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 capable of enhancing muscle-specific expression of genes. The present invention also relates to expression constructs, vectors, and cells comprising such muscle-specific regulatory nucleic acid sequences, as well as methods for their use. The regulatory nucleic acid sequences are particularly useful in gene therapy applications, but are also useful in other areas, such as bioprocessing and biotechnology.

Description

[Technical field]

[0001] The present invention relates to regulatory nucleic acid sequences, in particular muscle-specific synthetic promoters, elements thereof, and other such nucleic acid sequences capable of enhancing muscle-specific expression of genes. The present invention also relates to expression constructs, vectors, and cells comprising such muscle-specific regulatory nucleic acid sequences, and methods of using the same. The regulatory nucleic acid sequences are particularly useful in gene therapy applications, but are also useful in other areas such as bioprocessing and biotechnology. [Background technology]

[0002] The following discussion is presented to aid the reader in understanding the present disclosure, and is not intended to constitute any admission of content or pertinence of prior art.

[0003] In many areas, including gene therapy, it is desirable to provide regulatory nucleic acid sequences capable of driving 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 correct or enhance expression of various muscle proteins, such as dystrophin and sarcoglycan. 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 to express therapeutic proteins for the treatment of other conditions, such as congestive heart failure.

[0005] A variety of vectors have been used to deliver genes into muscle cells, including adenoviruses, retroviruses, lentiviruses, and adeno-associated viruses (AAV), as well as non-viral vectors such as plasmids. Adenovirus vectors have a relatively large cloning capacity and allow efficient transduction of some cells. However, adenovirus vectors face significant challenges in terms of the strong immune response they tend to induce. Retrovirus and lentivirus vectors stably integrate into the genome, which is associated with both advantages and disadvantages. Lentivirus vectors transduce both dividing and non-dividing cells, whereas most conventional retrovirus vectors transduce only 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 utility in a clinical context is less clear. AAV vectors are particularly attractive for gene therapy applications in muscle. AAV vectors display natural tropism for muscle cells, are capable of driving long-term expression of therapeutic payloads, and elicit minimal immune responses. Despite the ability of some gene therapy vectors to preferentially transduce muscle cells, off-target transduction still occurs. Several phase 1 and phase 2 clinical trials have been reported for the use of AAV serotypes 1, 2, and chimeric 2.5 for the treatment of Duchenne muscular dystrophy (DMD) and alpha-1 antitrypsin deficiency (D.E. Bowles, S. W.J.McPhee, C. Li, S.J. Gray, J.J. Samulski, A.S. Camp, J. Li, B. Wang, P.E. Monahan, J.E. Rabinowitz, J.C. Grieger, La. Govindasamy, M. Agbandje-McKenna, X. Xiao, and R.J.Samulski、「Phase 1 gene therapy for Duchenne Muscular Dystrophy using a translational optimised AAV vector」、Molecular Therapy、20、443~455(2012);M. L. Brantly、J. D. Chulay、L. Wang、C. Mueller、M. Humphries、L. T. Spencer、F. Rouhani、T. J. Conlon、R. Calcedo、M. R. Berts、C. Spencer、B. J. Byrne、J. M. Wilson、T. R. Flotte、「Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAVl-AAT gene therapy」、Proceedings of National Academy of Sciences of United States of America 106、16363~16368 (2009);T. R. Flotte、M. L. Brantly、L. T. Spencer、B. J. Byrne、C. T. Spencer、D. J. Baker、M. Humphries、「15 Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1 -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))。.

[0006] It is desirable to provide a system that regulates gene expression in a muscle-specific manner. Ideally, such a system would be highly specific to muscle (thereby avoiding or minimizing off-target expression in non-target tissues) and also potent, i.e., driving high expression levels in muscle. Furthermore, it may be desirable to provide a system that regulates gene expression primarily in a skeletal muscle-specific manner or primarily in a cardiac muscle-specific manner. Such a system may be incorporated into expression constructs and expression vectors for gene-specific expression desired for the treatment of skeletal or cardiac muscle diseases or disorders. It is hypothesized that the use of cis-acting regulatory elements provides both specificity and activity. Typically, this concerns cis-regulatory enhancer sequences, i.e., nucleic acid sequences that act in cis to increase the activity of a promoter.

[0007] A variety of muscle-specific promoters are known in the art and are typically derived from genes expressed primarily in muscle, such as those encoding desmin, skeletal actin, cardiac α-actin, muscle creatine kinase (CKM), myosin heavy and light chains, and troponin T / I. The C5-C12 promoters represent known synthetic promoters.

[0008] Regulatory sequences of short length are particularly desirable to minimize the proportion of the gene therapy vector occupied by the regulatory sequence, which is particularly important for gene therapy vectors with limited capacity (payload), such as AAV vectors. In addition, it is desirable to provide a strong promoter, but in many cases, it is desirable for the skilled artisan to be able to select an appropriate promoter with the desired capacity, for example, from a wide range of promoters with diverse capabilities. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 6,200,560 [Patent Document 2] U.S. Patent No. 6,221,349

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[0011] There remains a need in the art for regulatory nucleic acids capable of driving the expression of muscle-specific genes. In particular, there is a need for muscle-specific regulatory sequences of small size (e.g., promoters, cis-regulatory modules, cis-regulatory elements, and minimal or proximal promoter elements) that can be incorporated into expression constructs and vectors for muscle-specific expression of a desired gene (e.g., a therapeutic transgene in the context of gene therapy). Furthermore, there is a need for muscle-specific regulatory sequences of small size that are primarily active in skeletal or cardiac muscle and that can be incorporated into expression constructs and vectors for skeletal or cardiac muscle-specific expression of a desired gene. [Means for solving the problem]

[0012] In a first aspect of the present invention, a) a synthetic muscle-specific promoter comprising or consisting of a sequence according to any one of SEQ ID NOs: 1 to 29, 66, or a functional variant thereof; or b) A synthetic muscle-specific promoter comprising or consisting of a cis-regulatory module (CRM) comprising a sequence according to any one of SEQ ID NOs: 30 to 47, or a functional variant thereof. is provided.

[0013] 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 any one of SEQ ID NOs:1-29, 66.

[0014] In some embodiments, the synthetic muscle-specific CRM comprises 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: 30-47. In some embodiments, a synthetic muscle-specific cis-regulatory module (CRM) in accordance with the invention comprises: - CRE0119 (SEQ ID NO: 48) or a functional variant thereof; - CRE0127 (SEQ ID NO: 49) or a functional variant thereof; - CRE0137 (SEQ ID NO: 50) or a functional variant thereof; - CRE0138 (SEQ ID NO: 51) or a functional variant thereof; - CRE0139 (SEQ ID NO: 52) or a functional variant thereof; - CRE0143 (SEQ ID NO: 53) or a functional variant thereof; - CRE0145 (SEQ ID NO: 54) or a functional variant thereof; - CRE0077 (SEQ ID NO: 55) or a functional variant thereof; - DES_MT_enhancer_48bp (SEQ ID NO: 56) or a functional variant thereof; - CRE0075 (SEQ ID NO: 57) or a functional variant thereof; - CRE0083 (SEQ ID NO: 58) or a functional variant thereof; - Ch2EnhMYL1_3_v1 (SEQ ID NO: 59) or a functional variant thereof; - CRE0050 (SEQ ID NO: 60) or a functional variant thereof; - CRE0031 (SEQ ID NO: 67) or a functional variant thereof; and - CRE0069 (SEQ ID NO: 61) or a functional variant thereof The present invention comprises two or more operably linked cis-regulatory elements (CREs) selected from the group consisting of:

[0015] In some embodiments, the synthetic muscle-specific promoter according to b) comprises a CRM as specified above operably linked to a promoter element (typically a minimal promoter or a proximal promoter), the proximal promoter preferably being a muscle-specific proximal promoter.

[0016] In some embodiments, a synthetic muscle-specific promoter according to the invention comprises: - BG_mp (SEQ ID NO: 62) or a functional variant thereof; - SCP1 (SEQ ID NO: 63) or a functional variant thereof; - CRE0070 (SEQ ID NO: 64) or a functional variant thereof; - CRE0037 (SEQ ID NO: 68) or a functional variant thereof; and - CRE0053 (SEQ ID NO: 65) or a functional variant thereof operably linked to at least one promoter element selected from the group consisting of: - CRE0119 (SEQ ID NO: 48) or a functional variant thereof; - CRE0127 (SEQ ID NO: 49) or a functional variant thereof; - CRE0137 (SEQ ID NO: 50) or a functional variant thereof; - CRE0138 (SEQ ID NO: 51) or a functional variant thereof; - CRE0139 (SEQ ID NO: 52) or a functional variant thereof; - CRE0143 (SEQ ID NO: 53) or a functional variant thereof; - CRE0145 (SEQ ID NO: 54) or a functional variant thereof; - CRE0077 (SEQ ID NO: 55) or a functional variant thereof; - DES_MT_enhancer_48bp (SEQ ID NO: 56) or a functional variant thereof; - CRE0075 (SEQ ID NO: 57) or a functional variant thereof; - CRE0083 (SEQ ID NO: 58) or a functional variant thereof; - Ch2EnhMYL1_3_v1 (SEQ ID NO: 59) or a functional variant thereof; - CRE0050 (SEQ ID NO: 60) or a functional variant thereof; - CRE0031 (SEQ ID NO: 67) or a functional variant thereof; and - CRE0069 (SEQ ID NO: 61) or a functional variant thereof The present invention comprises at least one cis-regulatory element (CRE) selected from the group consisting of:

[0017] Thus, the present invention provides a variety of synthetic muscle-specific promoters, and functional variants thereof. It is generally preferred that a synthetic promoter according to the present invention that is a variant of any one of SEQ ID NOs: 1-29, 66 retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter. Suitably, the activity is assessed using one of the examples described herein, although other methods may also be used.

[0018] In another aspect of the invention, a muscle-specific cis-regulatory element (CRE) is provided that comprises or consists of a sequence according to any one of SEQ ID NOs: 48-61, 67, or any functional variant thereof. In some embodiments, the muscle-specific CRE 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: 48-61, or 67, or any point therein.

[0019] It is generally preferred that a muscle-specific CRE according to the invention that is a variant of any one of SEQ ID NOs: 48-61, or 67 retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of a reference CRE. Suitably, said activity is assessed using one of the examples described herein, although other methods may also be used.

[0020] In another aspect of the invention, there is provided a synthetic promoter comprising a CRE according to any aspect of the invention.

[0021] In another aspect of the invention, a CRM is provided that comprises a sequence according to any one of SEQ ID NOs: 30-47, or functional variants thereof. In some embodiments, the 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: 30-47, or any point therein.

[0022] In some embodiments, the CRM comprises: - CRE0119 (SEQ ID NO: 48) or a functional variant thereof; - CRE0127 (SEQ ID NO: 49) or a functional variant thereof; - CRE0137 (SEQ ID NO: 50) or a functional variant thereof; - CRE0138 (SEQ ID NO: 51) or a functional variant thereof; - CRE0139 (SEQ ID NO: 52) or a functional variant thereof; - CRE0143 (SEQ ID NO: 53) or a functional variant thereof; - CRE0145 (SEQ ID NO: 54) or a functional variant thereof; - CRE0077 (SEQ ID NO: 55) or a functional variant thereof; - DES_MT_enhancer_48bp (SEQ ID NO: 56) or a functional variant thereof; - CRE0075 (SEQ ID NO: 57) or a functional variant thereof; - CRE0083 (SEQ ID NO: 58) or a functional variant thereof; - Ch2EnhMYL1_3_v1 (SEQ ID NO: 59) or a functional variant thereof; - CRE0050 (SEQ ID NO: 60) or a functional variant thereof; - CRE0031 (SEQ ID NO: 67) or a functional variant thereof; and - CRE0069 (SEQ ID NO: 61) or a functional variant thereof The present invention comprises two or more operably linked cis-regulatory elements (CREs) selected from the group consisting of:

[0023] In a further aspect of the invention there is provided a minimal or proximal promoter comprising or consisting of a sequence according to any one of SEQ ID NOs: 62-65, 68, or a functional variant thereof. In another aspect of the invention there is provided a synthetic promoter comprising said minimal or proximal promoter, suitably a synthetic muscle-specific promoter comprising said minimal or proximal promoter. Suitably the functional variant comprises a sequence which is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 62-65 or 68. As indicated above "promoter element" may be used to refer to a minimal promoter or a proximal promoter.

[0024] The CRE, CRM, minimal / proximal promoter, and synthetic promoter of the present invention may be active in a variety of muscle tissues, particularly, but not exclusively, in skeletal and / or cardiac muscle. A CRE, CRM, promoter element, or synthetic promoter that is active in at least one muscle tissue type, or at least one muscle cell type, may be referred to as "muscle-specific." For convenience, a muscle-specific CRE, muscle-specific CRM, muscle-specific promoter element, or muscle-specific synthetic promoter may be further subdivided into subspecies depending on whether the CRE, CRM, promoter element, or synthetic promoter is primarily active in skeletal muscle or cardiac muscle.

[0025] In some embodiments, the cis-regulatory elements, CRMs, promoter elements, and synthetic promoters of the present invention are skeletal muscle specific. Cis-regulatory elements, CRMs, promoter elements, and synthetic promoters that are primarily active in skeletal muscle and less active or inactive in cardiac muscle are referred to as "skeletal muscle specific." Non-limiting examples of skeletal muscle are quadriceps, diaphragm, tibialis anterior, and soleus.

[0026] In some embodiments, the cis-regulatory elements, CRMs, promoter elements, and synthetic promoters of the invention are cardiac muscle specific. Cis-regulatory elements, CRMs, promoter elements, and synthetic promoters of the invention that are primarily active in cardiac muscle and have little or no activity in skeletal muscle are referred to as "cardiac muscle specific."

[0027] In some embodiments, the cis-regulatory elements, CRMs, promoter elements, and synthetic promoters of the invention are both skeletal-specific and cardiac muscle-specific.

[0028] In some embodiments, skeletal muscle-specific CREs, CRMs, promoter elements, and synthetic promoters may be preferred, as these CREs, CRMs, promoter elements, and synthetic promoters may be preferred promoters when activity is required in skeletal muscle, but with little or no activity in the heart (myocardium). Examples of synthetic muscle-specific promoters designed to be active primarily in skeletal muscle (skeletal muscle-specific synthetic promoters) include SP0497, SP0498, SP0499, SP0500, SP0501, SP0502, SP0503, SP0504, SP0505, SP0506, SP0507, SP0508, SP0509, SP0510, SP0511, SP0512, SP0513, SP0514, SP0515, SP0516, SP0517, SP0518, SP0519, SP0520, SP0521, SP0522, SP4169, SP0523, and SP0524. Examples of preferred synthetic skeletal muscle-specific promoters are SP0498, SP0500, SP0505, SP0508, SP0509, SP0513, SP0519, SP0522, and SP0524. Skeletal muscle-specific promoters can be active in fast and / or slow muscles. In some embodiments, CRE, CRM, promoter elements, and synthetic promoters that are active in fast muscles and are skeletal muscle-specific may be preferred. In some embodiments, CRE, CRM, promoter elements, and synthetic promoters that are active in slow muscles and are skeletal muscle-specific may be preferred. In some embodiments, CRE, CRM, promoter elements, and synthetic promoters that are active in both slow and fast muscles and are skeletal muscle-specific may be preferred. Examples of skeletal muscle-specific promoters designed to be active in slow muscles are SP0500, SP0501, and SP0514.

[0029] A skeletal muscle-specific promoter may be primarily active in skeletal muscle, but may also have low activity in cardiac muscle. Examples of synthetic muscle-specific promoters that are designed to be primarily active in skeletal muscle, but are also expected to have low activity in cardiac muscle, include SP0497, SP0498, SP0499, and SP0512.

[0030] A skeletal muscle-specific promoter may be primarily active in skeletal muscle, but may also have activity in cardiac muscle. Examples of synthetic muscle-specific promoters that are designed to be primarily active in skeletal muscle, but are also expected to have activity in cardiac muscle, include SP0502, SP0515, SP0521, SP4169, SP0522, SP0523, and SP0524.

[0031] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of a sequence according to any one of SEQ ID NO:1 (SP0497), SEQ ID NO:4 (SP0500), SEQ ID NO:5 (SP0501), SEQ ID NO:10 (SP0506), SEQ ID NO:12 (SP0508), SEQ ID NO:14 (SP0510), SEQ ID NO:18 (SP0514), SEQ ID NO:23 (SP0519), SEQ ID NO:24 (SP0520), SEQ ID NO:25 (SP0521), and SEQ ID NO:26 (SP4169) is particularly preferred. In some embodiments, a synthetic muscle-specific promoter comprising or consisting of a sequence according to any one of SEQ ID NO:4 (SP0500), SEQ ID NO:14 (SP0510), SEQ ID NO:18 (SP0514), and SEQ ID NO:23 (SP0519) is particularly preferred. In some embodiments, certain muscle-specific promoters are more highly expressed in certain muscles than in other muscles, e.g., cardiac muscle, skeletal muscle, with SEQ ID NO:18 (SP0514) and SEQ ID NO:23 (SP0519) being particularly preferred.

