Regulating nucleic acid sequences

Abstract

The present invention relates to regulatory nucleic acid sequences capable of enhancing muscle-specific expression of a gene, in particular muscle-specific promoters, elements thereof and other such nucleic acid sequences. In particular, the present invention relates to regulatory nucleic acid sequences capable of enhancing muscle-specific expression but with low liver-specific expression. The invention also relates to expression constructs, vectors, and cells comprising such muscle-specific regulatory nucleic acid sequences, as well as methods of their use. The regulatory nucleic acid sequences are particularly useful for gene therapy applications, but can also be used in other fields, such as bioprocessing and biotechnology.

Description

Technical Field

[0001] The present invention relates to regulatory nucleic acid sequences capable of enhancing muscle-specific expression of genes, particularly muscle-specific promoters, elements thereof, and other such nucleic acid sequences. In particular, the present invention relates to regulatory nucleic acid sequences capable of enhancing muscle-specific expression but having low expression in other tissues, such as the liver. The present invention also relates to expression constructs, vectors, and cells comprising such regulatory nucleic acid sequences, as well as methods of using the same. The regulatory nucleic acid sequences are particularly useful for gene therapy applications, but may also be used in other fields, such as bioprocessing and biotechnology. Background Art

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

[0003] In many fields, including gene therapy, there is a need to provide regulatory nucleic acid sequences that can drive gene expression to produce proteins or nucleic acid expression products in desired cells, tissues or organs.

[0004] The expression of therapeutic genes in muscle is very attractive for gene therapy. Gene therapy in muscle has the potential to correct or enhance the expression of various muscle proteins such as dystrophin and sarcoglycan. This can be used to treat conditions such as muscular dystrophy, for example Duchenne muscular dystrophy (DMD). Muscle can also be used as a platform for expressing therapeutic proteins to treat other conditions.

[0005] Various vectors have been used to deliver genes to muscle cells, such as adenoviruses, retroviruses, lentiviruses, and adeno-associated viruses (AAV), as well as non-viral vectors such as plasmids. Adenoviral vectors have a relatively large cloning capacity and can efficiently transduce some cells. However, they face significant challenges given that they tend to elicit a strong immune response. Retroviral and lentiviral vectors stably integrate into the genome, which has both advantages and disadvantages. Lentiviral vectors can transduce both dividing and non-dividing cells, but most conventional retroviral vectors can only transduce dividing cells, which limits their use in non-dividing muscle cells. Plasmid DNA can be used to transfer genes to muscle cells in vitro, but their potential utility in clinical settings is unclear.

[0006] AAV vectors are particularly attractive for gene therapy applications in muscle. AAV vectors exhibit a natural tropism for muscle cells, driving long-term expression of therapeutic payloads while eliciting minimal immune responses. Although some gene therapy vectors are able to preferentially transduce muscle cells, off-target transduction does occur. Several phase 1 and 2 clinical trials using AAV serotypes 1, 2, and chimeric 2.5 for the treatment of Duchenne muscular dystrophy (DMD) and alpha-1 antitrypsin deficiency have been reported (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. Brieger, L. Govindasamy, M. Agbandje-McKenna, X. Xiao and R. J. Samulski, 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 the National Academy of Sciences of the United States of America 106, 16363-16368 (2009); virus alpha 1-antitrypsin(rAAV2-CB-hAAT)gene vector to AAT-deficient adults. Human genetherapy 15,93-128(2004); TRFlotte, BCTrapnell,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 2clinical trialof a recombinant adeno-associated viralvector 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,HumanTreg responses allow sustained recombinant adeno-associated virus-mediatedtransgene expression.The Journalof clinicalinvestigation 123,5310-5318(2013))。.

[0007] One of the off-target effects that has been consistently observed in clinical trials of AAV therapeutic products is liver toxicity. Recently, the U.S. Food and Drug Administration has suspended Audentes Therapeutics' Phase II gene therapy trial for X-linked myotubular myopathy due to deaths caused by progressive liver dysfunction (High-dose AAV genetherapy deaths. Nat Biotechnol 38, 910 (2020). https: / / doi.org / 10.1038 / s41587-020-0642-9). The dose these patients received in this Phase II trial was the highest dose of any AAV-based gene therapy to date (3×10 14 vg / kg). Similarly, two deaths due to acute liver failure have been reported after treatment with Zolgensma, a gene therapy for spinal muscular atrophy (High-dose AAV gene therapy deaths. Nat Biotechnol 38, 910 (2020). https: / / doi.org / 10.1038 / s41587-020-0642-9).

[0008] The use of cis-acting regulatory elements has been proposed to provide muscle specificity and activity. Typically, this involves a cis-regulatory enhancer sequence, a nucleic acid sequence that acts in cis to increase promoter activity. Various muscle-specific promoters are known in the art and are typically obtained 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 C5-12 promoter represents a known synthetic muscle promoter.

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

[0010] Furthermore, different expression profiles within different muscle tissues may be desirable in different diseases. For example, a muscle-specific promoter with higher expression in skeletal muscle may be beneficial for myopathy treatment, while a promoter with higher expression in cardiac muscle may be beneficial for cardiomyopathy treatment. For certain diseases, such as DMD, high expression in different muscle groups may be desirable.

[0011] Finally, short regulatory sequences are ideal to minimize the proportion of the gene therapy vector occupied by regulatory sequences. This is particularly important for gene therapy vectors with limited capacity (payload), such as AAV vectors. Summary of the Invention

[0012] In a first aspect of the present invention, a synthetic muscle-specific cis-regulatory module (CRM) is provided, which comprises CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof and at least one additional regulatory element (such as CRE or detargeting element).

[0013] In some embodiments, the at least one additional regulatory element is selected from the group consisting of a CRE, a CRM, an inducible or repressible element, a boundary control element, an insulator, a locus control region, a response element, a binding site, a terminal repeat segment, a response site, a stabilizing element, a destabilizing element, a detargeting element, a liver detargeting element, an intron, a UTR, and a splicing element, etc., as long as they do not render the CRM non-functional.

[0014] In some embodiments, the at least one additional regulatory element is a CRE, an inducible or repressible element, or a detargeting element. In some embodiments, the at least one additional regulatory element is a CRE. In some embodiments, the at least one additional regulatory element is a detargeting element. In some embodiments, the at least one detargeting element is a liver detargeting element.

[0015] The additional regulatory elements may be additional regulatory elements disclosed herein, or may be other additional regulatory elements. In some embodiments, the additional regulatory elements may be selected from the group consisting of: CRE0145 (SEQ ID NO: 10), DES_MT_enhancer 48 bp (SEQ ID NO: 11), liver detargeting sequence 1 (SEQ ID NO: 13), liver detargeting sequence 2 (SEQ ID NO: 14), liver detargeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA target sequence 1 (SEQ ID NO: 16), Mir122 miRNA target sequence 2 (SEQ ID NO: 19), tMCK SA / SD intron (SEQ ID NO: 29), and MVM truncated intron (SEQ ID NO: 30). In some embodiments, the additional regulatory elements can be selected from: CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), liver de-targeting sequence 1 (SEQ ID NO: 13), liver de-targeting sequence 2 (SEQ ID NO: 14), liver de-targeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA target sequence 1 (SEQ ID NO: 16) and Mir122 miRNA target sequence 2 (SEQ ID NO: 19).

[0016] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element are operably linked.

[0017] In some embodiments, the at least one additional regulatory element is 20 nucleotides or more, preferably 30 nucleotides or more, more preferably 40 nucleotides or more, even more preferably 50 nucleotides or more, and most preferably 60 nucleotides or more in length.

[0018] In some embodiments, the CRM according to the first aspect of the invention retains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the activity of the CRM consisting of SEQ ID NO: 8. The activity of the CRM consisting of SEQ ID NO: 8 is as described in application PCT / GB2022 / 051611, in particular page 9, lines 1-11 and Figure 5 、 6 , 17 and 18, which are incorporated herein by reference.

[0019] In some embodiments, the CRM according to the first aspect of the invention comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8.

[0020] In some embodiments, the at least one additional regulatory element is located 5' relative to CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof. In some embodiments, the at least one additional regulatory element is located 3' relative to CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof. In some embodiments, the at least one additional regulatory element is located between CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof.

[0021] In some preferred embodiments, the functional variants of CRE0145 and DES_MT_enhancer_48bp comprise sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0022] In some embodiments, the synthetic muscle-specific cis-regulatory module (CRM) comprises CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element, wherein the additional regulatory element is a liver de-targeting element selected from the group consisting of:

[0023] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0024] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0025] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0026] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0027] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0028] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof.

[0029] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and additional regulatory elements are operably linked.

[0030] The regulatory element may be a regulatory element disclosed herein, or may be another regulatory element. In some embodiments, the regulatory element or its functional variant involved herein may be selected from: CRE0145, DES_MT_enhancer_48bp, liver de-targeting sequence 1, liver de-targeting sequence 2, liver de-targeting sequence 3, ZBTB20 binding site, Mir122 miRNA target sequence 1, and Mir122 miRNA target sequence 2.

[0031] In some embodiments, the synthetic muscle-specific CRM comprises a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0032] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 1;

[0033] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 2;

[0034] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 3;

[0035] -CRE0145, liver detargeting sequence 3, DES_MT_enhancer_48bp, and liver detargeting sequence 3;

[0036] - CRE0145, DES_MT_enhancer_48bp and ZBTB20 binding site; and

[0037] -CRE0145, ZBTB20 binding site, DES_MT_enhancer_48bp and ZBTB20 binding site.

[0038] In some embodiments, the regulatory elements are present in the CRM in the order listed and are adjacent to each other. In some embodiments, the regulatory elements are operably linked.

