic muscle-specific promoter comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1 to 4.

6. A synthetic muscle-specific promoter comprising a liver-detargeting element, optionally the liver-detargeting element comprising: - SEQ ID NO: 15 or a functional variant thereof; - SEQ ID NO: 13 or a functional variant thereof; - SEQ ID NO: 17 or a functional variant thereof; or - SEQ ID NO: 14 or a functional variant thereof, and optionally the liver detargeting element comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13-15 and 17.

7. below: - CRE0145 (SEQ ID NO: 10) or a functional variant thereof, - DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, - SCP1 (SEQ ID NO: 12) or a functional variant thereof or CRE0053 (SEQ ID NO: 26) or a functional variant thereof; and - at least one additional regulatory element and optionally at least one additional regulatory element is selected from the group consisting of: - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof; - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof; - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; - ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof a liver detargeting element selected from the group consisting of: an intron selected from the tMCK SA / SD intron (SEQ ID NO: 29) and the MVM cleavage intron (SEQ ID NO: 30); and Optionally, the synthetic muscle-specific promoter comprises or consists of a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0530 (SEQ ID NO: 27), or SP0531 (SEQ ID NO: 28), or any one of SEQ ID NOs: 1-4, 27-28.

8. 8. An expression cassette comprising the synthetic muscle-specific promoter of any one of claims 5 to 7 operably linked to a sequence encoding an expression product, or an expression cassette comprising the synthetic muscle-specific promoter comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, operably linked to an expression product and a target sequence for miR122.

9. 9. A vector comprising the synthetic muscle-specific promoter of any one of claims 5 to 7 or the expression cassette of claim 8, wherein the vector is an AAV, adenoviral, retroviral or lentiviral vector, as appropriate.

10. A gene therapy AAV vector comprising an expression cassette, the expression cassette comprising a synthetic promoter operably linked to a sequence encoding an expression product and a target sequence for miR122, optionally the target sequence for miR122 comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 19 or a functional variant thereof, optionally the target sequence for miR122 comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO:

19.

11. A virion comprising the vector of claim 9 or 10.

12. 12. A pharmaceutical composition comprising the synthetic muscle-specific promoter of any one of claims 5 to 7, the expression cassette of claim 8, the vector of claim 9 or 10, or the virion of claim 11.

13. 13. The synthetic muscle-specific promoter of any one of claims 5 to 7, the expression cassette of claim 8, the vector of claim 9 or 10, the virion of claim 11, or the pharmaceutical composition of claim 12 for use in therapy.

14. A cell comprising the synthetic muscle-specific promoter of any one of claims 5 to 7, the expression cassette of claim 8, the vector of claim 9 or 10, or the virion of claim 11.

15. 13. The synthetic muscle-specific promoter of any one of claims 5 to 7, the expression cassette of claim 8, the vector of claim 9 or 10, the virion of claim 11, or the pharmaceutical composition of claim 12 for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

16. 10. A method for producing an expression product ex vivo, comprising providing an expression cassette according to claim 8 in a muscle cell and expressing the expression product present in the expression cassette.

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