[0032] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 4 (SP0500) or a functional variant thereof is particularly preferred. SP0500 is primarily active in certain muscles, such as cardiac muscle, but activity is also observed in skeletal muscle; SP0500 is highly expressed in cardiac muscle compared to skeletal muscle (see, e.g., Figures 5, 6, and 12). Thus, SP0500 is a strong cardiac muscle-specific promoter and is less than 270 nucleotides in length.

[0033] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 27 (SP0522) or a functional variant thereof is particularly preferred. SP0522 is primarily active in cardiac muscle, but activity is also observed in skeletal muscle; SP0522 is highly expressed in cardiac muscle compared to skeletal muscle (see, for example, Figures 5, 6, and 17). Thus, SP0522 is a strong cardiac muscle-specific promoter and is less than 240 nucleotides in length.

[0034] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 29 (SP0524) or a functional variant thereof is particularly preferred. SP0524 is active in cardiac muscle and skeletal muscle; SP0524 exhibits comparable expression levels in cardiac muscle and skeletal muscle (see, for example, Figures 5, 6, and 18). Thus, SP0524 is a muscle-specific promoter that is less than 250 nucleotides in length.

[0035] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO:22 (SP0518) or a functional variant thereof is preferred. SP0518 is active in skeletal muscle, but activity is also observed in cardiac muscle.

[0036] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 11 (SP0507) or a functional variant thereof is preferred. SP0507 is active in skeletal and cardiac muscle.

[0037] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 18 (SP0514) or a functional variant thereof is preferred. SP0514 is active in cardiac muscle, and activity is also observed in skeletal muscle; however, SP0514 is highly expressed in cardiac muscle compared to skeletal muscle (see, e.g., Figures 5, 6, and 14).

[0038] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO: 23 (SP0519) or a functional variant thereof is preferred. SP0519 is active in skeletal muscle, and activity is also observed in cardiac muscle; however, SP0519 has increased expression in some skeletal muscle types compared to cardiac muscle (see, e.g., Figures 5, 6, and 16).

[0039] In some embodiments, synthetic muscle-specific promoters comprising or consisting of SEQ ID NO:4 (SP0500) or SEQ ID NO:27 (SP0522), or functional variants thereof, are particularly preferred. SP0500 and SP0522 are primarily active in cardiac muscle, but also have activity in skeletal muscle.

[0040] In some embodiments, synthetic muscle-specific promoters comprising or consisting of SEQ ID NO:4 (SP0500), SEQ ID NO:27 (SP0522), and SEQ ID NO:29 (SP0524), or functional variants thereof, are particularly preferred.

[0041] In some embodiments, synthetic muscle-specific promoters comprising or consisting of SEQ ID NO:4 (SP0500), SEQ ID NO:27 (SP0522), SEQ ID NO:22 (SP0518), and SEQ ID NO:29 (SP0524), or functional variants thereof, are particularly preferred.

[0042] In some embodiments, synthetic muscle-specific promoters comprising or consisting of SEQ ID NO:4 (SP0500), SEQ ID NO:27 (SP0522), SEQ ID NO:22 (SP0518), SEQ ID NO:29 (SP0524), SEQ ID NO:11 (SP0507), SEQ ID NO:18 (SP0514), and SEQ ID NO:23 (SP0519), or functional variants thereof, are particularly preferred.

[0043] In some embodiments, a synthetic muscle-specific promoter comprising or consisting of SEQ ID NO:66 (SP0321) or a functional variant thereof is particularly preferred. SP0321 is expected to be active in muscle. SP0321 is also expected to be active in lung. Thus, SP0321 is expected to be both a muscle-specific promoter and a lung-specific promoter.

[0044] In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a higher activity in the diaphragm than CK8, CK7, or CMV. In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a higher activity in the tibialis anterior than CK8, CK7, or CMV. In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a higher activity in the heart than CK8, CK7, or CMV. In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a higher activity in the quadriceps than CK8, CK7, or CMV. In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a higher activity in the soleus than CK8, CK7, or CMV. In some embodiments, a synthetic muscle-specific synthetic promoter according to the invention has a lower activity in the liver than CK8, CK7, or CMV. In some embodiments, a high degree of activity is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% or more activity, or at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more times activity compared to a control, e.g., CK8, CK7, or CMV. Exemplary methods for testing the activity of synthetic promoters can be found in Example 2 and Example 3.

[0045] In some embodiments, a synthetic muscle-specific promoter in accordance with the invention is also active in other tissues or cells, hi some embodiments, a synthetic muscle-specific promoter in accordance with the invention is also active in one or more of the following groups of tissues or cells: CNS, liver, kidney, spleen, lung, and duodenum.

[0046] In some embodiments, a synthetic muscle-specific promoter according to the invention comprises one of the combinations of a CRE or a functional variant thereof operably linked to a promoter element or a functional variant thereof as set forth in Table 5 below.

[0047] In any of the combinations of CREs or functional variants thereof disclosed herein, the listed CREs may be present in any order. In some preferred embodiments, the CREs are present in the order listed (i.e., in order from upstream to downstream, with reference to their position in relation to the operably linked promoter element or gene). In any of the combinations of CREs or functional variants thereof disclosed herein, some or all of the listed CREs may be suitably located adjacent to each other within the CRM (i.e., without any intervening CREs or other regulatory elements). The CREs may be contiguous or non-contiguous (i.e., they may be located immediately adjacent to each other or may be separated by a spacer or other sequence). In some embodiments, it is preferred that some or all of the CREs are contiguous. In some preferred embodiments, the CREs or functional variants thereof are provided adjacent to each other in the order listed. For example, a synthetic muscle-specific CRM may include CRE0077 immediately upstream of CRE0075, etc. In some embodiments, the promoter element is downstream of the CRE and is typically adjacent to the proximal CRE. The promoter element may be contiguous with the adjacent CRE or may be separated by a spacer.

[0048] In a further aspect of the invention there is provided an expression cassette comprising the synthetic muscle-specific promoter of any aspect of the invention, or the synthetic skeletal muscle-specific promoter, operably linked to a sequence, suitably a gene, e.g. a transgene, that encodes an expression product. In some embodiments, the expression product is a therapeutic expression product.

[0049] The therapeutic expression product may be a therapeutic expression product useful in the treatment of any condition where expression in muscle may be useful, for example for the treatment of a muscle condition, or where secretion of a therapeutic expression product from muscle is desired. The therapeutic expression product may be a therapeutic expression product useful in the treatment of a cardiovascular condition, or heart disease and disorders, such as heart failure or CHF. The therapeutic expression product may be a therapeutic expression product useful in the treatment of a skeletal muscle condition, disease, or disorder, such as any type of muscular dystrophy.

[0050] The sequence encoding the therapeutic expression product may be one or more genes that replace the function of one or more genes that are impaired or non-functional in an autosomal dominant or recessive disease, an X-linked dominant or recessive disease, or a Y-linked dominant or recessive disease. The sequence encoding the therapeutic expression product may be one or more genes that code for the wild-type replacement counterpart of a protein that is impaired or non-functional in an autosomal dominant or recessive disease, an X-linked dominant or recessive disease, or a Y-linked dominant or recessive disease. The sequence encoding the therapeutic expression product may be a gene that code for the wild-type replacement counterpart of a protein that is impaired or non-functional in an autosomal recessive disease.

[0051] The sequence encoding the therapeutic expression product may be a gene found in the human genome or a synthetic gene. Suitably, the therapeutic expression product may be a gene found in the human genome. The sequence encoding the therapeutic expression product may be the dysferlin gene (DYSF gene) and the therapeutic expression product may be a dysferlin protein. Mutations in the DYSF gene that impair the function of the dysferlin protein result in dysferlinopathy. Examples of dysferlinopathy caused by mutations in the DYSF gene include Miyoshi myopathy, type 2B limb-girdle muscular dystrophy, and distal myopathy. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, and the sequence encoding the therapeutic expression product is the DYSF gene, may be useful in the treatment of dysferlinopathies such as Miyoshi myopathy, type 2B limb-girdle muscular dystrophy, and distal myopathy.

[0052] The sequence encoding the therapeutic expression product can be the dystrophin gene (DMD gene), and the therapeutic expression product can be a dystrophin protein. Mutations in the DMD gene that impair the function of the dystrophin protein result in Becker muscular dystrophy or Duchenne muscular dystrophy, which are X-linked recessive muscular dystrophic disorders characterized by muscle weakness. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, and the sequence encoding the therapeutic expression product is the DMD gene, can be useful in the treatment of Becker muscular dystrophy or Duchenne muscular dystrophy.

[0053] The DMD gene is the largest known gene in humans (about 2.4 million base pairs). Thus, the full-length DMD gene is too large to be packaged into some viral vectors with limited capacity (payload), such as AAV vectors. A short form of the DMD gene (called mini-dystrophin) has been used to address this issue. However, even mini-dystrophin is still quite large (e.g., 3.5-4 kB), making it still difficult to package into AAV. Thus, within the expression cassette, synthetic muscle-specific promoters that are short in length (e.g., less than 400 nucleotides in length, less than 350 nucleotides in length, preferably less than 300 nucleotides in length, even more preferably less than 290 nucleotides in length, and most preferably less than 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 70, 68 nucleotides in length) may be particularly preferred, in which case the sequence encoding the therapeutic expression product is the DMD gene or a mini-variant of the DMD gene (mini-dystrophin).

[0054] In some embodiments, an expression cassette comprises a synthetic muscle-specific promoter according to any aspect of the invention or a synthetic skeletal muscle-specific promoter operably linked to a short form (mini-dystrophin) of the DMD gene. In some embodiments, an expression cassette comprises a synthetic muscle-specific promoter according to SEQ ID NO:29 (SP0524), or a functional variant thereof, operably linked to a short form (mini-dystrophin) of the DMD gene.

[0055] The sequence encoding the therapeutic expression product may be a miRNA or snRNA that targets and reduces the expression of the endogenous DUX4 gene. Gain-of-function mutations in the DUX4 gene result in loss of repression of the DUX4 transcription factor, which results in deleterious gene expression changes that cause facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant muscular dystrophic disorder characterized by progressive muscle dysfunction. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the present invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding the therapeutic expression product is a miRNA or snRNA that targets and reduces the expression of the endogenous DUX4 gene, may be useful in the treatment of FSHD.

[0056] The sequence encoding the therapeutic expression product may be an miRNA or snRNA that targets and reduces expression of the endogenous myotonin protein kinase gene (DMPK gene). Mutations in the DMPK gene result in myotonic dystrophy type 1 (DM1), an autosomal dominant muscular dystrophic disorder characterized by impaired muscle function. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding a therapeutic expression product is an miRNA or snRNA that targets and reduces expression of the endogenous DMPK gene, may be useful in treating DM1. In some embodiments, the expression cassette may comprise one or more synthetic muscle-specific promoters or synthetic skeletal muscle-specific promoters according to any aspect of the invention operably linked to an miRNA or snRNA that targets and reduces expression of the endogenous DMPK gene and a wild-type replacement DMPK gene. In some embodiments, an expression cassette can include one or more synthetic muscle-specific promoters or synthetic skeletal muscle-specific promoters according to any aspect of the invention operably linked to miRNA or snRNA that target and reduce expression of the endogenous DMPK gene, and the wild-type replacement MBNL1 gene. In some embodiments, an expression cassette can include one or more synthetic muscle-specific promoters or synthetic skeletal muscle-specific promoters according to any aspect of the invention operably linked to miRNA or snRNA that target and reduce expression of the endogenous DMPK gene, the wild-type replacement DMPK gene, and the wild-type replacement MBNL1 gene.

[0057] The sequence encoding the therapeutic expression product may be an miRNA or snRNA that targets and reduces expression of the endogenous cellular nucleic acid binding protein gene (CNBP gene). Gain-of-function mutations in the CNBP gene result in myotonic dystrophy type 2 (DM2), an autosomal dominant muscular dystrophic disorder characterized by impaired muscle function. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding the therapeutic expression product is an miRNA or snRNA that targets and reduces expression of the endogenous CNBP gene, may be useful in the treatment of DM2.

[0058] The sequence encoding the therapeutic expression product may be an miRNA or snRNA that targets and reduces the expression of the endogenous poly(A) binding protein 1 gene (PABPN11 gene). Gain-of-function mutations in the CNBP gene result in oculopharyngeal muscular dystrophy, an autosomal dominant or autosomal recessive muscular dystrophic disorder. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding the therapeutic expression product is an miRNA or snRNA that targets and reduces the expression of the endogenous PABPN1 gene, may be useful in the treatment of oculopharyngeal muscular dystrophy. In some embodiments, the expression cassette may comprise one or more synthetic muscle-specific promoters or synthetic skeletal muscle-specific promoters according to any aspect of the invention operably linked to an miRNA or snRNA that targets and reduces the expression of the endogenous PABPN1 gene and the synthetic replacement PABN1 gene. Suitably, the synthetic replacement gene is modified or codon optimised so as not to be targeted by miRNA or snRNA that reduce expression of the endogenous PABPN1 gene.

[0059] The sequence encoding the therapeutic expression product may be the ClC-1 ion channel gene (CLCN1 gene) and the therapeutic expression product may be the ClC-1 ion channel. Mutations in the CLCN1 gene result in myotonia congenita, an autosomal dominant or autosomal recessive channelopathy affecting skeletal muscle. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding the therapeutic expression product is the CLCN1 gene, may be useful in the treatment of myotonia congenita.

[0060] The sequence encoding the therapeutic expression product is a voltage-gated sodium channel, Na v 1.4 gene (SCN4A gene), and the therapeutic expression product is a voltage-gated sodium channel Na v 1.4. Mutations in the SCN4A gene result in paramyotonia congenita, potassium-induced myotonia, hyperkalemic periodic paralysis, and hypokalemic periodic paralysis. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the present invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding a therapeutic expression product is the SCN4A gene, may be useful in the treatment of paramyotonia congenita, potassium-induced myotonia, hyperkalemic periodic paralysis, and hypokalemic periodic paralysis.

[0061] The sequence encoding a therapeutic expression product may be the SEPN1 gene or the RYR1 gene. Mutations in the SEPN1 gene or in the RYR1 gene result in Multi / Minicore disease. An expression cassette comprising a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding a therapeutic expression product, wherein the sequence encoding a therapeutic expression product is the SEPN1 gene or the RYR1 gene, may be useful in the treatment of Multi / Minicore disease.

[0062] Suitably, the sequence encoding the therapeutic expression product may be a synthetic gene. A synthetic gene may be a gene found in the human genome that has been shortened, for example to allow packaging in an AAV vector. A synthetic gene may be a gene found in the human genome that has been optimized for rapid translation, for example a codon-optimized construct, or alternatively an artificial construct. Codon optimization is well understood by those skilled in the art and refers to adjusting synonymous codons to match the codon bias of the host organism in order to improve gene expression and increase the efficiency of translation. A synthetic gene may be a gene found in the human genome that has been modified to localize its expressed protein specifically within the cell, for example a gene modified to include a nuclear localization signal, so that a protein with this signal is imported into the cell nucleus by nuclear transport. A synthetic gene may be a gene found in the human genome that has been modified to be secreted more efficiently, for example a gene modified to include a secretion signal, so that a protein with this signal is targeted for translocation across the endoplasmic reticulum membrane and secretion into the environment. In some embodiments, a synthetic gene may be a gene found in the human genome that has been modified to reduce the immunogenicity of its expression product, for example by removal of B-cell epitopes. A synthetic gene may be a gene found in the human genome that has been modified to increase or decrease its function. A synthetic gene may be a gene found in the human genome that has been modified to prevent it from being silenced by a specific miRNA or a specific snRNA. Any gene found in the human genome may be modified by one or more of the modifications described above, resulting in a synthetic gene. In some embodiments, the modified gene is any one of the genes listed in any of the aspects herein.

[0063] The sequence encoding the therapeutic expression product can be a synthetic dysferlin gene (DYSF gene), and the therapeutic expression product can be a synthetic dysferlin protein. The sequence encoding the therapeutic expression product can be a truncated synthetic dysferlin gene (DYSF gene). The sequence encoding the therapeutic expression product can be a truncated synthetic dysferlin gene (DYSF gene) to allow packaging in an AAV vector. The sequence encoding the therapeutic expression product can be a truncated synthetic dysferlin gene (DYSF gene) to less than 6 kb, less than 5.5 kb, less than 5 kb, less than 4.5 kb, less than 4 kb, less than 3.5 kb, or less than 3 kb. The sequence encoding the therapeutic expression product can be a synthetic dysferlin-like molecule truncated to be suitable for packaging in a single AAV vector. The sequence encoding the therapeutic expression product can be Nano-Dysferlin, as detailed in Table 1 (Table 2) and Figure 1A of (Llanga et al., 2017).

[0064] In some preferred embodiments, the expression cassette comprises a muscle-specific promoter according to any aspect of the invention operably linked to Nano-Dysferlin as detailed in Table 1 and FIG. 1A of (Llanga et al., 2017).

[0065] The therapeutic 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 that encodes a protein that inhibits phosphatase activity, such as a transcription factor. The modulator can be a regulatory sequence that is integrated into or near an endogenous nucleic acid that encodes a protein that inhibits phosphatase activity. The modulator can be a nucleic acid that can result in a nucleic acid modulator of gene expression, such as an siRNA.