[0039] In any combination of regulatory elements disclosed herein or their functional variants, the listed regulatory elements may be present in any order. In some preferred embodiments, the regulatory elements are present in the order listed (i.e., according to their position relative to an operably connected promoter element or gene, in an order from upstream to downstream).

[0040] In any combination of regulatory elements or functional variants thereof disclosed herein, some or all of the listed regulatory elements may be suitably placed adjacent to each other in the CRM (i.e., without any intervening regulatory elements). The regulatory elements may be continuous or discontinuous (i.e., they may be placed immediately adjacent to each other, or they may be separated by spacers or other sequences). In some preferred embodiments, the regulatory elements or functional variants thereof are provided in the order listed and are adjacent to each other. For example, a synthetic muscle-specific CRM may comprise CRE0145, DES_MT_enhancer_48bp immediately upstream, liver detargeting sequence 1 immediately upstream, and so on. In some embodiments, it is preferred that some or all of the regulatory elements are continuous.

[0041] In some embodiments of the invention, the synthetic muscle-specific CRM comprises a CRM selected from the group consisting of CRM_SP0525 (SEQ ID NO:20), CRM_SP0526 (SEQ ID NO:21), CRM_SP0527 (SEQ ID NO:22) and CRM_SP0528 (SEQ ID NO:9), or a functional variant of any one thereof. Suitably, the functional variant of any said CRM comprises a sequence that is at least 70% identical to a reference synthetic muscle-specific CRM, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference synthetic muscle-specific CRM (SEQ ID NO: 20, 21, 22 and 9).

[0042] In a second aspect of the present invention, there is provided a synthetic muscle-specific promoter comprising the CRM according to the first aspect.

[0043] In some preferred embodiments, the CRM is operably linked to a promoter element. In some preferred embodiments, the promoter element is SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0044] In some embodiments, the synthetic muscle-specific promoter comprises a synthetic muscle-specific CRM comprising CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element, optionally a liver detargeting element, wherein the liver detargeting element is selected from the group consisting of:

[0045] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0046] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0047] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; and

[0048] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0049] Operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0050] In some embodiments, the synthetic muscle-specific cis-regulatory module (CRM) comprises CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and a ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof.

[0051] In some embodiments, a synthetic muscle-specific promoter is provided, comprising:

[0052] A synthetic muscle-specific CRM comprising a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0053] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 1;

[0054] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 2;

[0055] -CRE0145, DES_MT_enhancer_48bp and liver detargeting sequence 3;

[0056] -CRE0145, DES_MT_enhancer_48bp and ZBTB20 binding site;

[0057] -CRE0145, liver detargeting sequence 3, DES_MT_enhancer_48bp, and liver detargeting sequence 3; and

[0058] -CRE0145, ZBTB20 binding site, DES_MT_enhancer_48bp and ZBTB20 binding site;

[0059] Operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0060] In some embodiments, the regulatory elements are present in the CRM in the order listed and are adjacent to each other.

[0061] In any combination of regulatory elements disclosed herein or their functional variants, the listed regulatory elements may be present in any order. In some preferred embodiments, the regulatory elements are present in the order listed (i.e., according to their position relative to an operably connected promoter element or gene, in an order from upstream to downstream).

[0062] In some embodiments, a synthetic muscle-specific promoter is provided, which comprises or consists of a cis-regulatory module (CRM), wherein the CRM consists of CRM_SP0525 (SEQ ID NO: 20), CRM_SP0526 (SEQ ID NO: 21), CRM_SP0527 (SEQ ID NO: 22) or CRM_SP0528 (SEQ ID NO: 9) or a functional variant thereof, or comprises CRM_SP0525 (SEQ ID NO: 20), CRM_SP0526 (SEQ ID NO: 21), CRM_SP0527 (SEQ ID NO: 22) or CRM_SP0528 (SEQ ID NO: 9) or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a CRM consisting of or comprising CRM_SP0525 (SEQ ID NO: 20), CRM_SP0526 (SEQ ID NO: 21), CRM_SP0527 (SEQ ID NO: 22), or CRM_SP0528 (SEQ ID NO: 9), or a functional variant thereof, operably linked to a promoter element. The promoter element can be a minimal promoter or a proximal promoter. The proximal promoter is preferably a muscle-specific proximal promoter. The promoter element can be a promoter element that is broadly active across different tissues and cell types. In some embodiments, the promoter element is SCP1 (SEQ ID NO: 12). The promoter element can be a minimal promoter. In some embodiments, the promoter element is CRE0053 (SEQ ID NO: 26).

[0063] In some embodiments, a synthetic muscle-specific promoter is provided, comprising or consisting of a sequence according to any one of SEQ ID NOs: 1-4 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a promoter selected from the group consisting of SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3) and SP0528 (SEQ ID NO: 4), or a functional variant of any one thereof. Suitably, the functional variant of any of said promoters comprises a sequence that is at least 70% identical to a reference synthetic muscle-specific promoter, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference synthetic muscle-specific promoter (SEQ ID NO: 1-4).

[0064] In a third aspect, a synthetic muscle-specific promoter is provided, comprising or consisting of:

[0065] - CRE0145 or a functional variant thereof,

[0066] -DES_MT_enhancer_48bp or its functional variant,

[0067] - SCP1 or a functional variant thereof, or CRE0053 or a functional variant thereof; and

[0068] - at least one additional regulatory element, such as a detargeting element, optionally a liver detargeting element.

[0069] In some embodiments, the at least one additional regulatory element is selected from the group consisting of a CRE, a CRM, an inducible or repressible element, a boundary control element, an insulator, a locus control region, a response element, a binding site, a terminal repeat segment, a response site, a stabilizing element, a destabilizing element, a detargeting element, a liver detargeting element, an intron, a UTR, and a splicing element, etc., as long as they do not render the synthetic muscle-specific promoter non-functional.

[0070] In some embodiments, the at least one additional regulatory element is selected from the group consisting of: a CRE, a CRM, an inducible or repressible element, a detargeting element, a UTR (e.g., a 5' and / or 3' UTR), and an intron. In some embodiments, the at least one additional regulatory element is selected from the group consisting of: a CRE, a CRM, an inducible or repressible element, a detargeting element, a UTR (e.g., a 5' and / or 3' UTR), and an intron, as long as they do not render the synthetic muscle-specific promoter non-functional.

[0071] The additional regulatory element can be a CRE or a detargeting element. In some embodiments, the detargeting element is a liver detargeting element. In some embodiments, the liver detargeting element is selected from the group consisting of:

[0072] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0073] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0074] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0075] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0076] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0077] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof.

[0078] In some embodiments, the synthetic muscle-specific promoter comprises SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4.

[0079] In some embodiments, the at least one additional regulatory element can be selected from: CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11), liver de-targeting sequence 1 (SEQ ID NO: 13), liver de-targeting sequence 2 (SEQ ID NO: 14), liver de-targeting sequence 3 (SEQ ID NO: 17), ZBTB20 binding site (SEQ ID NO: 15), Mir122 miRNA target sequence 1 (SEQ ID NO: 16), Mir122 miRNA target sequence 2 (SEQ ID NO: 19), tMCK SA / SD intron (SEQ ID NO: 29), MVM truncated intron (SEQ ID NO: 30).

[0080] In some embodiments, the at least one additional regulatory element can be a UTR (e.g., a 5' and / or 3' UTR) or an intron. In some embodiments, the at least one additional regulatory element can be an intron. In some embodiments, the intron is the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncated intron (SEQ ID NO: 30).

[0081] In some embodiments, the synthetic muscle-specific promoter comprises SP0530 (SEQ ID NO: 27) or SP0531 (SEQ ID NO: 28) or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequences 27-28.

[0082] In some preferred embodiments, CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, SCP1 or a functional variant thereof (or CRE0053 or a functional variant thereof) and at least one additional regulatory element are operably linked.

[0083] In some embodiments, the at least one additional regulatory element is 20 nucleotides or more, preferably 30 nucleotides or more, more preferably 40 nucleotides or more, even more preferably 50 nucleotides or more, and most preferably 60 nucleotides or more in length.

[0084] In some embodiments, the synthetic muscle-specific promoter according to the third aspect of the invention retains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the activity of the synthetic promoter consisting of SEQ ID NO: 7. The activity of the synthetic muscle-specific promoter consisting of SEQ ID NO: 7 is as described in application PCT / GB2022 / 051611, in particular page 9, lines 7-11 and Figure 5 、 6 and 18, which are incorporated herein by reference.

[0085] In some embodiments, the synthetic muscle-specific promoter according to the third aspect of the invention comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the synthetic muscle-specific promoter consisting of SEQ ID NO:7.

[0086] In some embodiments, the at least one additional regulatory element is located 5' relative to the other regulatory elements. In some embodiments, the at least one additional regulatory element is located 3' relative to the other regulatory elements. In some embodiments, the at least one additional regulatory element is located between the other regulatory elements.

[0087] In some embodiments, a synthetic muscle-specific promoter is provided, comprising:

[0088] CRE0145 or a functional variant thereof, DES_MT_enhancer_48bp or a functional variant thereof, and at least one additional regulatory element, wherein the additional regulatory element is a liver de-targeting element and is selected from the group consisting of:

[0089] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0090] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0091] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0092] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0093] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0094] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof, or wherein the additional regulatory element is an intron and is selected from the group consisting of: tMCK SA / SD intron (SEQ ID NO: 29) or MVM truncated intron (SEQ ID NO: 30),

[0095] Optionally operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof, or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0096] In some embodiments, the synthetic muscle-specific promoter comprises SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0530 (SEQ ID NO: 27), or SP0531 (SEQ ID NO: 28), or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, 27-28.