[0066] The therapeutic expression product may be an inhibitor of protein phosphatase 1 (PP1), e.g., I-1 polypeptide. Type 1 phosphatases include, but are not limited to, PP1cα, PP1cβ, PP1cδ, and PP1cγ. Phosphatase inhibitor 1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatases. Increasing the level or activity of I-1 may restore β-adrenergic responsiveness in failing human cardiomyocytes. Suitably, the I-1 protein may be constitutively active, such as an I-1 protein in which threonine 35 is replaced with glutamic acid rather than aspartic acid. The therapeutic expression product may be any one or more of the inhibitors selected from phosphatase inhibitor 2 (PP2), which is an endogenous nuclear inhibitor of protein phosphatase 1; okadaic acid or calyculin; and nippl.

[0067] The therapeutic expression product can be any protein that modulates cardiac activity, such as a type 1 phosphatase inhibitor, e.g., I-1, or a sarcoplasmic reticulum Ca2+ ATPase (SERCA), e.g., SERCA1 (e.g., 1a or 1b), SERCA2 (e.g., 2a or 2b), or SERCA3.

[0068] The therapeutic expression product can be a nucleic acid sequence encoding a mutant form of a 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 where at least one amino acid is constitutively unphosphorylated or mimics the unphosphorylated state in the mutant form. The therapeutic expression product can be adenyl cyclase 6 (AC6; also referred to as adenyl cyclase VI), S100A1, β-adrenergic receptor kinase-ct (βARKct), sarco / endoplasmic reticulum (SR) Ca-ATPase (SERCA2a), IL-18, VEGF, VEGF activator, urocortin, and B-cell lymphoma 2 (Bcl2)-associated agnoprotein 3 (BAG3).

[0069] The therapeutic expression product can be an inhibitor of a cytokine, such as an IL-18 inhibitor. The therapeutic expression product can encode beta-adrenergic signaling proteins (beta-ASPs), including beta-adrenergic receptors (beta-Ars), G-protein receptor kinase inhibitors (GRK inhibitors), and adenyl cyclase (Acs), to enhance cardiac function.

[0070] The therapeutic 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.

[0071] In a further aspect, a vector is provided comprising a synthetic muscle-specific promoter, a skeletal muscle-specific promoter or an expression cassette according to the invention. 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, suitably 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 noted to achieve efficient transduction in cardiac and skeletal muscles, and thus AAV9 and its derivatives represent one non-limiting example of a suitable AAV vector. In some embodiments, the rAAV vector is an AAV3b serotype, including but not limited to AAV3b265D virion, AAV3b265D549A virion, AAV3b549A virion, AAV3bQ263Y virion, or AAV3bSASTG virion (i.e., a virion comprising an AAV3b capsid, including a Q263A / T265 mutation). In some embodiments, the virion may be a theoretical haploid, or a chimera or any mutant, such that the capsid may be tailored for increased renewal in a desired location, e.g., heart or skeletal muscle. Other capsids may include capsids from any of the known AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV7, AAV10, etc. In some preferred embodiments, the AAV vector is AAV2i8. In some embodiments, the AAV vector is AAV9.

[0072] A vector according to the invention can be an AAV vector comprising a nucleic acid encoding a therapeutic expression product for the treatment of muscular dystrophy, where the nucleic acid is operably linked to a skeletal muscle-specific promoter or a muscle-specific promoter.

[0073] The vector according to the invention can be an AAV vector comprising a nucleic acid encoding a therapeutic expression product for the treatment of heart failure, where the nucleic acid is operably linked to a muscle-specific promoter, which can be a myocardial-specific promoter.

[0074] In a further aspect, a virion (viral particle) is provided that comprises the vector according to the present invention, suitably a viral vector. In some embodiments, the virion is an AAV virion. Suitable virions are described above.

[0075] In a further aspect, there is provided a pharmaceutical composition comprising a synthetic muscle-specific promoter, a synthetic skeletal muscle-specific promoter, an expression cassette, a vector, or a virion in accordance with the invention.

[0076] In a further aspect, there is provided a synthetic muscle-specific promoter, a synthetic skeletal muscle-specific promoter, an expression cassette, a vector, a virion, or a pharmaceutical composition according to the invention for use in therapy, i.e. prevention or treatment of a medical condition or disease. Suitably for use in the treatment of a subject in need thereof. Suitably, the condition or disease is associated with abnormal gene expression in a muscle cell (myocyte) or tissue, optionally with abnormal gene expression. Suitably, the condition or disease is associated with abnormal gene expression in skeletal muscle or skeletal tissue. Suitably, the condition or disease is associated with abnormal gene expression in cardiomyocytes or cardiac tissue. Suitably, there is provided a synthetic muscle-specific promoter, a synthetic skeletal muscle-specific promoter, an expression cassette, a vector, a virion, or a pharmaceutical composition according to the invention for use in expressing a therapeutic expression product in skeletal and / or cardiac muscle.

[0077] In one embodiment, the disease may be a skeletal muscle disease or disorder. In one embodiment, the disease may be a muscular dystrophy, such as Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy type 1, myotonic muscular dystrophy type 2, or oculopharyngeal muscular dystrophy. In one embodiment, the disease may be a myotonia, such as myotonia congenita, paramyotonia congenita, potassium-induced myotonia, hyperkalemic periodic paralysis, or hypokalemic periodic paralysis. In one embodiment, the disease may be a congenital muscle disorder selected from nemaline myopathy, multi / minicore disease, and centronuclear myopathy. In one embodiment, the disease may be selected from mitochondrial myopathies, periodic paralysis, inflammatory myopathies, metabolic myopathies, Brodie's myopathies, and hereditary inclusion body myopathies. Suitably, the use is for gene therapy, preferably for use in the treatment of diseases involving abnormal gene expression. Suitably, the gene therapy involves expression of a therapeutic expression product in muscle cells or muscle tissue, suitably skeletal muscle cells or tissue, and / or cardiomyocytes or cardiac tissue.

[0078] In one embodiment, the disease may be a cardiovascular condition or a heart disease and a heart disorder. In one embodiment, the disease may be a heart failure, such as congestive heart failure. In one embodiment, the disease may be selected from ischemia, arrhythmia, myocardial infarction (MI), abnormalities of cardiac contraction, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormalities of Ca2+ metabolism, and combinations thereof. In some embodiments, the disease may be selected from the group of congestive heart failure, cardiomyopathy, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, dysfunction of the conduction system, dysfunction of the coronary arteries, and pulmonary hypertension. In some embodiments, the disease may be selected from congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, cardiac arrhythmias, Danon disease, muscular dystrophy, muscle mass disorders, muscle degeneration, infectious myocarditis, drug-induced or toxin-induced myopathy, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease. Suitably, the use is for gene therapy, preferably for use in the treatment of diseases involving abnormalities in gene expression. Suitably, the gene therapy involves expression of a therapeutic expression product in muscle cells or muscle tissue, suitably expression of a therapeutic expression product in cardiac muscle cells or cardiac tissue, and / or suitably expression of a therapeutic expression product in skeletal muscle cells or skeletal muscle tissue.

[0079] In some embodiments, the methods and compositions disclosed herein may be used to treat a subject with myocardial injury, where the subject with myocardial injury has heart failure. In such embodiments, the subject with heart failure has a classification equivalent to class III or higher in the New York Heart Association (NYHA) classification system. In some embodiments, the subject with heart failure has a cardiovascular or cardiac disease selected from any of left ventricular remodeling, peripheral arterial occlusive disease (PAOD), dilated cardiomyopathy (DCM) including idiopathic dilated cardiomyopathy (IDCM), coronary artery disease, ischemia, arrhythmias, myocardial infarction (MI), cardiac contractility abnormalities, acute (decompensated) heart failure (AHF), Ca2+ metabolic abnormalities, myocardial ischemia, atherosclerosis, myocardial injury, idiopathic cardiomyopathy, genetic disorder-induced myocardial injury, cardiac arrhythmias, Danon disease, muscular dystrophy, muscle mass abnormalities, muscle degeneration, infectious myocarditis, drug-induced or toxin-induced myopathy, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease, and pulmonary hypertension.

[0080] In some embodiments, the methods and compositions disclosed herein may be used to treat a subject with a myocardial disorder, where the subject with a myocardial disorder has non-ischemic heart failure and / or a non-ischemic myocardial disorder, including, but not limited to, an acquired myocardial disorder, which is a myocardial disorder acquired as a result of an infection or toxin, or a congenital myocardial disorder or a genetic disorder with a cardiac condition.In some embodiments, the subject with a congenital cardiomyopathy or genetic disorder with cardiac symptoms is arrhythmogenic right ventricular cardiomyopathy, atrial myxoma, familial, atrial septal defect ostium primum, atrial septal defect sinus venosus, Barth syndrome, muscular dystrophy, Buerger's disease, cardioencephalomyopathy, chromosome 1p36 deletion syndrome, congenital generalized lipodystrophy type 4, congenital heart block, dilated cardiomyopathy, Duchenne muscular dystrophy (DMD), Fabry disease, familial atrial fibrillation, familial dilated cardiomyopathy, familial hypertrophic cardiomyopathy, familial progressive cardiac conduction defect, familial thoracic aortic aneurysm, and aortic resection, fibromuscular dysplasia, Friedreich's ataxia, Gaucher disease, glycogen storage disease (types 2, 3, or 4), His bundle tachycardia, Hurler syndrome, hypoplastic left heart syndrome, histiocytic cardiomyopathy of childhood, intracranial arteriovenous malformations, isobutyl-CoA dehydrogenase deficiency, kallikrein hypertension, Kawasaki disease, Kearns-Sayre syndrome, left ventricular noncompaction, limb-girdle muscular dystrophies (types 1B, 2E, 2F, 2M, 2C, and 2D), localized systemic sclerosis, type 1 long QT syndrome, lymphedema and cerebral arteriovenous abnormalities, lymphocytic vasculopathy inflammation, microcephaly-cardiomyopathy; mitochondrial encephalomyopathy lactic acidosis stroke-like episodes, mitochondrial triprotein deficiency, myotonic dystrophy type 1, neonatal stroke, Noonan syndrome types 1, 2, 3, 4, 5, and 6, perinatal cardiomyopathy, Peters Plus syndrome, PGM1-CDG, PHACE syndrome, phospholamban Arg14 deletion, postural orthostatic tachycardia syndrome, primary carnitine deficiency, progressive familial heart block (progressive familial heart block types 1A, 1B, and 2), pseudoaldosteronism type 2, pulmonary arterial hypertension , pulmonary atresia with intact ventricular septum, pulmonary atresia with ventricular septal defect, pulmonary valve stenosis, pulmonary vein stenosis, pulmonary artery stenosis, renal function deficiency hypertension, retinal artery aortic aneurysm with supravalvular pulmonary stenosis, right ventricular hypoplasia, sarcoidosis, Saengers syndrome, situs inversus, sudden arrhythmic death syndrome, supravalvular aortic stenosis, Swyer syndrome, TANGO2-associated metabolic encephalopathy and arrhythmia, TARP syndrome, tetralogy of Fallot, Timothy syndrome, tricuspid atresia, Vici syndrome, VLCAD deficiency, and Williams syndrome.

[0081] In some embodiments, the methods and compositions disclosed herein may be used to treat a subject with myocardial injury, where the subject with myocardial injury has an ischemic myocardial injury.

[0082] Suitably, the subject in need of treatment will exhibit symptoms characteristic of a skeletal muscle condition, for example muscular dystrophy as discussed above. Medical uses typically include ameliorating symptoms exhibited by a subject in need thereof by expressing a therapeutic amount of a therapeutic product. In some embodiments, the expression cassette comprises a gene encoding dysferlin or synthetic dysferlin operably linked to a skeletal muscle specific promoter or a muscle specific promoter. Treatment suitably includes expressing a therapeutic amount of dysferlin or synthetic dysferlin in skeletal muscle tissue of said subject. Suitably, expressing a therapeutic amount of dysferlin or synthetic dysferlin in skeletal muscle tissue alleviates symptoms of dysferlinopathy in the subject. Suitably, expressing a therapeutic amount of dysferlin or synthetic dysferlin in skeletal muscle tissue may alleviate skeletal muscle weakness and atrophy.

[0083] Suitably, the subject in need of treatment will present symptoms characteristic of a cardiovascular condition, such as heart disease or heart failure as discussed above. The medical use typically involves expressing a therapeutic amount of a therapeutic product to improve the symptoms presented by the subject in need thereof. In some embodiments, the expression cassette comprises a gene encoding a PP1 inhibitor operably linked to a muscle-specific promoter. The treatment suitably involves expressing a therapeutic amount of a PP1 inhibitor in the cardiac tissue of said subject. Suitably, expression of a therapeutic amount of a PP1 inhibitor in the cardiac tissue alleviates the symptoms of the subject's heart failure or heart disorder. Suitably, expression of a therapeutic amount of a PP1 inhibitor in the cardiac tissue may alleviate cardiac remodeling, improve physical exercise capacity, or improve cardiac contraction. Suitably, expression of a therapeutic amount of a PP1 inhibitor in the cardiac tissue may result in myocyte shortening, a decrease in the time constant for relaxation, and an accelerated calcium signal decay, an improvement in end-systolic pressure-volume relationship, and combinations thereof.

[0084] In a further aspect, a cell is provided comprising a synthetic muscle-specific promoter, synthetic skeletal 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 can be a muscle cell, optionally where the cell is a human muscle cell. Suitably, a human skeletal muscle cell or a human cardiomyocyte cell. The synthetic muscle-specific promoter, synthetic skeletal muscle-specific promoter, expression cassette, vector, or virion of the invention can be episomal or within the genome of the cell.

[0085] In a further aspect, there is provided a synthetic muscle-specific CRE, CRM, synthetic muscle-specific promoter, synthetic skeletal muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition described herein for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

[0086] In a further aspect, a method for producing an expression product is provided, the method comprising providing a synthetic muscle-specific or skeletal muscle-specific expression cassette of the invention into a muscle cell and expressing the gene present in the expression cassette. The method can be an in vitro method, an ex vivo method, or an in vivo method. In some embodiments, the method is a bioprocessing method. In one embodiment, the muscle cell is a skeletal muscle cell. In one embodiment, the muscle cell is a cardiomyocyte.

[0087] In a further aspect, a method of expressing a therapeutic transgene in a muscle cell is provided, comprising introducing into the muscle cell a synthetic or skeletal muscle-specific expression cassette, vector, or virion as described herein. In one embodiment, the muscle cell is a skeletal muscle cell. In one embodiment, the muscle cell is a cardiomyocyte.

[0088] In a further 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 comprising a sequence encoding a therapeutic product operably linked to a promoter according to the invention, as described herein; - expressing a therapeutic amount of a therapeutic product in the muscle of said subject; A method is provided that includes:

[0089] A therapeutic product is a product used to prevent, alleviate, cure, or positively modify a physiological process or disease.

[0090] In one embodiment, the muscle is a skeletal muscle cell or skeletal muscle tissue. In one embodiment, the muscle is a cardiac muscle cell or cardiac muscle tissue. Suitably, the subject treatment comprises expression of a therapeutic amount of a therapeutic product in the skeletal and / or cardiac muscle.

[0091] In some embodiments, the method comprises: - introducing into the muscle of the subject an expression cassette, vector, virion, or pharmaceutical composition described herein that includes a gene encoding a therapeutic product; - expressing a therapeutic amount of a therapeutic product in the muscle of said subject; Includes.

[0092] In one embodiment, the muscle is a skeletal muscle cell or skeletal muscle tissue. In one embodiment, the muscle is a cardiac muscle cell or cardiac muscle tissue. Suitably, the method comprises expression of a therapeutic amount of a therapeutic product in the skeletal and / or cardiac muscle of said subject.

[0093] Suitably, the method comprises administering to a subject a vector, virion or pharmaceutical composition as described herein. In some preferred embodiments, the vector is a gene therapy viral vector, preferably an AAV vector.

[0094] In some embodiments, the method of treatment comprises administering to the subject a viral vector comprising a sequence encoding a therapeutic product operably linked to a promoter according to the invention. In some preferred embodiments, the viral vector is administered to the subject by antegrade epicardial coronary artery injection (AECAI). In some particularly preferred embodiments, the viral gene therapy vector is administered to the subject by antegrade epicardial coronary artery injection (AECAI) via percutaneous femoral access. In some embodiments, the subject has heart failure or congestive heart failure. In some embodiments, the method of treatment of the subject is a method for the treatment of heart failure or congestive heart failure.

[0095] Further features and embodiments of the invention are described under the following paragraphs. Any feature or embodiment in any paragraph may be combined with any other feature or embodiment, or with any aspect of the invention, in any workable combination.

[0096] In some embodiments, a synthetic muscle-specific promoter comprises two or more promoter elements. Synthetic promoters comprising two or more promoter elements are referred to herein as "tandem promoters." CRE0138, designated herein as CRE, comprises the transcription start site of the TNNI2 gene and would be expected to act similarly as a promoter element. Thus, for example, SP0508 may be considered a tandem promoter since it comprises promoter elements CRE0138 and CRE0053. Similarly, SP0519 may be considered a tandem promoter since it comprises promoter elements CRE0138 and BG mp.