[0097] In some embodiments, a muscle-specific promoter as described above is operably linked to one or more additional regulatory elements. The additional regulatory elements can, for example, enhance expression compared to a muscle-specific promoter not operably linked to the additional regulatory elements. Generally, it is preferred that the additional regulatory elements do not substantially reduce the specificity of the muscle-specific promoter.

[0098] For example, the synthetic muscle-specific promoter according to the present invention may be operably linked to sequences encoding UTRs (eg, 5' and / or 3' UTRs) and / or introns, etc.

[0099] In a fourth aspect of the present invention, a synthetic muscle-specific promoter SP0524 (SEQ ID NO: 7) operably linked to an intron is provided. In some embodiments, the intron is the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncated intron (SEQ ID NO: 30). In some embodiments, the synthetic muscle-specific promoter comprises or consists of SP0530 (SEQ ID NO: 27) or SP0531 (SEQ ID NO: 28) or a functional variant thereof of any one thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-28.

[0100] In a fifth aspect of the present invention, a synthetic muscle-specific promoter is provided, comprising a liver detargeting element. In some embodiments, a synthetic muscle-specific promoter is provided, comprising a minimal ZBTB20 binding site, suitably comprising SEQ ID NO: 15 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic muscle-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic muscle-specific promoter comprising a liver detargeting element has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% reduced expression in the liver compared to an equivalent synthetic muscle-specific promoter that does not comprise the liver detargeting element. In some embodiments, addition of the liver detargeting element to the synthetic muscle-specific promoter does not affect its muscle-specific expression.In some preferred embodiments, the muscle-specific promoter consists of or comprises SEQ ID NOs: 1-137, 342-367, 424-453 and 478-509 or functional variants thereof in PCT / GB2020 / 053371, which is incorporated herein by reference. In some preferred embodiments, the muscle-specific promoter consists of SEQ ID No: 1-137, 342-367, 424-453 and 478-509 or their functional variants and the following sequence in PCT / GB2020 / 053371, or comprises SEQ ID No: 1-137, 342-367, 424-453 and 478-509 or their functional variants and the following sequence in PCT / GB2020 / 053371: .

[0101] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15;

[0102] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13;

[0103] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14; or

[0104] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17.

[0105] In some preferred embodiments, the muscle-specific promoter consists of SEQ ID NO: 1-29, 66 or its functional variant in PCT / GB2022 / 051611, or comprises SEQ ID NO: 1-29, 66 or its functional variant in PCT / GB2022 / 051611, which is incorporated herein by reference. In some preferred embodiments, the muscle-specific promoter consists of SEQ ID NO: 1-29, 66 or its functional variant in PCT / GB2022 / 051611 and the following sequence, or comprises SEQ ID NO: 1-29, 66 or its functional variant in PCT / GB2022 / 051611 and the following sequence:

[0106] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15;

[0107] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13;

[0108] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14; or

[0109] - a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17.

[0110] In a sixth aspect of the present invention, a synthetic CNS-specific promoter is provided, which comprises a liver de-targeting element. In some embodiments, a synthetic CNS-specific promoter is provided, which comprises a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic CNS-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the synthetic CNS-specific promoter comprising a liver detargeting element has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% reduced expression in the liver compared to an equivalent synthetic CNS-specific promoter that does not comprise the liver detargeting element. In some embodiments, the addition of a liver detargeting element to the synthetic CNS-specific promoter does not affect its CNS-specific expression.

[0111] In a seventh aspect of the present invention, a synthetic kidney-specific promoter comprising a liver de-targeting element is provided. In some embodiments, a synthetic kidney-specific promoter comprising a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof is provided. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic kidney-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic kidney-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the synthetic kidney-specific promoter comprising a liver detargeting element has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% reduced expression in the liver compared to an equivalent synthetic kidney-specific promoter that does not comprise the liver detargeting element. In some embodiments, the addition of a liver detargeting element to the synthetic kidney-specific promoter does not affect its kidney-specific expression.

[0112] In an eighth aspect of the present invention, a synthetic lung-specific promoter is provided, which comprises a liver de-targeting element. In some embodiments, a synthetic lung-specific promoter is provided, which comprises a minimal ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the synthetic lung-specific promoter comprises SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the synthetic lung-specific promoter comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, a synthetic lung-specific promoter comprising a liver detargeting element has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% reduced expression in the liver compared to an equivalent synthetic lung-specific promoter that does not comprise the liver detargeting element. In some embodiments, addition of the liver detargeting element to the synthetic lung-specific promoter does not affect its lung-specific expression.

[0113] In a ninth aspect of the invention, an expression cassette is provided comprising a synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention, said promoter being operably linked to a sequence encoding an expression product (suitably a gene, such as a transgene).

[0114] In some embodiments, the expression product is a therapeutic expression product. In some preferred embodiments, the therapeutic expression product is suitable for treating a disease or condition related to abnormal gene expression, optionally in muscle (i.e., muscle disease), optionally in cardiac muscle and / or skeletal muscle. In some preferred embodiments, the therapeutic expression product includes those for treating muscle disease.

[0115] The therapeutic expression product can be a therapeutic expression product for treating any condition in which muscle expression may be useful, for example for treating a muscle condition or a condition in which secretion of the therapeutic expression product from muscle may be desired.

[0116] In the tenth aspect, an expression cassette is provided, comprising a synthetic muscle-specific promoter, wherein the synthetic muscle-specific promoter comprises CRE0145 or a functional variant thereof and DES_MT_enhancer_48bp or a functional variant thereof, wherein the synthetic promoter is operably linked to a sequence encoding an expression product and a target sequence of miR122.

[0117] In some embodiments, the synthetic muscle-specific promoter further comprises SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0118] In some preferred embodiments, the expression cassette comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product and a target sequence of miR122.

[0119] In some preferred embodiments, the target sequence of miR122 comprises SEQ ID NO: 16 or SEQ ID NO: 19 or a functional variant thereof, or consists of SEQ ID NO: 16 or SEQ ID NO: 19 or a functional variant thereof. In some preferred embodiments, the target sequence of miR122 comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 19, or consists of a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 19. NO:19 is composed of a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0120] In the eleventh aspect, a vector is provided, comprising a synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or an expression cassette according to the ninth or tenth aspect of the present invention. In some embodiments, the vector is a gene therapy vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a gene therapy vector, suitably an AAV vector, an adenoviral vector, a retroviral vector or a lentiviral vector. AAV vectors are particularly interesting. The AAV vector can be selected from the group consisting of AAV2, AAV6, AAV8, AAV9, BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8 and AAV2G9 or derivatives thereof. It has been noted that AAV serotype 9 (AAV9) achieves efficient transduction in cardiac and skeletal muscle, and therefore AAV9 and its derivatives represent a non-limiting example of suitable AAV vectors. In some embodiments, the rAAV vector is an AAV3b serotype, including but not limited to AAV3b265D virions, AAV3b265D549A virions, AAV3b549A virions, AAV3bQ263Y virions, or AAV3bSASTG virions (i.e., virions comprising an AAV3b capsid containing a Q263A / T265 mutation). In some embodiments, the virion can be a reasonable haploid, or a chimera or any mutant, such as a capsid that is customized to increase uptake at a desired location, such as the heart. Other capsids can include capsids from any known AAV serotype, including AAV1, AAV3, AAV4, AAV5, AAV7, AAV10, and the like. In some preferred embodiments, the AAV vector is AAV2i8.

[0121] The vector according to the present invention may be an AAV vector comprising a nucleic acid encoding a therapeutic expression product for treating heart failure, wherein the nucleic acid is operably linked to a muscle-specific promoter.

[0122] In a twelfth aspect of the present invention, a gene therapy vector is provided, comprising a synthetic promoter comprising SEQ ID NO: 15 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 13 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 14 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising SEQ ID NO: 17 or a functional variant thereof. In some embodiments, the gene therapy vector comprises a synthetic promoter comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

[0123] In some preferred embodiments, the gene therapy vector is a viral vector, such as a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus (AAV) vector. In some preferred embodiments, the vector is an AAV vector. Suitable AAV vectors are as described above.

[0124] In a thirteenth aspect, a gene therapy AAV vector is provided, comprising an expression cassette comprising a synthetic promoter operably linked to a sequence encoding an expression product and a target sequence for miR122. In some embodiments, the synthetic promoter is a muscle-specific promoter. In some embodiments, the synthetic promoter is a muscle-specific promoter according to any one of the first to fifth aspects of the present invention.

[0125] In some embodiments, the gene therapy AAV vector is AAV2. AAV2 has been noted to achieve efficient transduction in the liver and muscle. In some embodiments, the gene therapy AAV vector is AAV9. AAV9 has been noted to achieve efficient transduction in muscle and the CNS and is currently used in clinical settings. In some embodiments, the gene therapy AAV vector is AAV5. AAV5 has been noted to achieve efficient transduction in the lungs. In some embodiments, the gene therapy AAV vector is AAV8. AAV8 has been noted to achieve efficient transduction in muscle and is currently used in clinical settings.

[0126] In some embodiments, the target sequence of miR122 is expected to reduce liver expression of gene therapy AAV vectors. In some embodiments, the target sequence of miR122 comprises or consists of Mir122 miRNA target sequence 1. In some embodiments, the target sequence of miR122 comprises SEQ ID NO: 16 or a functional variant thereof, or consists of SEQ ID NO: 16 or a functional variant thereof. In some embodiments, the target sequence of miR122 comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16, or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16. In some embodiments, the target sequence of miR122 comprises or consists of Mir122 miRNA target sequence 2. In some embodiments, the target sequence of miR122 comprises or consists of SEQ ID NO: 19. In some embodiments, the target sequence of miR122 comprises or consists of SEQ ID NO: 19 at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence, or consists of SEQ ID NO: 19 at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence.