[0097] In some embodiments, the tandem promoter may include a promoter element immediately upstream of another promoter element. In some embodiments, the tandem promoter may include one or more CREs upstream of one or each of the promoter elements. In some embodiments, the tandem promoter may include one or more CREs between the promoter elements. In some embodiments, any one of the synthetic muscle-specific promoters disclosed herein may be operably linked to additional promoter elements. It will be appreciated that any other synthetic promoter disclosed herein may be further operably linked to any promoter element disclosed herein. [Brief description of the drawings]

[0098] [Figure 1] FIG. 1 shows the average activity of synthetic muscle-specific promoters according to some embodiments of the present invention in H9C2 cell lines differentiated into myocardial tubes. Error bars are standard deviation. CBA and CK8 are control promoters. The y-axis is luciferase activity (relative light units). [Diagram 2]FIG. 1 shows the average activity of synthetic muscle-specific promoters according to some embodiments of the present invention in H9C2 cell lines differentiated into myocardial tubes. Error bars are standard deviation. CBA and CK8 are control promoters. Average activity was normalized relative to the control promoter CBA. The y-axis is luciferase activity (relative light units). [Diagram 3] Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, and control promoters CK8, CMV, and CK7 in the diaphragm. Saline controls (background) were also included in the experiment. The saline controls (background) were subtracted from the luciferase expression by each promoter, and this value was then divided by the vector copy number of each promoter in the diaphragm. [Figure 4] Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, and control promoters CK8, CMV, and CK7 in the tibialis anterior (TA) muscle. Saline controls (background) were also included in the experiment. The saline controls (background) were subtracted from the luciferase expression by each promoter, and this value was then divided by the vector copy number of each promoter in the diaphragm. [Diagram 5] Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, and control promoters CK8, CMV, and CK7 in the heart. Saline controls (background) were also included in the experiment. The saline controls (background) were subtracted from the luciferase expression by each promoter, and this value was then divided by the vector copy number of each promoter in the diaphragm. [Figure 6]Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, and control promoters CK8, CMV, and CK7 in quadriceps muscle. Saline controls (background) were also included in the experiment. The saline controls (background) were subtracted from the luciferase expression by each promoter, and this value was then divided by the vector copy number of each promoter in the diaphragm. [Figure 7] Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, control promoters CK8, CMV, CK7, and saline control in soleus muscle. Vector copy number was not obtained in soleus muscle, and the graph shows luciferase expression by each promoter. [Figure 8] Figure 1 shows the in vivo activity of synthetic muscle-specific promoters SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, and control promoters CK8, CMV, and CK7 in liver. Saline control (background) was subtracted from luciferase expression from each promoter, and this value was then divided by the vector copy number of each promoter in the diaphragm. [Figure 9] FIG. 1 shows the in vivo activity of the control promoter CK8 in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 10] FIG. 1 shows in vivo activity of the control promoter CMV in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 11] FIG. 1 shows the in vivo activity of the control promoter, CK7, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 12]FIG. 1 shows in vivo activity of the synthetic muscle-specific promoter, SP0500, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 13] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter, SP0507, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 14] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter, SP0514, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 15] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter SP0518 in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 16] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter, SP0519, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 17] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter, SP0522, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. [Figure 18] FIG. 1 shows the in vivo activity of the synthetic muscle-specific promoter, SP0524, in diaphragm, tibialis anterior (TA), quadriceps, heart, and liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] CRE and its functional variants: Disclosed herein are various CREs that can be used to construct muscle-specific promoters. These CREs are generally derived from genomic promoter and enhancer sequences, but are used herein in a context quite different from their natural genomic environment. In general, CREs constitute a small portion of much larger genomic regulatory domains that control the expression of the genes with which they are normally associated. Surprisingly, these CREs, many of which are quite small, can be isolated from their normal environment and were found to retain muscle-specific regulatory activity when used to construct various synthetic promoters. This is surprising, since there is no reason to expect that a given CRE would retain the observed activity level when removed from its natural environment, since removal of regulatory sequences from their natural context in the genome, which is complex and "three-dimensional," often results in a significant loss of activity. Many combinations of these CREs have been examined and found to be highly effective in enhancing muscle-specific promoter activity when combined with minimal and proximal promoters. It is noted that the CRE sequences of the present invention can be altered without causing substantial loss of activity. Functional variants of CRE can be prepared by modifying the sequence of CRE under conditions that avoid significant detrimental modifications to the activity of CRE. With the information presented in this disclosure in mind, the modification of CRE to produce functional variants is straightforward. Furthermore, this disclosure presents a methodology for easily evaluating the functionality of any given CRE variant. Below, examples of functional variants are discussed.

[0100] CRE0145 is a functional variant of CRE0050, and vice versa, since CRE0145 is a truncated form of CRE0050.

[0101] The relatively small size of certain CREs according to the present invention is advantageous because it allows the CREs, and more particularly the promoters that contain them, to be incorporated into vectors while occupying a minimal amount of the vector's payload, which is particularly important when the CREs are used in vectors that have limited capacity, such as AAV-based vectors.

[0102] The CRE of the present invention includes certain muscle-specific TFBSs. In general, it is desired that these muscle-specific TFBSs maintain functionality within the functional variants of the CRE. Those skilled in the art are well aware that TFBS sequences can vary but retain functionality. With this in mind, sequences for TFBSs are typically illustrated by consensus sequences, from which there is typically some variation. Further information about the variations that occur within a TFBS can be illustrated using position weight matrices (PWMs), which represent the frequency with which a given nucleotide is typically found at a given position within a consensus sequence. Details about TF consensus sequences and associated position 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 the CRE within any given TFBS in a manner that retains and possibly even increases the functionality of the CRE. With this in mind, the skilled artisan is provided with ample guidance as to how, for any given TF, the TFBS can be modified while retaining the ability to bind to the desired TF; for example, the Jaspar system scores putative TFBSs based on their similarity to a given PWM. Furthermore, scanning of the CRE against all PWMs from the JASPAR database is also possible to identify / analyze all TFBSs. Of course, the skilled artisan can find further guidance in the literature, and routine experiments can also be used to confirm the binding of TFs to putative TFBSs in any variant CRE. It will be apparent that significant sequence modifications can be made within the CRE, and even within the TFBSs within the CRE, while retaining function.

[0103] Synthetic muscle-specific CRM and its functional variants: Disclosed herein are a variety of 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 variety of suitable minimal promoters or muscle-specific proximal promoters.

[0104] A functional variant of a CRM includes a sequence that varies from the reference CRM element, but that substantially retains activity as a muscle-specific CRM. One of skill in the art will appreciate that it is possible to vary the sequence of a CRM while retaining its ability to recruit the appropriate muscle-specific transcription factors (TFs), thereby enhancing expression. A functional variant of a CRM can include substitutions, deletions, and / or insertions compared to the reference CRM, under conditions that do not render the CRM substantially non-functional.

[0105] In some embodiments, a functional variant of a CRM may be considered to be a CRM that substantially retains 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 retains 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 retains 100% of its activity (compared to a reference promoter comprising an unmodified CRM).

[0106] Suitably, a functional variant of a CRM retains a significant level of sequence identity to the reference CRM. Suitably, the functional variant comprises a sequence that is at least 70% identical to the reference CRM, more preferably at least 80%, 90%, 95% or 99% identical to the reference CRM.

[0107] Retention of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an otherwise identical promoter containing a CRM substitution under equivalent conditions. Suitable assays for assessing muscle-specific promoter activity are disclosed herein, e.g., in the Examples.

[0108] In some embodiments, a functional variant of a given CRM may include one or more functional variants of the CREs present in a reference CRM. For example, a functional variant of a given CRM may include one, two, three, four, five, or six functional variants of the CREs present in a reference CRM.

[0109] In some embodiments, a functional variant of a given CRM may 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. Typically, it is preferred that the CREs are present in the same order as in the reference CRM (thus, a functional variant of a CRM suitably contains the same permutation of the CREs specified in the reference CRM).

[0110] In some embodiments, the functional variant of a given CRM may include one or more additional CREs in addition 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 other CREs. In general, it is preferred that the functional variant of a given CRM includes the same CRE (or a functional variant thereof) and does not include additional CREs.

[0111] A functional variant of a given CRM may include one or more additional regulatory elements compared to a reference CRM, for example, a functional variant of a given CRM may include inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, responsive sites, stabilizing elements, destabilizing elements, splicing elements, and the like, under conditions that do not render the CRM substantially non-functional.

[0112] A functional variant of a given CRM may contain additional spacers between adjacent CREs, and if one or more spacers are present in the reference CRM, said one or more spacers may be long or short in the reference CRM.

[0113] It will be apparent that the CRMs disclosed herein, or functional variants thereof, may be combined with any suitable promoter elements to yield a synthetic muscle-specific promoter in accordance with the present invention.

[0114] In many cases, short promoter sequences are preferred, especially for use in situations where the capacity of the vector (e.g., viral vectors such as AAV) is limiting. Thus, in some embodiments, the synthetic muscle-specific CRM has a length of 250 nucleotides or less, e.g., 220, 200, 180, 150, 100, 75, 60, 50 nucleotides or less. In some particularly preferred embodiments, the synthetic muscle-specific CRM has a length of 200 nucleotides or less.

[0115] Promoter elements and their functional variants: The CREs and CRMs of the present invention may be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters, collectively referred to as promoter elements.

[0116] A functional variant of a promoter element includes a sequence that varies from a reference promoter element but substantially retains activity as a muscle-specific promoter element. One of skill in the art will appreciate that it is possible to vary the sequence of a promoter element while retaining its ability to drive expression. A functional variant of a promoter element may include substitutions, deletions, and / or insertions compared to the reference promoter element, provided that the promoter element is not substantially non-functional.

[0117] In some embodiments, a functional variant of a promoter element may be considered to be a promoter element that substantially retains its activity when substituted for a reference promoter element in a synthetic promoter. For example, a muscle-specific synthetic promoter comprising a functional variant of a given promoter element preferably retains 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 retains 100% of its activity (compared to a reference promoter comprising an unmodified promoter element).

[0118] Suitably, a functional variant of a promoter element retains a significant level of sequence identity to the reference promoter element. Suitably, the functional variant comprises a sequence that is at least 70% identical to the reference promoter element, more preferably at least 80%, 90%, 95% or 99% identical to the reference promoter element.

[0119] Retention of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an otherwise identical promoter containing replacement promoter elements under equivalent conditions. Suitable assays for assessing muscle-specific promoter activity are disclosed herein, e.g., in the Examples.

[0120] 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 includes a sequence that varies from the reference synthetic muscle-specific promoter but substantially retains muscle-specific promoter activity. One of skill in the art will appreciate that the sequence of a synthetic muscle-specific promoter can be varied to provide muscle-specific expression of an operably linked sequence (e.g., an open reading frame) while retaining its ability to recruit appropriate muscle-specific transcription factors (TFs) and recruit RNA polymerase II. A functional variant of a synthetic muscle-specific promoter can include substitutions, deletions, and / or insertions compared to the reference promoter, provided that the substitutions, deletions, and / or insertions do not render the synthetic muscle-specific promoter substantially non-functional compared to the reference promoter.

[0121] Thus, in some embodiments, a functional variant of a synthetic muscle-specific promoter may be considered to be a variant that substantially retains the muscle-specific promoter activity of a reference promoter. For example, a functional variant of a synthetic muscle-specific promoter preferably retains at least 70% of the activity of the reference promoter, more preferably 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 retains 100% of its activity.

[0122] Functional variants of a synthetic muscle-specific promoter often retain a significant level of sequence similarity to the reference synthetic muscle-specific promoter. In some embodiments, the functional variant comprises a sequence that is at least 70% identical to the reference synthetic muscle-specific promoter, and more preferably at least 80%, 90%, 95%, or 99% identical to the reference synthetic muscle-specific promoter.

[0123] Activity in the functional variants can be assessed by comparing expression of a suitable reporter under control of a reference synthetic muscle-specific promoter with expression of a suitable reporter under control of the putative functional variant under comparable conditions. Suitable assays for assessing muscle-specific promoter activity are disclosed herein, e.g., in the Examples.

[0124] A functional variant of a given synthetic muscle-specific promoter may include one or more functional variants of the CREs present in a reference synthetic muscle-specific promoter. For example, a functional variant of a given CRM may include one, two, three, four, five, or six of the CREs present in the reference CRM. Functional variants of CREs are discussed above.

[0125] A functional variant of a given synthetic muscle-specific promoter may contain functional variants of promoter elements, or different promoter elements, when compared to a reference synthetic muscle-specific promoter.

[0126] 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.

[0127] 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 CRM, 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 other CREs.

[0128] A functional variant of a given synthetic muscle-specific promoter may include one or more additional regulatory elements compared to a reference synthetic muscle-specific promoter. For example, a functional variant of a given CRM may include inducible elements, intronic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, responsive sites, stabilizing elements, destabilizing elements, splicing elements, and the like, under conditions that do not render the promoter substantially non-functional.

[0129] A functional variant of a given synthetic muscle-specific promoter may contain additional spacers between adjacent CRE and promoter elements, and if one or more spacers are present in the reference synthetic muscle-specific promoter, said one or more spacers may be longer or shorter in the reference synthetic muscle-specific promoter. Examples of functional variants are provided below.

[0130] SP0522 is a functional variant of SP0502 and vice versa, as SP0522 is a short form of SP0502. SP0523 is a functional variant of SP0515 and vice versa, as SP0523 is a short form of SP0515. SP0524 is a functional variant of SP0521 and vice versa, as SP0524 is a short form of SP0521.

[0131] It will be apparent that a synthetic muscle-specific promoter of the invention may comprise a synthetic muscle-specific promoter of the invention and additional regulatory sequences. For example, a synthetic muscle-specific promoter of the invention may comprise one or more additional CRMs, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, responsive sites, stabilizing elements, destabilizing elements, splicing elements, and the like, provided that the promoter is not substantially non-functional.

[0132] The preferred synthetic muscle-specific promoters of the present invention exhibit muscle-specific promoter activity 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 promoter or RSV promoter in muscle cells. While in many cases high levels of promoter activity are preferred, this is not always the case, and thus in some cases more moderate levels of expression may be preferred. In some cases, it is desirable to have promoters with different levels of activity across an available range, allowing expression levels to be adjusted in light of requirements; the present disclosure presents promoters that are expected to provide such a range of activity. The activity of a given synthetic muscle-specific promoter of the invention relative to CBA 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 promoter or the RSV promoter when the two promoters are performed under equivalent conditions in otherwise equivalent expression constructs.

[0133] In some embodiments, the synthetic muscle-specific promoters of the invention are 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, suitably the SPc5-12 promoter (Gene Ther. 2008 Nov;15(22):1489-99).

[0134] Preferred 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 optionally 5% or less of CMV-IE, or 1% or less of CMV-IE.

[0135] In many cases, short promoter sequences are preferred, especially for use in situations where the capacity of the vector (e.g., viral vectors such as AAV) is limiting. Thus, in some embodiments, the synthetic muscle-specific promoter has a length of 300 nucleotides or less, e.g., 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 70, 68 nucleotides or less. In some embodiments, the synthetic muscle-specific promoter has a length of 300 nucleotides or less, preferably 290 nucleotides or less, more preferably 280 nucleotides or less, even more preferably 270 nucleotides or less. In some embodiments, the synthetic muscle-specific promoter has a length of 260 nucleotides or less, preferably 250 nucleotides or less, more preferably 240 nucleotides or less, even more preferably 230 nucleotides or less.

[0136] Particularly preferred synthetic muscle-specific promoters are those that are short and exhibit high levels of activity.

[0137] Synthetic muscle-specific expression cassette: The present invention also provides a synthetic muscle-specific expression cassette of the invention comprising a synthetic muscle-specific promoter operably linked to a sequence encoding an expression product, suitably a gene (e.g., a transgene).

[0138] A gene typically encodes a desired expression product of the gene, such as a polypeptide (protein) or RNA. A gene can be a full-length cDNA or genomic DNA sequence, or any fragment, subunit, or mutant thereof, that has at least some desired biological activity.

[0139] 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).

[0140] In some preferred embodiments of the invention, the gene encodes a therapeutic expression product, preferably a therapeutic polypeptide suitable for use in the treatment of a disease or condition associated with abnormalities in gene expression in cardiac muscle, optionally, skeletal muscle and / or muscle.

[0141] In some embodiments, the therapeutic expression product comprises a therapeutic expression product useful for the treatment of a muscle disease. The terms "muscular disease" or "muscle disease" are in principle understood by those skilled in the art. The term "muscular disease" relates to a disease suitable for treatment and / or prevention by administration of an active compound to muscle, in particular to muscle cells. In some embodiments, the muscle disease is a skeletal muscle disease. In some embodiments, the muscle disease is a cardiac muscle disease.

[0142] In some embodiments, the therapeutic expression product is a therapeutic expression product that is useful in the treatment of Duchenne muscular dystrophy, hi some embodiments, the therapeutic expression product is the DMD gene or a functional variant thereof.

[0143] In some embodiments, the therapeutic expression product is a therapeutic expression product that is useful in the treatment of heart failure or congestive heart failure.

[0144] In some embodiments, the muscle disease is vascular disease, muscular dystrophy, cardiomyopathy, myotonia, muscle atrophy, myoclonus-dystonia (susceptibility gene: SGCE), mitochondrial myopathy, rhabdomyolysis, fibromyalgia, and / or myofascial pain syndrome.