[0127] In some embodiments, the gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest) and a miR122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, the gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest) and a Mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0128] In some embodiments, the gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, another expression product (e.g., a second gene of interest), and a mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, the gene therapy AAV vector comprises a muscle-specific promoter operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, another expression product (e.g., a second gene of interest), and a Mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0129] In some embodiments, the gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest) and a mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, the gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest) and a Mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0130] In some embodiments, the gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, another expression product (e.g., a second gene of interest), and a mir122 miRNA target sequence 1 (SEQ ID NO: 16). In some embodiments, the gene therapy AAV vector comprises SP0524 (SEQ ID NO: 7) operably linked to a sequence encoding an expression product (e.g., a gene of interest), a T2A peptide, another expression product (e.g., a second gene of interest), and a Mir122 miRNA target sequence 2 (SEQ ID NO: 19). In some preferred embodiments, the elements are present in the order listed and are adjacent to each other.

[0131] In a fourteenth aspect, a virion (virion) is provided, comprising a vector according to the present invention, suitably a viral vector. In some embodiments, the virion is an AAV virion. Suitable virions are as described above.

[0132] In a fifteenth aspect, a pharmaceutical composition is provided, comprising the synthetic muscle-specific promoter, expression cassette, vector or virion according to the present invention.

[0133] In the 16th aspect, there is provided a muscle-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to the synthesis of the present invention, which is used in therapy, i.e., preventing or treating a medical condition or disease. In some embodiments, the muscle-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to the synthesis of the present invention is used in the therapy of a subject in need thereof. Suitably, the patient's condition or disease are related to abnormal gene expression, optionally related to abnormal gene expression in muscle cells (myoblasts) or tissues. Suitably, the patient's condition or disease are related to abnormal gene expression in cardiomyocytes or cardiac tissues. Suitably, the patient's condition or disease are related to abnormal gene expression in skeletal muscle or tissues. Suitably, there is provided a muscle-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to the synthesis of the present invention, which is used to express therapeutic expression products in skeletal muscle and / or myocardium.

[0134] In one embodiment, the disease may be a cardiovascular condition or heart disease and 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), abnormal cardiac contractility, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormal Ca 2+Metabolism and combinations thereof. In some embodiments, the disease can be selected from the group consisting of congestive heart failure, cardiomyopathy, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, conduction system dysfunction, coronary artery dysfunction, pulmonary heart hypertension. In some embodiments, the disease can be selected from the group consisting of congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, arrhythmia, muscular dystrophy, muscle mass abnormalities, muscle degeneration, infectious myocarditis, drug- or toxin-induced muscle abnormalities, allergic myocarditis, autoimmune endocarditis, and congenital heart disease.

[0135] Suitably, the use is for gene therapy, preferably for treating a disease involving abnormal gene expression. Suitably, the gene therapy involves expression of a therapeutic expression product in muscle cells or tissue, suitably in cardiomyocytes or cardiac tissue, suitably in skeletal muscle cells or tissue, or suitably in cardiac and skeletal cells and tissue.

[0136] Suitably, the subject in need of therapy will show symptoms of cardiovascular conditions (for example, heart disease or heart failure as described above) or skeletal muscle condition characteristics. Medical uses generally include improving the symptoms shown by the subject in need thereof by expressing a therapeutic amount of a therapeutic product. In some embodiments, the expression cassette comprises a gene encoding a protein phosphate (PP1) inhibitor, which is operably connected to a myocardial-specific promoter or a cardiac-selective promoter. Suitably, the therapy includes expressing a therapeutic amount of a PP1 inhibitor in the subject's cardiac tissue. Suitably, expressing a therapeutic amount of a PP1 inhibitor in cardiac tissue can alleviate the symptoms of the subject's heart failure or cardiac disease. Suitably, expressing a therapeutic amount of a PP1 inhibitor in cardiac tissue can weaken cardiac remodeling, improve exercise capacity or improve cardiac contractility. Suitably, expressing a therapeutic amount of a PP1 inhibitor in cardiac tissue can cause myocardial cell shortening, reduce the diastolic time constant, and accelerate calcium signal decay, improve end-systolic pressure dimension relationship and a combination thereof.

[0137] In a seventeenth aspect, a cell comprising a synthetic muscle-specific promoter, expression cassette, vector, or virion of the present invention is provided. 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 wherein the cell is a human muscle cell. Suitably, the cell can be a human skeletal muscle cell or a human cardiac muscle cell. The synthetic muscle-specific promoter, expression cassette, vector, or virion of the present invention can be episomal or can be in the genome of the cell.

[0138] In an eighteenth aspect, there is provided a synthetic muscle-specific CRM, a synthetic muscle-specific promoter, an expression cassette, a vector, a virion or a pharmaceutical composition as described herein for use in the preparation of a pharmaceutical composition for treating a medical condition or disease.

[0139] In a nineteenth aspect, a method for producing an expression product is provided, the method comprising providing a synthetic muscle-specific expression cassette of the present invention in a muscle cell and expressing the expression product, suitably a gene, present in the synthetic muscle-specific expression cassette. The method can be in vitro or ex vivo, or it can be in vivo. In some embodiments, the method is a bioprocessing method. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell.

[0140] In a twentieth aspect, a method for expressing an expression product in a muscle cell is provided, the method comprising introducing a synthetic muscle-specific expression cassette, vector, or virion as described herein into the muscle cell. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. The method can be in vitro or ex vivo, or it can be in vivo.

[0141] In a twenty-first aspect, there is provided a method of treating a subject, preferably a human, in need thereof, the method comprising:

[0142] - administering to a subject an expression cassette, vector, virion or pharmaceutical composition as described herein, comprising a sequence encoding a therapeutic product operably linked to a synthetic muscle-specific promoter according to the invention; and

[0143] - expressing a therapeutic amount of a therapeutic product in the muscle of the subject. The method may be ex vivo or in vitro. The subject may be a mammal excluding humans.

[0144] In one embodiment, the muscle cells are cardiac muscle cells. In one embodiment, the muscle cells are skeletal muscle cells. Suitably, the method of treating a subject comprises expressing a therapeutic amount of a therapeutic product in cardiac and / or skeletal muscle.

[0145] In some embodiments, the method comprises:

[0146] - introducing into the muscle of a subject an expression cassette, vector, virion or pharmaceutical composition as described herein, comprising an expression product, suitably a gene, encoding a therapeutic product; and

[0147] - expressing a therapeutic amount of a therapeutic product in the muscle of said subject.

[0148] In one embodiment, the muscle cells are cardiac muscle cells. In one embodiment, the muscle cells are skeletal muscle cells. Suitably, the method comprises expressing a therapeutic amount of a therapeutic product in the cardiac and / or skeletal muscle of the subject.

[0149] Suitably, the method comprises administering to the subject a vector, virosome or pharmaceutical composition as described herein. In some preferred embodiments, the vector is a viral gene therapy vector, preferably an AAV vector.

[0150] In one embodiment, any aspect herein relating to a muscle-specific promoter may also relate to a CNS-, kidney- or lung-specific promoter as defined above.

[0151] Further features and embodiments of the invention will now be described in the following sections.Any feature or embodiment in any section may be combined with any other feature or embodiment, or with any aspect of the invention, in any feasible combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 Shown is the in vivo activity of the control promoter CK8 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of protein extracted per mg. CK8 has high activity in heart, gastrocnemius and tibialis anterior, and also shows activity in diaphragm and soleus. CK8 has lower activity in the liver (about 13 times lower than the muscle tissue with the lowest expression (diaphragm) in the liver; about 570 times lower than the muscle tissue with the highest expression (heart) in the liver).

[0153] Figure 2 Figure 3 shows the in vivo activity of control promoter CK7 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver). Error bars are standard deviations. The y axis is the luciferase activity (relative light units) of the protein extracted per mg. CK7 has high activity in gastrocnemius and tibialis anterior, and also shows activity in diaphragm, heart, soleus and liver (lower than about 2 times with the lowest expression muscle tissue (diaphragm); lower than about 100 times with the highest expression muscle tissue (gastrocnemius) in liver).

[0154] Figure 3The in vivo activity of the synthetic promoter SP0525 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver) is shown. Error bars are standard deviations. The y axis is the luciferase activity (relative light units) of the protein extracted per mg. SP0525 has high activity in the heart and also shows activity in skeletal muscle (gastrocnemius, soleus, tibialis anterior and diaphragm). SP0525 has lower activity in the liver (about 2 times lower than the muscle tissue with the lowest expression (diaphragm) in the liver; about 100 times lower than the muscle tissue with the highest expression (heart) in the liver).

[0155] Figure 4 The in vivo activity of the synthetic promoter SP0526 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior as well as non-muscle tissue (liver) is shown. Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of protein extracted per mg. SP0526 has high activity in heart and gastrocnemius, and also shows activity in diaphragm and tibialis anterior. SP0526 shows lower activity in soleus. SP0526 shows lower activity in liver (about 4 times lower than the muscle tissue with the lowest expression (soleus) in liver); about 980 times lower than the muscle tissue with the highest expression (heart) in liver).

[0156] Figure 5 Figure 3 shows the in vivo activity of the synthetic promoter SP0527 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior, as well as non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of protein extracted per mg. SP0527 has high activity in heart and gastrocnemius, and also shows activity in diaphragm and tibialis anterior. SP0527 shows lower activity in soleus. SP0527 shows lower activity in liver (about 4 times lower than the muscle tissue (soleus) with the lowest expression in liver); about 570 times lower than the muscle tissue (gastrocnemius) with the highest expression in liver).