[0145] In one embodiment, the disease may be a cardiovascular condition or a heart disease or disorder. In one embodiment, the disease may be heart failure, such as congestive heart failure. In one embodiment, the disease may be selected from ischemia, arrhythmia, myocardial infarction (MI), abnormalities in cardiac contraction, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormalities in Ca2+ metabolism, and combinations thereof. In some embodiments, the disease may be selected from the group of congestive heart failure, cardiomyopathy, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, dysfunction of the conduction system, dysfunction of the coronary arteries, and pulmonary hypertension. In some embodiments, the disease may be selected from congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, cardiac arrhythmias, Danon disease, muscular dystrophy, muscle mass disorders, muscle degeneration, infectious myocarditis, drug-induced or toxin-induced myopathy, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease.

[0146] In some embodiments, the cardiomyopathies are hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy, restrictive cardiomyopathy, left ventricular noncompaction, takotsubo cardiomyopathy, myocarditis, eosinophilic myocarditis, and ischemic 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). 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).

[0147] In some embodiments, the muscular disease is a vascular disease. The vascular disease can be coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery stenosis, or aortic aneurysm. In some embodiments, the muscular disease can be a cardiomyopathy. The cardiomyopathy can be hypertensive heart disease, heart failure (such as congestive heart failure), pulmonary heart disease, cardiac rhythm disorders, inflammatory heart disease (such as endocarditis, inflammatory cardiac hypertrophy, myocarditis), valvular heart disease, congenital heart disease, and rheumatic heart disease.

[0148] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (genetically affected: DMD), Becker muscular dystrophy (genetically affected: DMD), limb-girdle muscular dystrophy (subtypes and affected genes: 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), and LGM D2M (gene: FKTN), LGMD2N (gene: POMT2), LGMD20 (gene: POMGNT1), LGMD2Q (gene: PLEC1), congenital muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, distal muscular dystrophy (subtype genes and affected genes: Miyoshi myopathy (gene: DYSF), distal myopathy with onset in the tibialis anterior (gene: DYSF), Welander distal myopathy (gene: TIA1), Gauwa -Rain distal myopathy (gene: MYH7), Nonaka distal myopathy, type 1 hereditary inclusion body myositis, distal myopathy with vocal cord and pharyngeal weakness, ZASP-associated myopathy), facioscapulohumeral muscular dystrophy (subtypes and affected genes: type 1 (gene: DUX4), type 2 (gene: SMCHD1)), oculopharyngeal muscular dystrophy (affected gene: PABPN1), and / or myotonic dystrophy (subtypes and affected genes: DM1 (gene: DMPK) and DM2 (gene: ZNF9)).

[0149] In some embodiments, the myotonia is myotonia congenita (affected gene: CLCN1; subtypes: Thomsen, Becker), potassium-induced myotonia, and / or paramyotonia congenita (affected gene: SCN4A).

[0150] In some embodiments, the muscle 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.

[0151] In some embodiments, the disease can be hyperkalemic periodic paralysis or hypokalemic periodic paralysis.

[0152] In some embodiments, the disease can be a congenital myopathy selected from nemaline myopathy, multi / minicore disease, and centronuclear myopathy.

[0153] In some embodiments, the disease may be selected from inflammatory myopathies, metabolic myopathies, Brodie's myopathies, or hereditary inclusion body myopathies.

[0154] In some embodiments, the gene is a non-disease mediating variant, e.g., 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, TIA1, MYH7, DUX4, SMCHD, PABPN1, DMPK, MBNL1, ZNF9, CF The gene encodes a wild-type variant of at least one human gene selected from the group consisting of CN1, SCN4A, MYH7, TNNT2, TPM1, MYBPC3, PRKAG2, TNNI3, MYF3, TTN, MYF2, ACTC1, CSRP3, TGFB3, RYR2, TMEM43, DES, DSP, PKP2, DSG2, DSC2, JUP, CNBP, CLCN1, SEPN1, RYR1, and HYPP.

[0155] In some embodiments, the gene is 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, TIA1, MYH7, DUX4, SMCHD, PABPN1, DMPK, MBNL1, ZNF9, CFCN 1, SCN4A, MYH7, TNNT2, TPM1, MYBPC3, PRKAG2, TNNI3, MYF3, TTN, MYF2, ACTC1, CSRP3, TGFB3, RYR2, TMEM43, DES, DSP, PKP2, DSG2, DSC2, JUP, CNBP, CLCN1, SEPN1, RYR1, and HYPP.

[0156] Further exemplary muscle tissue related diseases include acid maltase deficiency (AMD), alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis (ALS), Andersen-Tawil syndrome, Becker muscular dystrophy (BMD), Becker congenital myotonia, Bethlem myopathy, cardiovascular disease, carnitine deficiency, carnitine palmityltransferase deficiency (CPT deficiency), central core disease (CCD), centronuclear myopathy, Charcot-Marie-Tooth disease (CMT), congenital myasthenic syndrome (CMS), congenital myotonic dystrophy, congestive heart failure, coagulation syndrome, and the like. Leigh disease (debranching enzyme deficiency), debranching enzyme deficiency, Degerines-Sottas disease (DSD), dermatomyositis (DM), endocrine myopathy, Eulenberg disease (congenital paramyotonia), Forbes disease (debranching 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, Gauerlein distal myopathy, Hauptmann-Tannhäuser MD (Emery-Dreifuss muscular dystrophy), hereditary inclusion body myositis, hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease) ), hyperthyroid myopathy, hypothyroid myopathy, inclusion body myositis (IBM), genetic myopathy, integrin-deficient congenital muscular dystrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome (LEMS), McArdle disease (phosphorylase deficiency), muscle metabolic disease, mitochondrial myopathy, Miyoshi-type distal myopathy, motor neuron disease, muscle-eye-brain disease, myasthenia gravis (MG), myoadenylate deaminase deficiency, fibrotic myopathy, myophosphorylase deficiency, congenital myotonia (MC), myotonic muscular dystrophy (MMD), myotubule Myopathy (MTM or MM), Nemaline myopathy, Intermediate 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), Pombe disease (Acid maltase deficiency), Progressive external ophthalmoplegia syndrome (PEO), Nemaline body disease (Nemaline myopathy), Spinal muscular atrophy (SMA),These include, but are not limited to, spinal-bulbar muscular atrophy (SBMA), Steinert disease (myotonic muscular dystrophy), Tarui disease (phosphofructokinase deficiency), Thomsen disease (congenital myotonia), Ullrich congenital muscular dystrophy, Walker-Warburg syndrome (congenital muscular dystrophy), Welander distal myopathy, and ZASP-associated myopathy.

[0157] In some embodiments, the muscle disease is a myocardial disease. In some embodiments, the muscle disease is congestive heart failure.

[0158] In some embodiments, useful expression products include dystrophin (including microdystrophin), beta 1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthetase I, alpha-1 antitrypsin, ornithine transcarbamylase, arcinococcate synthetase, arcinococcate lylase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovalyl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, and cystic fibrosis transmembrane conductance regulator (CFTR).

[0159] Still other useful expression products include enzymes useful in enzyme replacement therapy and in a variety of conditions resulting from deficiencies in enzyme activity, for example, enzymes containing mannose-6-phosphate may be utilized in treatments for lysosomal storage diseases (e.g., suitable genes including those encoding β-glucuronidase (GUSB)).

[0160] 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), neurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor beta), 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).

[0161] In some embodiments, useful therapeutic expression products include, but are 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), transforming growth factor alpha superfamily factor (TGFα), and the like. and hormones and growth and differentiation factors, including any one of the following: myelin, activin, inhibin, or bone morphogenetic proteins (BMPs), any one of any of BMP1-BMP15, any one of the heregulin / 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, the semaphorin / collapsin family, netrin 1 and netrin 2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0162] In some embodiments, useful expression products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) from IL-1 through IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor alpha and tumor necrosis factor beta, interferons (interferon alpha, interferon beta, and interferon gamma), stem cell factor, flk-2 / flt3 ligand, etc. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, 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 MHC molecules, class II MHC molecules, as well as engineered immunoglobulin molecules and engineered MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), degradation accelerating factor (DAF), CR1, CF2, and CD59.

[0163] In some embodiments, useful expression products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and / or lipid modulation, including 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 receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. Additionally, 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.

[0164] In some embodiments, useful expression products include expression products used to treat hemophilia, including hemophilia B (including factor IX), and hemophilia A (including factor VIII and variants thereof, such as heterodimeric light and heavy chains and the B deleted domain; including U.S. Pat. No. 6,200,560 and U.S. Pat. No. 6,221,349).

[0165] In some embodiments, a useful expression product may be a modulator of phosphatase activity, e.g., type 1 phosphatase activity. The modulator may be a protein that inhibits phosphatase activity, e.g., type 1 phosphatase activity. The modulator may be a nucleic acid that increases expression of an endogenous nucleic acid that encodes a protein that inhibits phosphatase activity, such as a transcription factor. The modulator may be a regulatory sequence that is integrated into or near an endogenous nucleic acid that encodes a protein that inhibits phosphatase activity. The modulator may be a nucleic acid that may result in a nucleic acid modulator of gene expression, such as an siRNA.

[0166] In some embodiments, a useful expression product may be an inhibitor of protein phosphatase 1 (PP1), e.g., I-1 polypeptide. Phosphatase inhibitor 1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatase. Increasing the level or activity of I-1 may restore beta-adrenergic responsiveness in failing human cardiomyocytes. Suitably, the I-1 protein may be constitutively active, such as an I-1 protein in which threonine 35 is replaced with glutamic acid rather than aspartic acid. The therapeutic expression product may be any one or more of the inhibitors selected from phosphatase inhibitor 2 (PP2), which is an endogenous nuclear inhibitor of protein phosphatase 1; okadaic acid or calyculin; and nippl.

[0167] In some embodiments, a useful expression product can be any protein that modulates cardiac activity, such as a type 1 phosphatase inhibitor, e.g., I-1, or a sarcoplasmic reticulum Ca2+ ATPase (SERCA), e.g., SERCA1 (e.g., 1a or 1b), SERCA2 (e.g., 2a or 2b), or SERCA3.

[0168] In some embodiments, a useful expression product can be a nucleic acid sequence encoding a mutant form of a 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 where at least one amino acid is constitutively unphosphorylated or mimics the unphosphorylated state in the mutant form. Therapeutic expression products can be adenyl cyclase 6 (AC6; also referred to as adenyl cyclase VI), S100A1, β-adrenergic receptor kinase-ct (βARKct), sarco / endoplasmic reticulum (SR) Ca-ATPase (SERCA2a), IL-18, VEGF, VEGF activator, urocortin, and B-cell lymphoma 2 (Bcl2)-associated athanogene 3 (BAG3).

[0169] In some embodiments, useful expression products can be inhibitors of cytokines, such as IL-18 inhibitors. Therapeutic expression products can be beta-adrenergic signaling proteins (beta-ASPs), including beta-adrenergic receptors (beta-Ars), G-protein receptor kinase inhibitors (GRK inhibitors), and adenyl cyclase (Acs), to enhance cardiac function.

[0170] In some embodiments, a useful 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.

[0171] In some embodiments, useful expression products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions, or amino acid substitutions. In some embodiments, the expression product may be a synthetic dysferlin protein, such as Nano-Dysferlin, which is a shortened form of wild-type dysferlin, detailed in Table 1 and FIG. 1A of (Llanga et al., 2017).

[0172] Further suitable expression products include microRNA (miRNA), interfering RNA, antisense RNA, ribozymes, and aptamers.

[0173] In some preferred embodiments, the expression product is an inhibitor of protein phosphatase 1 (PP1).

[0174] In some embodiments of the invention, the synthetic muscle-specific expression cassette comprises a gene useful for gene editing, for example a gene encoding a site-specific nuclease, such as 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-Cas). Suitably, the site-specific nuclease is adapted to edit the desired target genomic locus by creating a nick (typically a site-specific double-strand break) which is then repaired via non-homologous end joining (NHEJ) or homology-dependent repair (HDR) resulting in the desired edit. The edit can be a partial or complete repair of a gene that is dysfunctional or a 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.

[0175] In some embodiments of the invention, the synthetic muscle-specific expression cassette comprises a gene useful for gene modulation, e.g., a gene for a DNA binding protein fused to a gene repressor or gene activator, e.g., a zinc finger protein gene fused to a gene repressor or gene activator, or an endonuclease-deficient cas9 fused to a gene repressor or gene activator.

[0176] Suitably, the synthetic muscle-specific expression cassette comprises sequences providing for or encoding one or more, preferably all, of a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Suitably, the expression cassette comprises a nucleic acid encoding a post-transcriptional regulatory element. Suitably, the expression cassette comprises a nucleic acid encoding a polyA element.

[0177] Vectors and viral particles: The present invention further provides a vector comprising a synthetic muscle-specific promoter or an expression cassette according to the invention.

[0178] In some embodiments of the present invention, the vector is a plasmid. Such a plasmid may contain various 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 present invention, the vector is a viral vector.

[0179] In some embodiments of the invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic 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, pCMV-EGFP available from Clontech. Many other vectors are also well known and commercially available. For adenoviral vectors for mammalian cells, the pSV series and pCMV series vectors are particularly well known non-limiting examples. There are many well known yeast expression vectors, including, but not limited to, Yeast Integrating Plasmid (Yip) and Yeast Replicating Plasmid (Yrp). For plants, the Ti plasmid of Agrobacterium is an exemplary expression vector, and plant viruses such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus also provide suitable expression vectors.

[0180] In some preferred embodiments, the vector is a gene therapy vector. A variety of gene therapy vectors are known in the art, and can be mentioned AAV, adenoviral, retroviral, and lentiviral vectors. When the vector is a gene therapy vector, it preferably comprises a nucleic acid sequence operably linked to the synthetic muscle-specific promoter of the present invention, encoding a therapeutic product, suitably a therapeutic protein. The therapeutic protein can be a secreted protein. Non-limiting examples of secreted proteins are discussed above, but exemplary secreted 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, and the like.

[0181] In some embodiments of the present invention, the 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. In some preferred embodiments, the AAV has a serotype suitable for intramuscular transduction. In some embodiments, the AAV is selected from the group consisting of AAV2, AAV5, AAV6, AAV7, AAV8, AAV9BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8, and AAV2G9, or derivatives thereof. The use of single-stranded AAV vectors (ssAAV) is also encompassed herein, but in order to overcome one of the rate-limiting steps of AAV transduction (i.e., conversion from single-stranded AAV to double-stranded AAV), the AAV vector is preferably used as a self-complementary double-stranded AAV vector (scAAV). In some embodiments of the present invention, the AAV vector is a chimeric AAV vector, which means 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 effectively transduce skeletal muscle and particularly effectively transduce cardiac muscle, so AAV9 and its derivatives are particularly of interest for targeting skeletal muscle and cardiac muscle. AAV1, AAV6, AAV7, and AAV8 are also known to target skeletal muscle, so these AAV serotypes and their derivatives are also particularly of interest for targeting skeletal muscle. AAV1 and AAV8 are also known to target cardiac muscle, so these AAV serotypes and their derivatives are also particularly of interest for targeting cardiac muscle. In some embodiments, the rAAV vector is an AAV3b serotype, including 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 theoretically haploid, or chimeric or any mutant, such that the capsid can be tailored for increased renewal in a desired location, e.g., the heart. Other capsids can include capsids derived from any of the known AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV7, AAV10, etc.

[0182] The present invention further provides a recombinant virion (virus particle) comprising the above-described vector.

[0183] Pharmaceutical Compositions: The vectors or virions of the invention may be formulated into a pharmaceutical composition together with a pharma- ceutically acceptable excipient, i.e., one or more pharma- ceutically 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, or methods and uses thereof, are known in the art.

[0184] Thus, a further aspect of the invention provides a pharmaceutical composition comprising a vector or virion as described herein.

[0185] Therapeutic and Other Methods and Uses: The invention also provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the invention for use in treating a disease, preferably optionally a disease associated with abnormal gene expression in muscle (e.g., a genetic 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 a skeletal muscle disease. In one embodiment, the invention also provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the invention for use in treating a myocardial disease.

[0186] Implicated conditions, diseases, and therapeutic expression products are discussed above.

[0187] The invention also provides the synthetic muscle-specific promoters, expression cassettes, vectors, virions according to various aspects of the invention in use for the manufacture of a pharmaceutical composition for the treatment of any condition or disease mentioned herein.

[0188] The invention further provides a cell comprising a synthetic muscle-specific promoter, expression cassette, vector, virion according to various aspects of the invention. Suitably, the cell is a eukaryotic cell. The eukaryotic cell may suitably 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.

[0189] In some embodiments of the invention, the cells are ex vivo cells, e.g., in cell culture, hi other embodiments of the invention, the cells may be part of a tissue or a multicellular organism.

[0190] In a preferred embodiment, the cell is a muscle cell (myocyte), which may be an ex vivo or in vivo cell. In a preferred embodiment, the cell is a cardiomyocyte, which may be an ex vivo or in vivo cell. In an alternative preferred embodiment, the cell is a skeletal muscle cell, which may be an ex vivo or in vivo cell. The muscle cell may be a primary muscle cell or a cell from 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. in cell culture. Suitably, the cell is a human cell.

[0191] Skeletal muscle cells can be either fast-twitch or slow-twitch.