[0157] Figure 6Figure 3 shows the in vivo activity of the synthetic promoter SP0528 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior, as well as non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of protein extracted per mg. SP0528 has high activity in heart, tibialis anterior and gastrocnemius, and also shows activity in diaphragm. SP0528 shows lower activity in soleus. SP0528 shows lower activity in liver (about 2 times lower than the muscle tissue with the lowest expression (soleus) in liver); about 175 times lower than the muscle tissue with the highest expression (heart) in liver).

[0158] Figure 7 Shown is the in vivo activity of synthetic expression cassette 529 (SP0524+ expression product+mir122 target sequence) in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver). Error bars are standard deviations. The y axis is the luciferase activity (relative light units) of protein extracted per mg. Expression cassette 529 shows high activity in cardiac muscle and also shows activity in skeletal muscle (diaphragm, gastrocnemius, soleus and tibialis anterior). Expression cassette 529 shows lower activity in liver (about 13 times lower than the muscle tissue (soleus) with the lowest expression in liver); about 7520 times lower than the muscle tissue (heart) with the highest expression in liver).

[0159] Figure 8 The in vivo activity of synthetic promoter SP0530 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver) is shown. Error bar is standard deviation. Y axis is the luciferase activity (relative light unit) of the protein extracted per mg. SP0530 shows high activity in cardiac muscle, and also shows activity in skeletal muscle (diaphragm, gastrocnemius, soleus and tibialis anterior) and liver (activity ratio in liver has the lowest activity muscle tissue (soleus) low about 2 times; activity ratio in liver has the highest activity muscle tissue (heart) low about 1430 times).

[0160] Figure 9 Shown is the in vivo activity of synthetic promoter SP0531 in various muscle tissues including diaphragm, heart, gastrocnemius, soleus and tibialis anterior and non-muscle tissue (liver).Error bar is standard deviation.Y axis is the luciferase activity (relative light unit) of the protein extracted per mg.SP0531 shows high activity in diaphragm, and also shows activity (compared with the muscle tissue (gastrocnemius) with the highest activity, the activity in liver is low about 137 times).

[0161] Figure 10 Shown are saline controls (background) in various muscle tissues including diaphragm, heart, gastrocnemius, soleus, and tibialis anterior, as well as non-muscle tissue (liver). Error bars are standard deviations. The y-axis is luciferase activity (relative light units) per mg of extracted protein.

[0162] Figure 11 The vector copy number per diploid genome for the synthetic promoters and control promoters tested in the heart is shown. The y-axis is the vector copy number per diploid genome. Error bars are standard deviations.

[0163] Figure 12 The vector copy number per diploid gene for the synthetic promoters tested and the control promoter in liver is shown. The y-axis is the vector copy number per diploid genome. Error bars are standard deviations.

[0164] Figure 13 In vivo imaging using the IVIS Spectrum in vivo imaging system is shown. Luminescence is represented in color, with blue representing areas of low luminescence and red representing areas of intense luminescence, ranging from 0.2–2.1 p / sec / cm2 / Sr (photon emission from the subject or radiation). Luminescence detected in the lower half of the mouse primarily corresponds to skeletal muscle (including the soleus, tibialis anterior, and gastrocnemius muscles) and occasionally to viscera. Luminescence detected in the upper half of the mouse corresponds to a range of tissues, such as the liver, heart, and others.

[0165] Figure 14 Figure 3 shows the in vivo activity of the control promoter CK7 (low dose) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of the protein extracted per mg. CK7 shows high activity in quadriceps, gastrocnemius, tibialis anterior and psoas, and shows activity in diaphragm, soleus and heart. CK7 shows lower activity in liver (lower than the muscle tissue with the lowest expression by about 5 times).

[0166] Figure 15 Figure 3 shows the in vivo activity of the control promoter CK7 (medium dose) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of the protein extracted per mg. CK7 shows high activity in quadriceps, gastrocnemius, tibialis anterior and psoas, and shows activity in diaphragm, soleus and heart. CK7 shows lower activity in liver (about 80 times lower than the muscle tissue with the lowest expression).

[0167] Figure 16 Figure 2 shows the in vivo activity of the synthetic promoter SP0527 (low dose) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and non-muscle tissue (liver). Error bars are standard deviations. The y axis is the luciferase activity (relative light units) of the protein extracted per mg. SP0527 shows high activity in the heart and shows activity in diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus. SP0527 shows lower activity (approximately 10 times lower than the muscle tissue with the lowest expression) in the liver.

[0168] Figure 17 Figure 2 shows the in vivo activity of the synthetic promoter SP0527 (medium dose) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, as well as non-muscle tissue (liver). Error bars are standard deviations. The y axis is the luciferase activity (relative light units) of protein extracted per mg. SP0527 shows high activity in the heart and in the diaphragm, quadriceps, gastrocnemius and tibialis anterior. SP0527 also shows activity in the psoas and soleus. SP0527 shows lower activity in the liver (about 40 times lower than the muscle tissue with the lowest expression).

[0169] Figure 18 Shown is the in vivo activity of the synthetic promoter SP0527 (high dose) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and non-muscle tissue (liver). Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of protein extracted per mg. SP0527 shows high activity in the heart and in diaphragm, quadriceps, gastrocnemius and tibialis anterior. SP0527 also shows activity in psoas and soleus. SP0527 shows lower activity in the liver (about 950 times lower than the muscle tissue with the lowest expression).

[0170] Figure 19 Shown are saline controls (background) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas, and soleus, as well as non-muscle tissue (liver). Error bars are standard deviations. The y-axis is luciferase activity (relative light units) per mg of extracted protein.

[0171] Figure 20 The vector copies per diploid genome for the control promoter CK7 (low and medium doses), SP0527 (low, medium and high doses) and saline background in non-muscle tissue (liver) are shown. The y-axis is the vector copy number per diploid genome. The error bars are standard deviations.

[0172] Figure 21The in vivo activity of the synthetic promoter SP0527 (high dose) in various muscle tissues including the heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and in various non-muscle tissues including the liver, brain, lung, colon, spleen, testis and kidney is shown. Error bars are standard deviations. The y-axis is the luciferase activity (relative light units) of the protein extracted per mg. SP0527 shows high activity in the heart and in the diaphragm, quadriceps, gastrocnemius and tibialis anterior. SP0527 also shows activity in the psoas and soleus. SP0527 shows low activity in the brain, lung and kidney (compared with the muscle tissue with the lowest expression, the expression in the brain, lung and kidney is about 30-165 times lower). SP0527 shows lower activity in the liver, colon and testis (compared with the muscle tissue with the lowest expression, the expression in the liver, colon and testis is about 400-960 times lower). SP0527 showed the lowest expression in spleen (approximately 2100-fold lower expression in spleen compared to muscle tissue which had the lowest expression).

[0173] Figure 22 Shown are saline controls (background) in various muscle tissues including heart, diaphragm, quadriceps, gastrocnemius, tibialis anterior, psoas and soleus, and various non-muscle tissues including liver, brain, lung, colon, spleen, testis and kidney. Error bars are standard deviations. The y-axis is luciferase activity (relative light units) per mg of extracted protein. DETAILED DESCRIPTION

[0174] Muscle-specific, muscle-type-selective, and muscle-specific

[0175] The CREs, CRMs, promoter elements, and synthetic promoters of the present invention can be active in various muscle tissues, particularly, but not limited to, skeletal muscle and / or cardiac muscle. CREs, CRMs, promoter elements, and synthetic promoters of the present invention that are active in at least one muscle tissue type or at least one muscle cell type can be referred to as "muscle-specific." For simplicity, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention can be further subdivided into subtypes, depending on whether they are primarily active in skeletal muscle or cardiac muscle.

[0176] In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are primarily active in skeletal muscle and have less activity or no activity in cardiac muscle. These CREs, CRMs, promoter elements, and synthetic promoters of the present invention are referred to as "skeletal muscle specific" or "skeletal selective." In some embodiments, the CREs, CRMs, promoter elements, and synthetic promoters of the present invention are skeletal muscle specific or bone selective.

[0177] In some embodiments, the CREs, CRMs, promoter elements and synthetic promoters of the present invention are primarily active in cardiac muscle and have lower or no activity in skeletal muscle. These CREs, CRMs, promoter elements and synthetic promoters of the present invention are referred to as "cardiac muscle specific" or "cardiac selective". In some embodiments, the CREs, CRMs, promoter elements and synthetic promoters of the present invention are cardiac muscle specific or cardiac selective. Examples of muscle-specific promoters that are primarily active in cardiac muscle include SP0525 (SEQ ID NO: 1) and SP0530 (SEQ ID NO: 27). Examples of muscle-specific expression cassettes that are primarily active in cardiac muscle include expression cassette 529.

[0178] In some embodiments, muscle-specific CREs, CRMs, promoter elements, and synthetic promoters that are active in both skeletal and cardiac muscle are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is desired in both skeletal and cardiac muscle (in cardiac muscle). Examples of muscle-specific promoters that are active in both skeletal and cardiac muscle include SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0531 (SEQ ID NO: 28), and SP0524 (SEQ ID NO: 7).

[0179] In some embodiments, skeletal muscle-specific or skeletal-selective CREs, CRMs, promoter elements, and synthetic promoters are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is desired in skeletal muscle but little or no activity in the heart (in cardiac muscle).

[0180] Skeletal muscle specific or skeletal selective promoters may be active in fast and / or slow muscles. In some embodiments, skeletal muscle specific or skeletal selective CREs, CRMs, promoter elements, and synthetic promoters that are active in fast muscles may be preferred. In some embodiments, skeletal muscle specific or skeletal selective CREs, CRMs, promoter elements, and synthetic promoters that are active in slow muscles may be preferred. In some embodiments, skeletal muscle specific or skeletal selective CREs, CRMs, promoter elements, and synthetic promoters that are active in both slow and fast muscles may be preferred.

[0181] In some embodiments, cardiac-specific or cardiac-selective CREs, CRMs, promoter elements, and synthetic promoters are preferred. These CREs, CRMs, promoter elements, and synthetic promoters may be preferred when promoter activity is desired in the heart (in cardiac muscle) but little or no activity in skeletal muscle.

[0182] The cardiac-specific or cardiac-selective CREs, CRMs, promoter elements and synthetic promoters of the present invention can be active in various cells of the heart. The main cell types in the heart are ventricular cardiomyocytes, atrial cardiomyocytes, cardiac fibroblasts or endothelial cells (EC) in the heart, as well as perivascular cells and pacemaker cells. In addition, the myocardial-specific or cardiac-selective CREs, CRMs, promoter elements and synthetic promoters of the present invention can be active in various regions of the heart, for example, in any or all of the following cardiac regions: aortic arch (AA); aorta; cardiomyocytes (CM); endothelial or endocardial cells (EC); inferior vena cava (ICV); ventricular septum (IVS); left atrium (LA); left superior vena cava (LSCV); left ventricle (LV); outflow tract (OT); pulmonary artery (PO); proepicardial organ (PEO); pulmonary vein (PV); right atrium (RA); right superior vena cava (RSCV); right ventricle (RV); superior vena cava (SCV); cardiac smooth muscle cells (SM).

[0183] In some embodiments, skeletal muscle and cardiac muscle specific or cardiac selective CRE, CRM, promoter elements and synthetic promoters are preferred. When promoter activity is required in skeletal muscle and in the heart (in the cardiac muscle), these CRE, CRM, promoter elements and synthetic promoters may be preferred. This is especially beneficial for diseases such as DMD.

[0184] Cis-regulatory elements and their functional variants

[0185] Disclosed herein are various CREs that can be used to construct muscle-specific promoters. These CREs are typically derived from genomic promoter and enhancer sequences, but they are used herein in environments that are quite different from their natural genomic environments. Generally speaking, CREs constitute a small portion of a much larger genomic regulatory domain that controls the expression of genes with which they are typically associated. Surprisingly, it has been found that these CREs (many of which are very small) can be isolated from their normal environment and retain muscle-specific regulatory activity when used to construct various synthetic promoters. This is surprising because removing regulatory sequences from the complex and "three-dimensional" natural environment of the genome typically results in a significant loss of activity, so there is no reason to expect a given CRE to maintain the observed level of activity after being removed from its natural environment. Combinations of these CREs have been tested and found to be very effective in enhancing muscle-specific promoter activity when combined with minimal promoters and proximal promoters. It should be noted that the sequence of the CRE of the present invention can be altered without causing a significant loss of activity. Functional variants of CREs can be prepared by modifying the sequence of the CRE, provided that modifications that significantly impair CRE activity are avoided. Given the information provided in this disclosure, it is straightforward to modify CRE to provide functional variants. Furthermore, this disclosure provides a methodology for easily assessing the function of any given CRE variant. Each functional variant of CRE is discussed below.

[0186] The relatively small size of certain CREs according to the present invention is advantageous because it allows the CRE, and more specifically the promoter comprising them, to be provided in a vector while taking up a minimal amount of payload on the vector. This is particularly important when the CRE is used in a vector with limited capacity, such as an AAV-based vector.

[0187] The CRE disclosed herein contains certain muscle-specific transcription factor binding sites (TFBS). It is generally desired that these muscle-specific TFBSs remain functional in functional variants of the CRE. It is well known to those skilled in the art that TFBS sequences can vary but still retain function. In view of this, the sequences used for TFBSs are generally described by consensus sequences, from which there is generally a certain degree of variation. Further information about the variations that occur in the TFBS can be described using a position weight matrix (PWM), which represents the frequency with which a given nucleotide typically occurs at a given position in the consensus sequence. Details of the TF consensus sequence and the associated position weight matrix 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 in any given TFBS of the CRE in a manner that retains, or in some cases even increases, CRE function. In view of this, those skilled in the art have ample guidance on how to modify the TFBS of any given transcription factor (TF) while maintaining the ability to bind to the desired TF; for example, the Jaspar system will score the hypothetical TFBS based on its similarity to a given PWM. Furthermore, CREs can be scanned against all PWMs in the JASPAR database to identify / analyze all TFBSs. A skilled artisan can of course find additional guidance in the literature, and furthermore, routine experimentation can be used to confirm the binding of a TF to a putative TFBS in any variant CRE. It is readily apparent that significant sequence modifications can be made within a CRE, and even within a TFBS within a CRE, while retaining function.

[0188] Functional variants of CRE may comprise substitutions, deletions and / or insertions compared to a reference CRE, as long as they do not render the CRE substantially non-functional.

[0189] CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) are muscle-specific cis-regulatory elements (CREs). It has been found that these CREs provide significant muscle-specific enhancer activity when combined with appropriate promoter elements and / or when added to appropriate synthetic promoters.

[0190] Promoter elements and their functional variants

[0191] The CRM of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters. Minimal promoters and proximal promoters are collectively referred to as promoter elements.

[0192] Functional variants of promoter elements include sequences that differ from a reference promoter element but that substantially retain activity as a muscle-specific promoter element. A skilled artisan will appreciate that it is possible to alter the sequence of a promoter element while retaining its ability to promote expression. Functional variants of promoter elements may comprise substitutions, deletions, and / or insertions compared to a reference promoter element, as long as they do not render the promoter element substantially nonfunctional.

[0193] A functional variant of a promoter element can be considered 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 retains at least 90% of its activity, more preferably retains 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).

[0194] Suitably, the functional variant of a promoter element retains a significant level of sequence identity to a reference promoter element. Suitably, the functional variant comprises a sequence that is at least 70% identical to a reference promoter element, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference promoter element.

[0195] Retention of activity can be assessed by comparing expression of a suitable reporter gene under the control of a reference promoter with expression under equivalent conditions from an otherwise identical promoter comprising the replaced promoter element. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, for example in the Examples.

[0196] SCP1 (SEQ ID NO: 12) is a promoter element. It has been found that when combined with the cis-regulatory element CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11), this promoter element provides significant muscle-specific activity, as described in application PCT / GB2022 / 051611, in particular page 9, lines 7-11 and Figure 5 、 6 and 18, which are incorporated herein by reference.

[0197] CRE0053 (SEQ ID NO: 26) is a promoter element, suitably a minimal promoter. It has been found that when combined with the cis-regulatory element CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11), this promoter element provides significant muscle-specific activity, as described in application PCT / GB2022 / 051611, in particular page 9, lines 1-6 and Figure 5 、 6 and 17, which applications are incorporated herein by reference, suitably provide significant cardiomyocyte-specific or cardioselective activity.

[0198] Detargeting element and liver detargeting element

[0199] A detargeting element can be added to a synthetic promoter. In some embodiments, the addition of the detargeting element reduces expression of the synthetic promoter in a specific tissue or cell. In some embodiments, the addition of the detargeting element alters the expression profile of the synthetic muscle-specific promoter compared to a reference promoter that does not comprise the detargeting element. In some embodiments, the addition of the detargeting element alters the expression profile of the synthetic muscle-specific promoter in different muscle tissues compared to a reference promoter that does not comprise the detargeting element. In some embodiments, the detargeting element is a binding site for a protein that is highly expressed in a specific tissue or cell. In some embodiments, the detargeting element is a liver detargeting element. In some embodiments, the addition of the liver detargeting element alters the expression profile of the synthetic muscle-specific promoter in different muscle tissues compared to a reference promoter that does not comprise the liver detargeting element.

[0200] In one embodiment, the liver detargeting element reduces the expression of a synthetic promoter comprising the element in the liver by at least 10% compared to a suitable control. In one embodiment, the liver detargeting element reduces the expression of a synthetic promoter comprising the element in the liver by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a suitable control. As used herein, a "suitable control" refers to the expression level of a synthetic promoter in the liver that does not contain a detargeting element in an otherwise identical sample. One skilled in the art can assess the expression level of a given synthetic promoter (with or without a detargeting element) in the liver using standard techniques, for example, using a PCR-based assay to measure mRNA levels in hepatocytes and / or tissue samples.

[0201] The alpha-fetoprotein (AFP) gene is activated in the fetal liver but is repressed after birth. ZBTB20, a zinc finger protein, acts as a transcriptional repressor of AFP promoter-mediated activity in the liver (Xie Z, Zhang H, Tsai W, Zhang Y, Du Y, Zhong J, Szpirer C, Zhu M, Cao X, Barton MC, Grusby MJ, Zhang WJ. Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci US A. 2008 Aug 5; 105(31):10859-64. Doi:10.1073 / pnas.0800647105. Epub 2008 Jul 31. PMID:18669658; PMCID:PMC2504784). Different regions bound by ZBTB20 have been identified in the literature (Zhang, H., Cao, D., Zhou, L. et al., ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene. Sci Rep 5, 11979 (2015). https: / / doi.org / 10.1038 / srep11979). Notably, Zhang et al., 2015 teach to avoid reducing the size of the -151 / -53 region of AFP (which they identified as the region bound by ZBTB20) because this reduces promoter activity.