[0192] Cardiomyocytes may be selected from intracardiac ventricular cardiomyocytes, atrial cardiomyocytes, cardiac fibroblasts, or endothelial cells (ECs), as well as perivascular cells and pacemaker cells.

[0193] A synthetic muscle-specific promoter, expression cassette, or vector according to the invention may be inserted into the genome of the cell or may be episomal (e.g., present in an episomal vector).

[0194] In a further aspect, the invention provides a method for producing an expression product, comprising providing a synthetic muscle-specific expression cassette according to the invention (preferably in a vector as specified above) into a cell, preferably a muscle cell, and expressing the gene present in the synthetic muscle-specific expression cassette. The method comprises maintaining said muscle cell appropriately under conditions suitable for expression of the gene. In culture, this may comprise incubating the cell or tissue comprising the cell under suitable culture conditions. Expression may of course be in one or more cells in vivo, for example in the muscle of a subject. In one embodiment the muscle cell is a cardiomyocyte. In one embodiment the muscle cell is a skeletal muscle cell.

[0195] Suitably, the method comprises introducing a synthetic muscle-specific expression cassette into muscle cells. A wide variety of methods for transfecting muscle cells are known in the art. A preferred method for transfecting muscle cells is to transduce a viral vector, such as an AAV vector, containing the synthetic muscle-specific expression cassette into the cells.

[0196] It will be apparent to one skilled in the art that the synthetic muscle-specific promoters, expression cassettes, vectors or virions according to the various aspects of the invention may be used in gene therapy, and therefore the use of such nucleic acid constructs in gene therapy forms part of the present invention.

[0197] Thus, in some embodiments, the invention provides an expression cassette, vector or virion according to the invention for use in gene therapy in a subject, preferably gene therapy via muscle-specific expression of a therapeutic gene. Suitably gene therapy via skeletal muscle-specific expression of a therapeutic gene and / or cardiac muscle-specific expression of a therapeutic gene. The therapy may involve the treatment of a disease, suitably a disease as discussed above, such as a disease involving aberrant gene expression in muscle, via secretion of a therapeutic product from muscle cells.

[0198] The invention also provides a method for expressing a therapeutic transgene in a muscle cell, comprising the step of introducing into the muscle cell an expression cassette or vector according to the invention. The muscle cell may be an in vivo cell or an ex vivo cell. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell.

[0199] The present invention also provides a method of gene therapy for a subject, preferably a human, in need thereof, comprising the steps of: - administering to a subject (suitably introducing into the muscle of the subject) a synthetic muscle-specific expression cassette, vector, virion or pharmaceutical composition of the invention comprising a gene encoding a therapeutic product. Also provided is a method comprising:

[0200] In one embodiment, the muscle is cardiac muscle. In one embodiment, the muscle is skeletal muscle. In one embodiment, the muscle is cardiac and / or skeletal muscle. The method suitably comprises expressing a therapeutic amount of a therapeutic product from a gene in the muscle of said subject. Various conditions and diseases that may be treated are discussed above. In one embodiment, the muscle is cardiac muscle. In one embodiment, the muscle is skeletal muscle.

[0201] Genes encoding suitable therapeutic products are discussed above.

[0202] The method suitably comprises the step of administering to a subject a vector or virion according to the invention. Suitably the vector is a gene therapy viral vector, such as an AAV vector.

[0203] In some embodiments, the methods include administering to the subject a synthetic muscle-specific viral gene therapy vector (i.e., a viral gene therapy vector comprising a muscle-specific promoter described herein) that contains a gene encoding a therapeutic product.

[0204] In some embodiments, the method includes systemically administering the gene therapy viral vector. Systemic administration can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection). Preferred injection routes include intravenous, intramuscular, subcutaneous, intraarterial, intraarticular, intrathecal, and intradermal injection. In some preferred embodiments, the viral gene therapy vector is administered to the subject by antegrade epicardial coronary injection (AECAI). In some particularly preferred embodiments, the viral gene therapy vector is administered to the subject by antegrade epicardial coronary injection (AECAI) via percutaneous femoral access.

[0205] In some embodiments, the subject has heart failure. In some embodiments, the subject method of gene therapy is a method for the treatment of heart failure.

[0206] In some embodiments, the methods include administering to a subject via antegrade epicardial coronary artery infusion (AECAI) a synthetic muscle-specific viral gene therapy vector for the treatment of heart failure, where the vector includes a gene encoding a therapeutic product.

[0207] In some embodiments, the gene therapy viral vector is administered contemporaneously or sequentially with one or more additional therapeutic agents or one or more saturating agents designed to prevent clearance of the vector by the reticuloendothelial system.

[0208] If the vector is an AAV vector, the vector dosage is 1 x 10 10 gc / kg~1×10 15 The dosage may be at or above gc / kg, suitably at or above 1×10 12 gc / kg~1×10 14 gc / kg, appropriately 5×10 12 gc / kg~5×10 13 It can be gc / kg.

[0209] Generally, the "subject in need thereof" will be a mammal, preferably a primate, and more preferably a human. Typically, the "subject in need thereof" 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 a therapeutic product.

[0210] Gene therapy protocols for the expression of therapeutic genes in target cells in vitro and in vivo are well known in the art and will not be discussed in detail here. Briefly, gene therapy protocols include intramuscular, interstitial, airway instillation, endothelial application, intrahepatic parenchymal, and intravenous or intraarterial administration (e.g., intrahepatic artery, intrahepatic vein) of plasmid DNA vectors (naked plasmid DNA vectors or plasmid DNA vectors in liposomes) or viral vectors. A variety of devices have been developed to enhance the availability of DNA to target cells. A simple approach is to physically contact the target cells with a catheter or implantable material containing the relevant vector, while more complex approaches use jet injection devices and the like. Gene transfer into mammalian muscle cells has been performed using both ex vivo and in vivo procedures. Ex vivo methods typically require harvesting muscle cells, transduction in vitro with the appropriate expression vector, followed by reintroduction of the transduced muscle cells into the muscle. In vivo gene transfer has been achieved by intramuscular injection of DNA or viral vectors. Antegrade epicardial coronary artery injection can be used to inject DNA or viral vectors in close proximity to the heart.

[0211] According to some preferred embodiments, the methods set forth above may be used to treat a subject with a muscle-related disease as discussed above, such as muscular dystrophy or congestive heart failure.

[0212] Definitions and general points: Although the making and using of various embodiments of the invention are discussed in detail below, it should be appreciated that the invention presents many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not delimit the scope of the invention.

[0213] The discussion of the background to the invention herein is incorporated to explain the context of the invention and is not to be understood as an admission that any of the material mentioned was published, publicly known, or part of the general common knowledge in any country as of the priority date of any of the claims.

[0214] Throughout this disclosure, various publications, patents, and patent specification disclosures are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents that are specifically mentioned herein are incorporated by reference.

[0215] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that 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 son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins, eds., 1984); Transcription and Translation (Hames and Higgins, eds., 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); Methods in Enzymology (Abelson and Simon, eds., Academic Press, 1988). Press, Inc., New York), in particular volumes 154 and 155 (Wu et al., eds.) and volume 185, "Gene Expression Technology" (Goeddel, ed.); "Gene Transfer Vectors For Mammalian Cells" (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); "Immunochemical Methods in Cell and Molecular Biology" (Mayer and Walker, eds., Academic Press, London, 1987); "Handbook of Experimental Immunology", volumes I-IV (Weir and Blackwell, eds., 1986); and "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0216] In order to facilitate understanding of the present invention, a number of terms are defined or explained below. Terms used herein have meanings commonly understood by one of ordinary skill in the art in the areas pertaining to the present invention. Terms such as "a," "a," and "the" are not intended to refer to a singular entity only, but are intended to include a general class of which their specific examples may be used for illustration. Although the terminology used herein is used to describe specific embodiments of the present invention, their usage does not define the present invention unless outlined in the claims.

[0217] The term "muscle" is well understood by those skilled in the art. Preferably, the muscle is a skeletal muscle (including diaphragm) or cardiac muscle. The promoters of the present invention may be active in skeletal muscle and / or cardiac muscle. Preferably, the muscle is of a vertebrate, more preferably a mammal, even more preferably a human subject. Preferably, the muscle is a striated muscle.

[0218] The term "muscle cell" or "myocyte" in this application relates to a cell found in muscle (muscle tissue) or derived from muscle tissue. A muscle cell may be a primary cell or a cell line, such as C2C12 cells or H2K cells (skeletal muscle cell line) or H9C2 cells (cardiac cell line). A muscle cell may be in vivo (e.g., in muscle tissue) or in vitro (e.g., in 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 cardiac muscle (cardiomyocytes). The promoters of the present invention may be active in skeletal muscle cells and / or cardiac muscle cells.

[0219] The term "cis-regulatory element" or "CRE" is a term well known to those skilled in the art and refers to a nucleic acid sequence, such as an enhancer, promoter, insulator, or silencer, that can regulate or modulate the transcription of adjacent genes (i.e., genes in cis). CREs are found in the vicinity of the genes they regulate. CREs typically regulate gene transcription by binding to TFs, i.e., CREs contain TFBSs. A single TF can bind to many CREs and thus control the expression of many genes (can be pleiotropic). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene they regulate. An "enhancer" in this context is a CRE that enhances (i.e., upregulates) the transcription of the gene with which they are operatively associated, and can be found upstream, downstream, or even within an intron of the gene they regulate. Multiple enhancers can act cooperatively to regulate the transcription of one gene. "Silencer" in this context refers to a CRE that binds to a TF called a repressor, which acts to prevent or downregulate the transcription of a gene. The term "silencer" may also refer to a region in the 3' untranslated region of a messenger RNA that binds to a protein that inhibits the translation of this mRNA molecule, but this usage is significantly different from its use when describing a CRE. In general, the CRE of the present invention is a muscle-specific enhancer element, or a skeletal muscle-specific enhancer element (often referred to as a muscle-specific CRE, or a skeletal muscle-specific CRE, or a muscle-specific CRE enhancer, or a skeletal muscle-specific CRE enhancer, etc.). In this context, the CRE is preferably located 2500 nucleotides or less from the transcription start site (TSS), more preferably 2000 nucleotides or less from the TSS, more preferably 1500 nucleotides or less from the TSS, and suitably located 1000, 750, 500, 250, 200, 150, or 100 nucleotides or less from the TSS.The CREs of the present invention are preferably relatively short in length, preferably 250 nucleotides or less in length, for example, 200, 175, 150, 90, 80, 70, 60, or 50 nucleotides or less in length. The CREs of the present invention are typically administered 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 activity or skeletal muscle-specific activity of the promoter element. In any of the combinations of CREs or functional variants thereof disclosed herein, some or all of the listed CREs and promoter elements may be located adjacent to each other, suitably within the promoter (i.e., without the intervening CRE or other regulatory element). The CREs may be contiguous or non-contiguous (i.e., they may be located immediately adjacent to each other or separated by a spacer or other sequence). The CREs may be placed in any order. In some preferred embodiments, the CREs or functional variants thereof are administered adjacent to each other in the order listed. For example, a synthetic muscle-specific regulatory nucleic acid can include CRE0077 immediately upstream of CRE0075, etc. In some embodiments, it is preferred that some or all of the CREs are contiguous.

[0220] The term "cis-regulatory module" or "CRM" generally refers to a functional regulatory nucleic acid module that includes two or more CREs, and in the present invention, the CRE is typically a muscle-specific enhancer or a skeletal muscle-specific enhancer, so that the CRM is a synthetic muscle-specific regulatory nucleic acid or a synthetic skeletal muscle-specific regulatory nucleic acid. Thus, in the present application, the CRM typically includes multiple muscle-specific CREs or skeletal muscle-specific CREs. Typically, multiple CREs in a CRM act together (e.g., additively or synergistically) to enhance the transcription of a gene operably associated with a synthetic promoter that includes the CRM. There is extensive room to shuffle (i.e., rearrange the order), invert (i.e., reverse orientation), and change the spacing of the CREs in a CRM. Thus, functional variants of the CRM of the present invention include, inter alia, variants of the reference CRM, in which the CREs therein are shuffled and / or inverted, and / or the spacing between the CREs is changed.

[0221] As used herein, the term "promoter" generally refers to a region of DNA located upstream of a nucleic acid sequence to be transcribed, which is required for transcription to occur, i.e., induces transcription. Promoters allow for the proper activation or repression of transcription of the coding sequence under their control. Promoters typically contain 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. Many different promoters are known in the art.

[0222] In some cases, the term "promoter" or "composite promoter" is used herein to refer to a combination of a promoter with additional regulatory elements, such as regulatory sequences located immediately downstream of the transcription start site (TSS), such as the 5'UTR and / or the 5'UTR and an intron. Such sequences downstream of the TSS may contribute to regulating expression at the transcription and / or translation stages.

[0223] As used herein, the term "synthetic promoter" refers to a promoter that is not naturally occurring. In this context, a "synthetic promoter" typically comprises a CRE and / or CRM of the present invention operably linked to a minimal (or core) promoter, or a muscle-specific proximal promoter, or a skeletal muscle-specific proximal promoter (promoter element). The CRE and / or CRM of the present invention is used to enhance muscle-specific or skeletal muscle-specific transcription of a gene operably linked to the synthetic promoter. Although parts of the synthetic promoter may be naturally occurring (e.g., a minimal promoter, or one or more CREs within the promoter), the synthetic promoter as an entity is not naturally occurring.

[0224] As used herein, a "minimal promoter" (also known as a "core promoter") refers to a short DNA segment that is typically inactive or largely 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, including prokaryotic and eukaryotic genes. Examples of minimal promoters are discussed above and include the desmin minimal promoter, the dopamine beta-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 include the transcription start site (TSS) and elements immediately upstream of the RNA polymerase II binding site, as well as general transcription factor binding sites (often a TATA box). Minimal promoters may also include some elements downstream of the TSS, but these typically have little functionality without additional regulatory elements.

[0225] As used herein, the term "proximal promoter" refers to a minimal promoter plus at least some additional regulatory sequences, typically the proximal sequence upstream of a gene, which tends to contain the primary regulatory elements. The "proximal promoter" often spans about 250 base pairs upstream of the TSS and includes a specific TFBS. The proximal promoter may also include one or more regulatory elements, such as UTRs or introns, downstream of the TSS. In this case, the proximal promoter may be a naturally occurring muscle-specific proximal promoter or a skeletal muscle-specific proximal promoter, which may be suitably combined with one or more CREs or CRMs of the present invention. However, the proximal promoter may be a synthetic proximal promoter.

[0226] As used herein, "promoter element" refers to a minimal promoter or a proximal promoter, as defined above. In the context of the present invention, a promoter element is typically combined with one or more CREs to provide a synthetic muscle-specific promoter or a synthetic skeletal muscle-specific promoter of the present invention.

[0227] A "functional variant" of a CRE, CRM, promoter element, synthetic promoter, or other regulatory nucleic acid in the context of the present invention is a variant of the reference sequence that retains the ability to function in the same manner as a reference sequence, e.g., a muscle-specific CRE, or skeletal muscle-specific CRE, or a muscle-specific CRM, or a muscle-specific promoter, or a skeletal muscle-specific promoter. Alternative terms for such functional variants include "biological equivalent" or "equivalent."

[0228] It will be appreciated that the ability of a given CRE, CRM, promoter, or other regulatory sequence to function as a muscle-specific or skeletal muscle-specific enhancer is meaningfully determined by the ability of the sequence to bind to the same muscle-specific, cardiac, or skeletal muscle-specific TFs that bind to the reference sequence. Thus, in most cases, a functional variant of a CRE or CRM will contain TFBSs for most or all of the same TFs as a reference CRE, CRM, or promoter. It is preferred, but not essential, that the TFBSs of the functional variant are in the same relative position (i.e., order and overall position) as the reference CRE, CRM, or promoter. It is also preferred, but not essential, that the TFBSs of the functional variant are in the same orientation as the reference sequence (it will be noted that TFBSs are sometimes present in reverse orientation, e.g., as reverse complements, which are opposite sequences in the reference sequence). It is also preferred, but not essential, that the TFBSs of the functional variant are on the same strand as the reference sequence. Thus, in a preferred embodiment, the functional variant comprises TFBSs for the same TF in the same order, position, orientation and on the same strand as the reference sequence. It will also be appreciated that sequences between TFBSs (sometimes referred to as spacer sequences, etc.) are not critical to the function of the CRE or CRM. Such sequences may typically vary widely and their length may also be altered. However, in a preferred embodiment, the spacing (i.e., the distance between adjacent TFBSs) is substantially the same (e.g., does not vary by more than 20%, preferably does not vary by more than 10%, and more preferably is approximately the same) in the functional variant as the reference sequence. It will be apparent that in some cases, the functional variant of the CRE may be in the reverse orientation, e.g., the reverse complement of the CRE described above or its variant.

[0229] The level of sequence identity between a functional variant and a reference sequence can also be an indicator for retention of functionality. A high level of sequence identity in the TFBS of a CRE, CRM, or promoter is generally of higher importance than sequence identity in a spacer sequence (where there is little or no requirement for any conservation of sequence). However, given that the sequence of a functional TFBS does not have to match the consensus sequence exactly, it will be appreciated that even within a TFBS, a significant degree of sequence variation can be accommodated.