[0202] SEQ ID NO: 15 shows the minimal ZBTB20 binding site. Liver detargeting sequence 1 (SEQ ID NO: 13), liver detargeting sequence 2 (SEQ ID NO: 14), and liver detargeting sequence 3 (SEQ ID NO: 17) contain the minimal ZBTB20 binding site.

[0203] In some embodiments, the detargeting element is a binding site for a miRNA that is highly expressed in a specific tissue or cell. In some embodiments, the detargeting element is a liver detargeting element.

[0204] miR122 is highly expressed in the liver, and miR122 target sequences effectively reduce liver expression (Qiao C, Yuan Z, Li J, He B, Zheng H, Mayer C, Li J, Xiao X. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther. 2011 Apr; 18(4): 403-10. Doi: 10.1038 / gt.2010.157. Epub 2010 Dec 9. PMID: 21150938; PMCID: PMC3686499).

[0205] SEQ ID NO: 19 shows the minimal Mir122 miRNA target sequence. Mir122 miRNA target sequence 1 (SEQ ID NO: 16) contains three times the minimal Mir122 miRNA target sequence.

[0206] A functional variant of a detargeting element or a liver detargeting element comprises a sequence that differs from a reference detargeting element or a liver detargeting element but substantially retains activity as a detargeting element or a liver detargeting element. A skilled artisan will appreciate that it is possible to alter the sequence of a detargeting element while retaining its ability to reduce expression of a synthetic promoter in a specific tissue or cell. A skilled artisan will appreciate that it is possible to alter the sequence of a liver detargeting element while retaining its ability to reduce expression of a synthetic promoter in liver tissue or cells. A functional variant of a detargeting element or a liver detargeting element may comprise substitutions, deletions, and / or insertions as compared to a reference detargeting element or a liver detargeting element, so long as they do not render the detargeting element or the liver detargeting element substantially nonfunctional.

[0207] A functional variant of a detargeting element or a liver detargeting element can be considered a detargeting element or a liver detargeting element that substantially retains its activity when substituted for a reference detargeting element or a liver detargeting element in a synthetic promoter. For example, a muscle-specific synthetic promoter comprising a functional variant of a given detargeting element or a liver detargeting element preferably retains at least 80% of its activity, more preferably retains at least 90% of its activity, more preferably retains at least 95% of its activity, and even more preferably retains 100% of its activity (compared to a reference promoter comprising an unmodified detargeting element or a liver detargeting element).

[0208] Suitably, the functional variant of the detargeting element or liver detargeting element retains a significant level of sequence identity to the reference detargeting element or liver detargeting element. Suitably, the functional variant comprises a sequence that is at least 70% identical to the reference detargeting element or liver detargeting element, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference detargeting element or liver detargeting element.

[0209] Retention of activity can be assessed by comparing expression of a suitable reporter gene under the control of a reference promoter with expression of an otherwise identical promoter comprising the replaced detargeting element or liver detargeting element under equivalent conditions. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, for example in the Examples.

[0210] Synthetic muscle-specific CRM and its functional variants

[0211] Disclosed herein are various synthetic muscle-specific CRMs that can be used to construct synthetic muscle-specific promoters. The CRMs of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters.

[0212] Functional variants of CRMs include sequences that differ from a reference CRM element but that substantially retain activity as a muscle-specific CRM. A skilled artisan will appreciate that it is possible to alter the sequence of a CRM while retaining its ability to recruit appropriate muscle-specific transcription factors (TFs) and thereby enhance expression. Functional variants of CRMs may comprise substitutions, deletions, and / or insertions compared to a reference CRM, as long as they do not render the CRM substantially nonfunctional.

[0213] In some embodiments, a functional variant of a CRM can be considered 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 retains at least 90% of its activity, more preferably retains at least 95% of its activity, and even more preferably retains 100% of its activity (compared to a reference promoter comprising an unmodified CRM).

[0214] Suitably, the 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 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference CRM.

[0215] Retention of activity can be assessed by comparing expression of a suitable reporter gene under the control of a reference promoter with expression under equivalent conditions from an otherwise identical promoter comprising the replaced CRM. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, for example in the Examples.

[0216] In some embodiments, the functional variant of a given CRM may comprise a functional variant of one or more of the CREs present in the reference CRM. For example, the functional variant of a given CRM may comprise a functional variant of 1, 2, 3, 4, 5, or 6 of the CREs present in the reference CRM.

[0217] In some embodiments, a functional variant of a given CRM may comprise the same combination of CREs as a reference CRM, but the CREs may be present in a different order than in the reference CRM. It is generally preferred that the CREs are present in the same order as in the reference CRM (thus, suitably, a functional variant of a CRM comprises the same arrangement of CREs as listed in the reference CRM).

[0218] In some embodiments, a functional variant of a given CRM may comprise one or more additional CREs beyond the CRE present in the reference CRM. 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 they may be other CREs. Generally, it is preferred that a functional variant of a given CRM comprises the same CRE (or a functional variant thereof) and does not comprise an additional CRE.

[0219] Functional variants of a given CRM may comprise one or more additional regulatory elements compared to a reference CRM. For example, they may comprise inducible or repressible elements, intronic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, and splicing elements, etc., as long as they do not render the CRM substantially nonfunctional.

[0220] Functional variants of a given CRM may contain additional spacers between adjacent CREs, or, if present in the reference CRM, the spacer(s) may be longer or shorter (or absent) than in the reference CRM.

[0221] Obviously, a CRM or a functional variant thereof as disclosed herein may be combined with any suitable promoter element to provide a synthetic muscle-specific promoter according to the present invention.

[0222] In some embodiments, the synthetic muscle-specific CRM according to the present invention is operably linked to a promoter element to form a synthetic muscle-specific promoter. The promoter element can be a minimal promoter or a proximal promoter. The proximal promoter is preferably a muscle-specific proximal promoter. In some embodiments, the synthetic muscle-specific CRM according to the present invention is operably linked to promoter element SCP1 (SEQ ID NO: 12). In some embodiments, the synthetic muscle-specific CRM according to the present invention is operably linked to promoter element CRE0053 (SEQ ID NO: 26).

[0223] In a CRM, the regulatory elements are preferably present in the order listed and are preferably adjacent to each other (i.e., without any intervening regulatory elements). The regulatory elements can be continuous or discontinuous (i.e., they can be placed in close proximity to each other, or they can be separated by spacers or other sequences). In some embodiments, some or all of the regulatory elements can be CREs. CREs are preferably present in the order listed and are preferably adjacent to each other. CREs can be continuous or discontinuous (i.e., they can be placed in close proximity to each other, or they can be separated by spacers or other sequences). In some preferred embodiments, the regulatory elements or their functional variants are provided in the order listed and are adjacent to each other. For example, a synthetic skeletal muscle-specific or bone-selective CRM can comprise CRE0145, DES_MT_enhancer_48bp, and liver detargeting sequence 1, etc. In some embodiments, it is preferred that some or all of the CREs or regulatory elements are continuous.

[0224] In some embodiments, the synthetic muscle-specific CRM comprises one or more regulatory elements in addition to the above-mentioned regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more other regulatory elements according to the present invention or other regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more CREs according to the present invention or other CREs. In some embodiments, the one or more additional regulatory elements may be one or more promoter elements. In some embodiments, the one or more additional regulatory elements may be one or more de-targeting elements or liver de-targeting elements. In some embodiments, the one or more additional regulatory elements may be one or more introns.

[0225] In some embodiments, the synthetic muscle-specific CRM comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9, 20-22.

[0226] In some embodiments, the synthetic muscle-specific CRM is active in both skeletal and cardiac muscle. In some embodiments, the synthetic muscle-specific CRM is active primarily in skeletal muscle. In some embodiments, the synthetic muscle-specific CRM is active primarily in cardiac muscle.

[0227] It has been found that a CRM comprising CRE0145 and DES_MT_enhancer_48bp provides significant muscle-specific enhancer activity in both skeletal and cardiac muscle when combined with appropriate promoter elements, as described in application PCT / GB2022 / 051611, in particular page 9, lines 1-11 and Figure 5 、 6 , 17 and 18, which are incorporated herein by reference.

[0228] In many cases, shorter promoter sequences are preferred, particularly when the capacity of a vector (e.g., a viral vector such as AAV) is limited. Thus, in some embodiments, the length of the synthetic muscle-specific CRM is 300 nucleotides or less, such as 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 60, 50 nucleotides or less. In some embodiments, the length of the synthetic muscle-specific CRM is 270 nucleotides or less, preferably 220 nucleotides or less, more preferably 190 nucleotides or less, and most preferably 170 nucleotides or less.

[0229] Synthetic muscle-specific promoter and its functional variants

[0230] Disclosed herein are a variety of synthetic muscle-specific promoters. A functional variant of a reference synthetic muscle-specific promoter is a promoter comprising a sequence that is different from the reference synthetic muscle-specific promoter but substantially retains muscle-specific promoter activity. It will be appreciated by those skilled in the art that the sequence of a synthetic muscle-specific promoter can be altered while retaining its ability to recruit suitable muscle-specific transcription factors (TFs) and to recruit RNA polymerase II to provide muscle-specific expression of an operably linked sequence (e.g., an open reading frame). Compared to a reference promoter, a functional variant of a synthetic muscle-specific promoter can comprise substitutions, deletions, and / or insertions, as long as such substitutions, deletions, and / or insertions do not render the synthetic muscle-specific promoter substantially non-functional compared to the reference promoter.

[0231] Thus, in some embodiments, a functional variant of a synthetic muscle-specific promoter can be considered 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 retains at least 80% of its activity, more preferably retains at least 90%, 91%, 92%, 93%, 94% of its activity, more preferably retains at least 95%, 96%, 97%, 98% of its activity, and even more preferably retains 100% of its activity. In some embodiments, a functional variant of a synthetic muscle-specific promoter retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95% or 100% of the activity of the reference promoter.