[0230] The ability of one or more TFs to bind to TFBSs in a given functional variant may be determined by any relevant means known in the art, including, but not limited to, gel 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 technique is described in Sambrook et al., cited above. Many relevant articles describing this procedure are available, e.g., Hellman and Fried, Nat Protoc., 2007, 2(8):1849-1861.

[0231] "Muscle-specific" or "muscle-specific expression" refers to the ability of a cis-regulatory element, cis-regulatory module, or promoter to enhance or drive expression of a gene in a preferential or predominant manner in muscle cells (or muscle-derived cells) compared to other tissues (e.g., liver, kidney, spleen, heart, lung, and brain). The expression of the gene can be in the form of mRNA or protein. In a preferred embodiment, muscle-specific expression is such that expression in other (i.e., non-muscle) tissues or cells is negligible, i.e., expression is highly muscle-specific. For example, expression in muscle cells versus other cells is at least 75%, 80%, 85%, 90%, or 95%. "Myocardial specific" 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 in a preferential or predominant manner in myocardial compared to other tissues (e.g., spleen, liver, lung, and brain) and compared to skeletal muscle tissue. "Skeletal muscle specific" 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 in a preferential or predominant manner in skeletal muscle compared to other tissues (e.g., spleen, liver, lung, and brain) and compared to cardiac muscle tissue. A lower degree of specificity may be desired and is part of the invention.

[0232] The ability of a CRE, CRM, or promoter to function as a muscle-specific CRE, cardiac muscle-specific CRE, or skeletal muscle-specific CRE, muscle-specific CRM, cardiac muscle-specific CRM, or skeletal muscle-specific CRM, or muscle-specific promoter, cardiac muscle-specific promoter, or skeletal muscle-specific promoter, can be easily evaluated by a person skilled in the art. Thus, a person skilled in the art can easily determine whether any variant of the specific CRE, specific CRM, or specific promoter listed above maintains functionality (i.e., is a functional variant as defined above). For example, any given CRM to be evaluated may be operably linked to a minimal promoter (e.g., located upstream of a CMV-MP), and the ability of the cis-regulatory element to drive expression of a muscle-specific gene, cardiac muscle-specific gene, or skeletal muscle-specific gene (typically a reporter gene) is measured. Alternatively, a variant of a CRE or CRM may be substituted for a synthetic muscle-, cardiac-, or skeletal-specific promoter instead of a reference CRE or CRM, and the effect on muscle-, cardiac-, or skeletal-specific expression driven by the modified promoter may be determined and compared to the unmodified form. Similarly, the ability of a promoter to drive muscle-, cardiac-, or skeletal-specific expression may be readily assessed by one of skill in the art (e.g., as described in the Examples below). The expression level of a gene driven by a variant of a reference promoter may be compared to the expression level driven by the reference promoter. In some embodiments, where the level of muscle-, or skeletal-specific expression driven by a 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, the variant may be said to maintain functionality. Suitable nucleic acid constructs and reporter assays for assessing enhanced muscle-, cardiac, or skeletal muscle-specific expression can be readily constructed, and the examples set forth below demonstrate suitable methodologies.

[0233] Muscle-, cardiac-, or skeletal-specificity may be identified when expression of a gene (e.g., a therapeutic gene or a reporter gene) occurs preferentially or predominantly in muscle- or skeletal-derived cells. Preferential or predominant expression may be defined, for example, when expression levels are significantly higher in muscle-, cardiac-specific, or skeletal-derived cells than in other cell types (i.e., non-muscle-, non-cardiac-specific, or non-skeletal-derived cells). For example, expression in muscle-, cardiac-specific, or skeletal-derived cells is suitably at least 5-fold higher than in non-muscle or non-skeletal cells, preferably at least 10-fold higher than in non-muscle or non-skeletal cells, and in some cases may be 50-fold or higher. For convenience, muscle-specific expression may suitably be established via comparison of expression levels in a muscle cell line (e.g., a muscle-derived cell line such as C2C12 cells or H2K cells (skeletal muscle) or H9C2 cells (cardiac)) with 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). Myocardial-specific expression may suitably be established through comparison of expression levels in a cardiomyocyte cell line (e.g., a myocardial-derived cell line such as H9C2) or primary cardiomyocytes with 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 expression may be established, suitably, through comparison of expression levels in skeletal muscle-derived cells (e.g., C2C12 or H2K), or primary skeletal muscle cells, with 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 cardiomyocyte cell lines (e.g., H9C2).

[0234] The synthetic muscle-specific promoter, synthetic cardiac muscle-specific promoter, or synthetic skeletal muscle-specific promoter of the present invention preferably exhibits reduced expression in non-muscle-derived cells, suitably Huh7 cells, HEK-293 cells, HeLa cells, and / or A549 cells, when compared to non-tissue specific promoters such as CMV-IE. The synthetic muscle-specific promoter, synthetic cardiac muscle-specific promoter, or synthetic skeletal muscle-specific promoter of the present invention preferably has 50% or less, suitably 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less activity in non-muscle-derived cells, suitably in Huh7 cells, HEK-293 cells, HeLa cells, and / or A549 cells, of the promoter that is CMV-IE. In general, it is preferred that expression in non-muscle-derived cells is minimized, although in some cases this may not be necessary. A synthetic promoter of the invention may still be a muscle-specific promoter, for example, if it has high expression in one or two non-muscle cells, so long as it has generally high expression throughout the range of muscle cells relative to non-muscle cells. In some embodiments, a muscle-specific promoter causes a gene to be highly expressed in muscle cells by 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 percentage between 25% and 95%, compared to non-muscle cells.

[0235] The synthetic muscle-specific promoters of the invention are preferably suitable for promoting expression in muscle of a subject, e.g., suitable for driving muscle-specific expression of a transgene, preferably a therapeutic transgene. The synthetic skeletal muscle-specific promoters of the invention are preferably suitable for promoting expression in skeletal muscle of a subject, e.g., suitable for driving skeletal muscle-specific expression of a transgene, preferably a therapeutic transgene. Preferred synthetic muscle-specific promoters of the invention are suitable for promoting muscle-specific expression of a transgene and have an activity in muscle cells that is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity of a CBA promoter. In some embodiments, the synthetic muscle-specific promoters of the invention are suitable for promoting muscle-specific expression of a transgene at a level of at least 100% of the activity of the CBA promoter, preferably 150%, 200%, 300%, or 500% of the activity of the CBA promoter or the spc5-12 promoter. In some embodiments, the synthetic skeletal muscle-specific promoters of the invention are suitable for promoting skeletal muscle-specific expression of a transgene at a level of at least 100% of the activity of the Tnnt2 promoter or the Myl2 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the spc5-12 promoter. Such muscle-specific expression is suitably determined in muscle-derived cells, e.g., C2C12 cells or H2K cells (skeletal muscle) or H9C2 cells (heart) or primary muscle cells (suitably primary human myocytes).

[0236] The synthetic muscle-specific promoters, synthetic cardiac muscle-specific promoters, or synthetic skeletal muscle-specific promoters of the invention may also be capable of promoting muscle-specific, or skeletal muscle-specific 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 cells or H2K cells (skeletal muscle) or h9C2 cells (heart)).

[0237] As used herein, the term "nucleic acid" 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 phosphate group, e.g., one, two, or three phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include, among others, 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 (e.g., methylated), non-naturally occurring, or derivatized bases. The sugar groups may preferably include pentose (pentofuranose) or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, among others, as well as modified or substituted sugar groups, which are common in naturally occurring nucleic acids. Nucleic acids as contemplated herein may include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or mixtures 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, including 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 may be, for example, a naturally occurring nucleic acid, present in nature or isolated from nature; a non-naturally occurring nucleic acid, i.e., produced by recombinant DNA technology and / or partially or entirely chemically or biochemically synthesized, e.g., a recombinant nucleic acid. A "nucleic acid" may be double-stranded, partially double-stranded, or single-stranded. If single-stranded, the nucleic acid may be the sense strand or the antisense strand. In addition, a nucleic acid may be circular or linear.

[0238] "Isolated" when referring to a nucleic acid means a nucleic acid molecule that is devoid of all or part of the sequences with which it is normally associated in nature; or a sequence that is present in nature but has associated heterologous sequences; or a molecule that is dissociated from the chromosome.

[0239] Terms such as "identity" and "identical" refer to sequence similarity between two nucleic acid molecules, such as between two polymeric molecules, e.g., between two DNA molecules. Sequence alignment and sequence identity determination can be performed using the Basic Local Alignment Search Tool (BLAST), originally described by Altschul et al., 1990 (J Mol Biol, 215:403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden, 1999 (FEMS Microbiol Lett, 174:247-250).

[0240] Methods for aligning sequences for comparison are well known in the art. For various programs and alignment algorithms, see, e.g., 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 calculations can be found, for example, in Altschul et al. (1990), J. Mol. Biol., 215:403-10.

[0241] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and is also available on the Internet for use in conjunction with several sequence analysis programs. A description of how to use this program to determine sequence identity is available on the Internet under the "Help" section for BLAST™. For comparison of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program can be used, using default parameters. Nucleic acid sequences with even greater similarity to the reference sequence will show increasing percentage identity when evaluated by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0242] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm, with the following scoring parameters: match score: +2, mismatch score: -3; gap penalties: gap open: 5, gap extension: 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, including both matches and mismatches, multiplied by 100.

[0243] The term "hybridizing" means that two at least partially complementary nucleotide sequences anneal in a hybridization step. 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 remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridization is 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 edition, Cold Spring Harbor Laboratory Press, CSH, New York, but the skilled artisan will realize that a large number of different hybridization conditions can be designed as a function of known or predicted homology and / or length of the nucleic acid sequences. High stringency conditions for hybridization include high temperature and / or low sodium / salt concentration (salts include, for example, sodium in NaCl and Na citrate), and / or incorporation of formamide into the hybridization buffer, and / or lowering the concentration of compounds such as SDS (sodium dodecyl sulfate detergent) in the hybridization buffer, and / or excluding compounds (promoting molecular overcrowding), such as dextran sulfate or polyethylene glycol, from the hybridization buffer. By way of non-limiting example, representative salt and temperature conditions for stringent hybridization are 65° C., 1× SSC, 0.5% SDS. The abbreviation SSC refers to the buffer used in the nucleic acid hybridization solution. One liter of 20× stock (20× concentrated) SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate. A typical time to achieve hybridization is 12 hours.

[0244] 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 may also be used, provided that they are bound by the intended TF. Consensus sequences for various TFBSs known in the art are disclosed herein, and one of skill in the art may readily use this information to determine alternative TFBSs. Furthermore, the ability of a TF to bind to a given putative sequence may be readily determined by one of skill in the art through experimentation (e.g., by EMSA and other techniques well known in the art and discussed herein).

[0245] 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 sequences: A[CT]N{A}YR When considering the above, A means that A is always found at this position; [CT] represents C or T at this position; N represents any base at this position; {A} means that any base except A can be found at this position, Y represents any pyrimidine, and R denotes any purine.

[0246] "Synthetic" in this application refers to a nucleic acid molecule that is not naturally occurring. 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 such regulatory sequences), but they are present in a non-naturally occurring context. For example, a synthetic gene (or part 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, and combinations thereof.

[0247] As used herein, "complementary" or "complementarity" refers to Watson-Crick base pairing of two nucleic acid sequences. For example, the sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'. Complementarity between two nucleic acid sequences can be "partial" complementarity, where only some of the bases bind to their complements, or it can be complete complementarity, where every base in the sequence binds to its complementary base. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0248] In this application, "transfection" refers broadly to any process of deliberately introducing a nucleic acid into a cell, covers the introduction of viral and non-viral vectors, and includes or is synonymous with terms and processes such as transformation, transduction, etc. 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 gene guns (Klein et al. (1987), Nature, 327:70).

[0249] As used herein, the phrase "transgene" refers to an exogenous nucleic acid sequence. In one example, the transgene is a gene that encodes an industrially or pharma- ceutically useful compound, or a gene that encodes a desired trait. In yet another example, 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, e.g., a protein.

[0250] The term "vector" is well known in the art and is used herein to refer to a nucleic acid molecule, e.g., double-stranded DNA, into which a nucleic acid sequence according to the invention may be inserted. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all the necessary elements that allow the transcription of the inserted nucleic acid molecule and, preferably, the translation of the transcript into a polypeptide. A vector typically, once inside a host cell, the vector is capable of replicating independently of or simultaneously with the host chromosomal DNA; it contains all the necessary elements so that several copies of the vector and its inserted nucleic acid molecule can be made. A vector of the invention may be an episomal vector (i.e., not integrated into the genome of the host cell) or may be integrated into the genome of the host cell. This definition includes 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 derivatives thereof lacking bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors, or Sleeping Beauty (SB) vectors), and the like. Large vectors such as artificial chromosomes (bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or human artificial chromosomes (HAC)) can be used to accommodate large inserts. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis virus vectors, and the like. Typically, but not necessarily, viral vectors are replication-deficient because viral genes essential for replication have been lost from the viral vector, rendering it unable to reproduce in a given cell. However, some viral vectors may also be adapted to replicate specifically in given cells, e.g. cancer cells, but are typically used to induce (cancer) cell-specific (onco)lysis.Virosomes are a non-limiting example of a vector that contains both viral and non-viral elements, in particular, virosomes combine liposomes with inactivated HIV or inactivated influenza virus (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0251] As used herein, the terms "operably linked", "operably connected" or equivalent expressions refer to the arrangement of various nucleic acid elements relative to each other such that the elements are functionally connected and capable of interacting with each other in an intended manner. Such elements may include, without limitation, synthetic promoters, CREs (e.g., enhancers or other regulatory elements), CRMs, promoter elements, polyadenylation sequences, one or more introns and / or exons, and the coding sequence of the gene of interest to be expressed. When properly oriented or operably linked, the nucleic acid sequence elements can act together to modulate each other's activity, ultimately affecting the expression level of the expression product. "Modulating" means increasing, decreasing, or maintaining the activity level of a particular element. The position of each element relative to other elements may be expressed in terms of the 5' and 3' ends of each element, or in terms of their position upstream or downstream of another element or position (such as a TSS or promoter element), and the distance between any particular elements may be referred to by the number of nucleotides or base pairs intervening between the elements. As will be understood by those skilled in the art, "operably linked" implies functional activity and does not necessarily refer to a positional association in nature. Indeed, when used within a nucleic acid expression cassette, the CRE is typically placed immediately upstream of the promoter element (this is the general case, but should not be construed as a definitive restriction or exclusion of the position within the nucleic acid expression cassette), although in vivo this is not necessarily the case, e.g., a naturally occurring regulatory element sequence downstream of the gene whose transcription it affects can function in the same way as if it were placed upstream of the promoter. Thus, according to specific embodiments, the regulatory or enhancing effect of the regulatory element may be position-independent.

[0252] 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 elements, etc.). It can have essentially any sequence, provided that it does not prevent the functional nucleic acid sequence (e.g., cis-regulatory element) from functioning as desired (e.g., this can occur when it contains a silencer sequence of a desired transcription factor, preventing its binding, etc.). Typically, it is non-functional, since it is present only to space adjacent functional nucleic acid sequences from each other. In some embodiments, the spacer can be 75, 50, 40, 30, 30, or 10 nucleotides in length, or less. In some embodiments, one or more spacers can be recognition sites for one or more restriction enzymes.

[0253] As used herein, the term "pharmaceutically acceptable" means consistent with the art, compatible with the other ingredients of a pharmaceutical composition, and not deleterious to the recipient thereof.

[0254] Phrases such as "therapeutically effective amount" refer to a dosage or plasma concentration in a subject that results in expression of a therapeutic gene, e.g., intramuscularly, that produces a desired specific pharmacological effect. Although a therapeutically effective amount may not always be effective in treating a condition described herein, such an amount would still be considered a therapeutically effective amount by those skilled in the art. A therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the disease or condition being treated.

[0255] As used herein, the term "AAV vector" refers to the nucleic acid sequence of an AAV vector, which is well known in the art and generally includes a variety of nucleic acid sequences. As used herein, an AAV vector typically includes a heterologous nucleic acid sequence, not derived from AAV, as part of the vector. This heterologous nucleic acid sequence typically includes a promoter as disclosed herein, as well as other sequences of interest for genetic transformation of a cell. Generally, the heterologous nucleic acid sequence is flanked by at least one inverted terminal repeat (ITR), generally flanked by two AAV ITRs. "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle that is composed of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an AAV vector by an encapsidated polynucleotide. 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 as an "AAV vector." Thus, production of AAV virions or AAV particles necessarily includes production of the AAV vector as such a vector is contained within the AAV virion or AAV particle.

[0256] "Small interfering RNA" or "short interfering RNA" or siRNA is an RNA duplex with nucleotides targeted to a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. The 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 target 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 in length. In some embodiments, the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can contain a loop portion located between the two sequences that form the duplex. The loop may vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also contain a 3' or 5' overhang. In some embodiments, the overhang is a 3' or 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0257] As used herein, the term "microRNA" refers to any type of interfering RNA, including but not limited to endogenous microRNA and artificial microRNA (e.g., synthetic miRNA). Endogenous microRNA is a small RNA that is naturally encoded in the genome and can modulate the productive utilization of mRNA. Artificial microRNA can be any type of RNA sequence, other than endogenous microRNA, that can modulate the activity of mRNA. MicroRNA sequence can be an RNA molecule that is composed of any one or more of these sequences. MicroRNA (or "miRNA") sequences have been described in publications such as Lim et al., 2003, Genes & Development, 17, 991-1008; Lim et al., 2003, Science, 299, 1540; Lee and Ambrose, 2001, Science, 294, 862; Lau et al., 2001, Science 294, 858-861; Lagos-Quintana et al., 2002, Current Biology, 12, 735-739; Lagos-Quintana et al., 2001, Science, 294, 853-857; and Lagos-Quintana et al., 2003, RNA, 9, 175-179. Examples of microRNAs include any RNA fragment of a larger RNA, or are miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNA. See, for example, U.S. Patent Application Nos. 20050272923, 20050266552, 20050142581, and 20050075492. "MicroRNA precursor" (or "pre-miRNA") refers to a nucleic acid having a stem-loop structure incorporating a microRNA sequence therein."Mature microRNA" (or "mature miRNA") includes microRNA that is cleaved from a microRNA precursor ("pre-miRNA") or synthesized (e.g., synthesized in the laboratory by cell-free synthesis) and has a length of about 19 nucleotides to about 27 nucleotides, e.g., mature microRNAs can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt. Mature microRNAs can bind to target mRNAs and inhibit translation of the target mRNA.

[0258] The term "treatment" or "treating" refers to reducing, ameliorating, or eliminating one or more signs, symptoms, or effects of a disease or condition. Thus, as used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who is predisposed to or at risk of having the disease, but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0259] "Administration" of an agent to a subject includes any route that introduces or delivers the agent to a subject to perform its intended function. Administration can be by any suitable route, including oral, intranasal, intraocularly, ophthalmically, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration can be via antegrade epicardial coronary injection. Administration includes self-administration and administration by another person. Intramuscular administration is of particular interest in the present invention.

[0260] The terms "individual," "subject," and "patient" are used interchangeably and refer to any individual subject with a disease or condition requiring treatment. For purposes of this disclosure, a subject can be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or cow. EXAMPLES

[0261] The strength of synthetic muscle-specific or skeletal muscle-specific promoters according to certain embodiments of the invention was examined by operably linking them to the reporter gene luciferase. Expression cassettes containing the test muscle-specific or skeletal muscle-specific promoter and the luciferase gene were inserted into appropriate plasmids, and cells were then transfected with these plasmids to examine expression from the promoter in the cells.

[0262] Example 1 In vitro testing Materials and Methods DNA preparations were transfected into H9C2 (a rat BDIX cardiomyoblast cell line; available from ATCC) and assessed for transcriptional activity. The H9C2 cell line was used because previous experiments have shown it to be a good predictor of skeletal and cardiac muscle activity in vivo. The DNA preparation contained a synthetic promoter (e.g., SP0500) operably linked to luciferase.

[0263] Culture and transfection of H9C2 cells H9C2 is a rat BDIX cardiomyoblast cell line. H9C2 has cardiac muscle properties, e.g., myotubes formed at confluency are responsive to acetylcholine.

[0264] Cell maintenance H9C2 cells were cultured in T-75 flasks in DMEM (high glucose; D6546; Sigma) with 1% FBS (heat inactivated; Gibco: 10270-106; lot number: 42G2076K), 1% Glutamax (35050-038; Gibco), 1% penicillin-streptomycin solution (15140-122; Gibco). Cells were subcultured at the subconfluent stage (70-80%) to avoid the risk of cells becoming confluent and fusing to form myotubes.

[0265] For subculture during cell maintenance, the culture medium was removed and the cells were soaked in CaCl 2 Without MgCl 2 The flasks were washed twice with 5 ml of DPBS (14190-094; Gibco) without trypsin. The cells were dissociated from the flasks by incubating with 1 ml of Trypsin EDTA (25200-056; Gibco) for approximately 5 minutes. 4 ml of culture medium was then added to the flask and the mixture was gently pipetted up and down to help dissociate the cells from the flask surface. The cells were pelleted at 100g for 3 minutes. The supernatant was discarded and the cells were resuspended in 3 ml of culture medium. The cells were counted on a Countess automated cell counter and seeded at 1:3 to 1:10, i.e., 1 cm 2 Seed 1–3 × 10,000 cells per well and incubate at 37 °C, 5% CO. 2 and incubated at 4°C for 1 hour.

[0266] Cell transfection and differentiation H9C2 cells were harvested from two T-75 flasks at approximately 70-80% confluency by washing with DPBS, dissociating from the flask using 1 ml of trypsin-EDTA, rinsing from the flask surface with 4 ml of culture medium, and pelleting at 100 g for 3 min as described above. Cells were resuspended in 45 ml of culture medium and seeded into 48-well flat-bottom plates (300 μl per well) (353230; Corning) at a density of 40,000 cells per well. Cells in the 48-well plates were incubated at 37°C, 5% CO2 for 3 h. 2 and incubated at 4°C for 1 hour.

[0267] After 24 hours, the culture medium on the cells was replaced with 300 μl of antibiotic-free culture medium (i.e., DMEM (high glucose; D6546; Sigma) with 1% FBS (heat inactivated; Gibco: 10270-106; lot number: 42G2076K), 1% Glutamax (35050-038; Gibco)). 300 ng of DNA per well was transfected with ViaFect (E4981; Promega) in a total complex volume of 30 μl per well. After transfection, the plates were gently mixed and incubated at 37°C, 5% CO. 2 and incubated at 4°C for 1 hour.

[0268] After 24 hours, the culture medium was removed from the transfected cells and replaced with 300 μl of differentiation medium consisting of DMEM (high glucose; D6546; Sigma), 1% Glutamax (35050-038; Gibco), 1% FBS (heat inactivated; Gibco: 10270-106; Lot No.: 42G2076K), 1% penicillin-streptomycin solution (15140-122; Gibco), and 0.1% retinoic acid (Sigma: R2625). The plates were incubated at 37° C., 5% CO2 for 7 days to induce differentiation. After differentiation, the cell morphology was observed to confirm differentiation into myotubes.

[0269] The cells were then washed with 500 μl of DPBS and lysed with 100 μl of Luciferase Cell Culture Lysis 5× Reagent (E1531; Promega) diluted to 1× using Milli-Q water. The cell lysis reagent was pipetted up and down 10 times, and the plate was then vortexed at medium power for 30 minutes to promote cell lysis. The plate was sealed and stored at −80° C. before completing the luciferase assay. Data collected from luciferase assays after transfection in H9C2 cells are based on three technical replicates with one biological replicate.

[0270] Measurement of luciferase activity - Luciferase activity was measured using LARII (Dual Luciferase Reporter 1000 Assay System; Promega; E1980). - 24 hours after transfection, the medium was removed from the cells. - The cells were washed once in 300 μl of DPBS. - Cells were lysed using 100 μl of passive lysis buffer and incubated for 15 min with shaking. - Cell debris was pelleted by centrifugation of the plate in a benchtop centrifuge at full speed for 1 minute. - 10 μl of sample was transferred to a white 96-well plate and luminescence was measured by injection of 50 μl of LARII substrate into a BMG Labtech FLUOstar Omega plate reader.

[0271] The results from these cell cultures are shown in Figure 1. This figure shows that the synthetic promoters SP0500, SP0510, SP0514 and SP0519 show good activity in the muscle cell line H9C2. Other similar promoters described herein are expected to have the same or better performance.

[0272] Example 2 In vitro data The experiments were carried out as detailed in Example 1 above, however, in this example, the data collected from the luciferase assay following transfection in H9C2 cells is based on triplicate biological replicates, each of which is the average of triplicate technical replicates.

[0273] The results from these cell cultures are shown in Figure 2, where the results are normalized relative to CBA, which shows that the synthetic promoters SP0497, SP0500, SP0501, SP0506, SP0508, SP0510, SP0514, SP0519, SP0520, SP0521 and SP4169 show good activity in the muscle cell line H9C2. The promoters SP0498, SP0499, SP0502, SP0503, SP0504, SP0505, SP0507, SP0509, SP0511, SP0512, SP0513, SP0515, SP0516, SP0517, SP0518, SP0522, SP0523, and SP0524 were also experimentally tested in the H9C2 cell line and showed low activity (data not shown).

[0274] Example 3 In vivo data A selection of synthetic muscle-specific promoters were investigated in vivo (see, e.g., Figures 3-18).

[0275] Materials and Methods AAVs containing a synthetic promoter operably linked to luciferase (e.g., SP0500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524) were diluted in 0.9% saline and delivered via tail vein injection to 8-week-old male Balb / c mice (6 mice per group) at 1 × 10 per 200 μl per mouse. 11 vg. Six weeks after injection, mice were sacrificed. Diaphragm, heart, quadriceps, soleus, tibialis anterior (TA), and liver were harvested from each mouse. For vector copy number (VCN) analysis, samples were snap frozen in liquid nitrogen immediately after excision and stored at -80°C.

[0276] Proteins were extracted and quantified using the BCA Pierce Protein Assay Kit (ThermoFisher; 23225) according to the manufacturer's instructions.

[0277] Luciferase was quantified using the ONE-Glo Luciferase Assay System (Promega; E6120).

[0278] DNA was extracted and all samples and reagents were incubated at room temperature until completely melted and at equilibrium temperature. All samples and reagents were mixed thoroughly before use. ONE-Glo™ Reagent was added to each sample in equal volume and the samples were mixed thoroughly. After 3 minutes, samples were measured by luminometer to ensure complete cell lysis.

[0279] Vector copy number testing was performed by dual Taqman qPCR. DNA was extracted using DNeasy Blood & Tissue Kit (250) (QIAGEN; model number: 69506). For each sample, Taqman qPCR was performed using both the luciferase-specific primer / probe set and the GAPDH-specific primer / probe set:

[0280] [Table 1]

[0281] For analytical purposes, standard curves were used for luciferase and GAPDH. 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 / uL). The PCR cycle protocol was as follows: 95°C: 20 sec, PCR: 40 cycles, 95°C: 1 sec, 60°C: 20 sec. The ΔΔCt (threshold) of vector copy number (copy amount) per genome was calculated by subtracting the average vector copy number (copy amount) per genome for the saline samples.

[0282] result As shown in Figure 12, the synthetic muscle-specific promoter SP0500 showed high activity in cardiac muscle (heart). In addition, SP0500 showed activity in skeletal muscles (e.g., tibialis anterior, quadriceps, and diaphragm).

[0283] As shown in FIG. 13, the synthetic muscle-specific promoter, SP0507, exhibits activity in skeletal muscle (eg, tibialis anterior) and cardiac muscle (heart).

[0284] As shown in Figure 14, the synthetic muscle-specific promoter SP0514 exhibits activity in cardiac muscle (heart). In addition, SP0514 exhibits some activity in skeletal muscle (e.g., diaphragm and tibialis anterior).

[0285] As shown in Figure 15, the synthetic muscle-specific promoter SP0518 exhibits activity in skeletal muscle (e.g., tibialis anterior). In addition, SP0518 exhibits some activity in cardiac muscle.

[0286] As shown in Figure 16, the synthetic muscle-specific promoter SP0519 exhibits activity in skeletal muscle (e.g., tibialis anterior). In addition, SP0519 exhibits some activity in cardiac muscle.

[0287] As shown in Figure 17, the synthetic muscle-specific promoter SP0522 showed high activity in cardiac muscle (heart). In addition, SP0522 showed activity in skeletal muscle (e.g., tibialis anterior and diaphragm).

[0288] As shown in FIG. 18, the synthetic muscle-specific promoter, SP0524, showed high activity in cardiac (heart) and skeletal muscles (eg, tibialis anterior, diaphragm, and quadriceps).

[0289] As shown in Figure 3, the synthetic muscle-specific promoter SP0524 showed equal or greater activity in the diaphragm compared to the control promoters CMV and CK8. The synthetic muscle-specific promoters SP500, SP0518, and SP0522 showed equal or greater activity in the diaphragm compared to CK7.

[0290] As shown in FIG. 4, the synthetic muscle-specific promoter SP0524 exhibited similar activity in tibialis anterior muscle as the control promoters CMV, CK7, and CK8.

[0291] As shown in FIG. 5, the synthetic muscle-specific promoters, SP500, SP0522, and SP0524, showed equal or greater activity in the heart compared to the control promoters, CK8, CMV, and CK7.

[0292] As shown in FIG. 6, all tested synthetic promoters showed low activity in quadriceps muscle compared to the control promoters, CK8, CMV, and CK7.

[0293] As shown in Figure 7, the synthetic muscle-specific promoter SP0524 showed equal or greater activity in soleus muscle than the control promoters CK8 and CMV. The synthetic muscle-specific promoters SP0500 and SP0522 showed equal or greater activity than the control promoter CK7.

[0294] As shown in FIG. 8, the tested synthetic muscle-specific promoters, SP500, SP0507, SP0514, SP0518, SP0519, SP0522, and SP0524, showed reduced or similar activity in the control promoters, CK8 and CMV, compared to liver.

[0295] Bibliography Llanga, T. et al. (2017) ‘Structure-Based Designed Nano-Dysferlin Significantly Improves Dysferlinopathy in BLA / J Mice’, Molecular Therapy. Elsevier Ltd., 25(9), pp. 2150-2162. doi: 10.1016 / j.ymthe.2017.05.013.

[0296] Sequence information

[0297]

Table 2A

[0298]

Table 2B

[0299]

Table 2C

[0300]

Table 2D

[0301]

Table 3A

[0302]

Table 3B

[0303]

Table 4A

[0304]

Table 4B

[0305]

Table 5

[0306]

Table 6

Claims

1. a) A cis-regulatory module (CRM) comprising a sequence according to any one of SEQ ID NOs: 47, 30-46, or a functional variant thereof, wherein the functional variant comprises 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: 47, 30-46, or b) A sequence according to any one of SEQ ID NOs: 29, 27, 28, 1-26, 66, or a functional variant thereof, wherein the functional variant comprises 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: 29, 27, 28, 1-26, 66 comprising a synthetic muscle-specific promoter.

2. The synthetic muscle-specific promoter according to claim 1a), comprising the CRM as defined above, operably linked to a promoter element.

3. The synthetic muscle-specific promoter according to claim 1, wherein the functional variant retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter.

4. A muscle-specific cis-regulatory element (CRE) comprising a sequence according to any one of SEQ ID NOs: 54, 56, 48-53, 55, 57-61, 67, or a functional variant thereof, wherein the functional variant comprises 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: 54, 56, 48-53, 55, 57-61, or 67.

5. A synthetic muscle-specific promoter comprising the CRE according to claim 4.

6. An isolated minimal promoter or an isolated proximal promoter comprising a sequence according to any one of SEQ ID NOs: 63, 62, 65, 64, 68, or a functional variant thereof, wherein the functional variant comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 63, 62, 65, 64, or 68.

7. A synthetic muscle-specific promoter comprising the minimal promoter or proximal promoter according to claim 6.

8. An expression cassette comprising the synthetic muscle-specific promoter according to claim 1, operably linked to a sequence encoding an expression product.

9. A vector comprising the synthetic muscle-specific promoter according to claim 1.

10. The vector according to claim 9, which is an AAV vector, an adenovirus vector, a retrovirus vector, or a lentivirus vector.

11. A virion comprising the vector according to claim 10.

12. A pharmaceutical composition comprising the synthetic muscle-specific promoter according to any one of claims 1 to 3, 5 and 7, the expression cassette according to claim 8, the vector according to claim 9 or 10, or the virion according to claim 11.

13. A pharmaceutical composition for use in the treatment of a condition or disease, wherein the pharmaceutical composition comprises the synthetic muscle-specific promoter according to any one of claims 1 to 3, 5 and 7, the expression cassette according to claim 8, the vector according to claim 9 or 10, or the virion according to claim 11.

14. A cell comprising the synthetic muscle-specific promoter according to any one of claims 1 to 3, 5 and 7, the expression cassette according to claim 8, the vector according to claim 9 or 10, or the virion according to claim 11.

15. The pharmaceutical composition according to claim 12, for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

16. A method for producing an expression product ex vivo, comprising the steps of providing the synthetic muscle-specific expression cassette according to claim 8 into muscle cells and expressing the gene present in the synthetic muscle-specific expression cassette.

17. A method for expressing a therapeutic transgene in muscle cells ex vivo, comprising the step of introducing into the muscle cells the synthetic muscle-specific expression cassette according to claim 8, the vector according to claim 9 or 10, or the virion according to claim 11. A pharmaceutical composition for use in the treatment of a condition or disease in a subject in need thereof, preferably a human, wherein said composition comprises the expression cassette according to claim 8, the vector according to claim 9 or 10, or the virion according to claim 11, comprising a sequence encoding a therapeutic product operably linked to the promoter according to any one of claims 1 to 3, 5 and 7, wherein said expression cassette, vector, virion, or pharmaceutical composition is administered to a subject and a therapeutically effective amount of the therapeutic product is expressed intramuscularly in said subject and optionally a therapeutically effective amount of the therapeutic product is expressed within skeletal muscle and / or within cardiac muscle. The pharmaceutical composition according to claim 13, wherein said condition or disease is associated with abnormal gene expression, said disease is congestive heart failure, and / or said disease is cardiomyopathy.

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