[0232] Functional variants of synthetic muscle-specific promoters typically retain a significant level of sequence similarity to a reference synthetic muscle-specific promoter. In some embodiments, the functional variant comprises a sequence that is at least 70% identical to a reference synthetic muscle-specific promoter, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference synthetic muscle-specific promoter.

[0233] In one embodiment, the synthetic muscle-specific promoters described herein can increase the expression of, for example, a gene product driven by the promoter in muscle by at least 10% compared to a reference level. In one embodiment, the synthetic muscle-specific promoters described herein can increase the expression of, for example, a gene product driven by the promoter in muscle by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a reference level, or by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold or more compared to a reference level. As used herein, "reference level" refers to the expression level of a product (e.g., a promoter or a gene product driven by the promoter) in muscle of an otherwise identical sample driven by a non-synthetic muscle-specific promoter or a control muscle-specific promoter. One skilled in the art can assess the expression level of a given product in muscle using standard techniques, such as PCR-based assays or Western blots to measure mRNA or protein levels, respectively.

[0234] Activity in a functional variant can be assessed by comparing expression of a suitable reporter gene under the control of a reference synthetic muscle-specific promoter with expression of a putative functional variant under equivalent conditions. Suitable methods for assessing muscle-specific promoter activity are disclosed herein, for example in the Examples.

[0235] A functional variant of a given synthetic muscle-specific promoter may comprise a functional variant of one or more CREs present in a reference synthetic muscle-specific promoter. Functional variants of CREs are discussed above.

[0236] Functional variants of a given synthetic muscle-specific promoter may comprise functional variants of a CRM present in a reference synthetic muscle-specific promoter. Functional variants of CRMs are discussed above.

[0237] Functional variants of a given synthetic muscle-specific promoter may comprise functional variants of promoter elements, or different promoter elements compared to a reference synthetic muscle-specific promoter. Functional variants of promoter elements are discussed above.

[0238] A functional variant of a given synthetic muscle-specific promoter may comprise the same CREs as a reference synthetic muscle-specific promoter, but the CREs may be present in a different order than in the reference synthetic muscle-specific promoter.

[0239] Functional variants of a given synthetic muscle-specific promoter may comprise one or more additional CREs beyond the CRE present in the reference synthetic muscle-specific promoter. Additional CREs may be provided upstream of the CRE present in the reference synthetic muscle-specific promoter, downstream of the CRE present in the reference synthetic muscle-specific promoter, and / or between the CREs present in the reference synthetic muscle-specific promoter. The additional CREs may be the CREs disclosed herein, or they may be other CREs.

[0240] A functional variant of a given synthetic muscle-specific promoter may comprise additional spacers between adjacent CRE and promoter elements, or, if one or more spacers are present in a reference synthetic muscle-specific promoter, the one or more spacers may be longer or shorter than in the reference synthetic muscle-specific promoter (or it may be absent).

[0241] Obviously, the synthetic muscle-specific promoters of the present invention can comprise the CRMs of the present invention and additional regulatory elements. For example, they can comprise one or more additional CRMs, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, introns, and splicing elements, etc., so long as they do not render the promoter substantially nonfunctional.

[0242] 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 CBA, CK8, CK7 or RSV promoters in muscle cells. In many cases, higher levels of promoter activity are preferred, but not always; thus, in some cases, more moderate expression levels may be preferred. In some cases, it is desirable to have a range of promoters with different activity levels to tailor expression levels as needed; the present disclosure provides such a range of activities for promoters. By comparing muscle-specific expression of a reporter gene under the control of a synthetic muscle-specific promoter with expression of the same reporter gene under the control of a CBA, CK8, CK7, or RSV promoter, wherein both promoters are provided in otherwise identical expression constructs and under equivalent conditions, the activity of a given synthetic muscle-specific promoter of the present invention compared to CBA, CK8, CK7, or RSV can be assessed. Suitable methods for testing muscle-specific promoter activity can be found, for example, in the Examples.

[0243] Suitably, the synthetic muscle-specific promoter of the invention may be capable of increasing the expression of a gene (e.g. a therapeutic gene or a gene of interest) in a muscle or in a 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 relative to a known muscle-specific promoter, suitably the SPc5-12 promoter (Gene Ther. 2008 Nov; 15(22): 1489-99).

[0244] Suitably, the synthetic muscle-specific promoters of the present invention exhibit an activity in non-muscle cells (e.g., Huh7 and HEK293 cells) that is 50% or less of that of CMV-IE, preferably 25% or less of that of CMV-IE, more preferably 10% or less of that of CMV-IE, and in some cases 5% or less of that of CMV-IE, or 1% or less of that of CMV-IE.

[0245] In many cases, shorter promoter sequences are preferred, particularly when used in situations where the capacity of a vector (e.g., a viral vector such as AAV) is limited. Thus, in some embodiments, the length of the synthetic muscle-specific promoter is 400 nucleotides or less, such as 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250 nucleotides or less. In some embodiments, the length of the synthetic muscle-specific promoter is 390 nucleotides or less, more preferably 380 nucleotides or less, even more preferably 350 nucleotides or less, and most preferably 330 nucleotides or less. In some embodiments, the length of the synthetic muscle-specific promoter is 320 nucleotides or less, preferably 310 nucleotides or less, more preferably 280 nucleotides or less, and most preferably 250 nucleotides or less.

[0246] Particularly preferred synthetic muscle-specific promoters are those that are both short and exhibit high levels of activity.

[0247] In some embodiments, the synthetic muscle-specific promoter comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, 27-28.

[0248] It is generally preferred that the promoter according to the present invention is a functional variant of any of SEQ ID NOs: 1-4, 27-28, which 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, but other methods may also be used.

[0249] In some embodiments, the synthetic muscle-specific promoter is active in both skeletal and cardiac muscle. In some embodiments, the synthetic muscle-specific promoter is active primarily in skeletal muscle. In some embodiments, the synthetic muscle-specific promoter is active primarily in cardiac muscle.

[0250] It has been found that a synthetic promoter comprising CRE0145 and DES_MT_enhancer_48bp operably linked to SCP1 provides significant muscle-specific enhancer activity in both skeletal and cardiac muscle, as described in application PCT / GB2022 / 051611, in particular page 9, lines 7-11 and Figure 5 、 6 and 18, which are incorporated herein by reference.

[0251] It has been found that a synthetic promoter comprising CRE0145 and DES_MT_enhancer_48bp operably linked to CRE0053 provides significant cardiomyocyte-specific or cardioselective activity as described in application PCT / GB2022 / 051611, in particular page 9, lines 1-6 and Figure 5 、 6 and 17, which are incorporated herein by reference.

[0252] In the synthetic muscle-specific promoter, the regulatory elements are preferably present in the order listed and are preferably adjacent to each other (i.e., without any intervening regulatory elements). The regulatory elements can be continuous or discontinuous (i.e., they can be placed in close proximity to each other, or they can be separated by spacers or other sequences). In some embodiments, some or all of the regulatory elements can be CREs. The CREs are preferably present in the order listed and are preferably adjacent to each other. The CREs can be continuous or discontinuous (i.e., they can be placed in close proximity to each other, or they can be separated by spacers or other sequences). In some preferred embodiments, the regulatory elements or their functional variants are provided in the order listed and are adjacent to each other. The promoter element is located downstream of other regulatory elements and is typically adjacent to proximal regulatory elements. The promoter element can be adjacent to adjacent regulatory elements, or can be separated by spacers. In some embodiments, the promoter element is located downstream of the CRE and is typically adjacent to the proximal CRE. The promoter element can be adjacent to adjacent CREs, or can be separated by spacers.

[0253] In some embodiments, the promoter element is located downstream of other regulatory elements, and typically it is adjacent to proximal regulatory elements.The promoter element may be contiguous with adjacent regulatory elements, or may be separated by a spacer.

[0254] In some embodiments, the synthetic muscle-specific promoter comprises one or more regulatory elements in addition to the above-mentioned regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more other regulatory elements according to the present invention or other regulatory elements. In some embodiments, the one or more additional regulatory elements may be one or more other CREs according to the present invention or other CREs. In some embodiments, the one or more additional regulatory elements may be one or more promoter elements. In some embodiments, the one or more additional regulatory elements may be one or more de-targeting elements or liver de-targeting elements. In some embodiments, the one or more additional regulatory elements may be one or more introns.

[0255] Synthetic muscle-specific expression cassette

[0256] Also disclosed herein is a muscle-specific expression cassette. The muscle-specific expression cassette may comprise a synthetic muscle-specific promoter according to any aspect of the invention operably linked to a sequence encoding an expression product, suitably a gene (e.g., a transgene, such as a therapeutic transgene).

[0257] In some embodiments, the expression product is a therapeutic expression product. In some preferred embodiments, the therapeutic expression product is suitable for treating a disease or condition. In some embodiments, the disease or condition is associated with abnormal gene expression, optionally in muscle (i.e., muscle disease), optionally in cardiac muscle and / or skeletal muscle. In some preferred embodiments, the therapeutic expression product includes those for the treatment of a disease, suitably a muscle disease.

[0258] The term "muscle disease" is generally understood by those skilled in the art. This term relates to diseases that can be treated and / or prevented by administering an active compound to muscle, particularly muscle cells. In some embodiments, the muscle disease is a skeletal muscle disease. In some embodiments, the muscle disease is a cardiac muscle disease. In some embodiments, the muscle disease is a skeletal and cardiac muscle disease.

[0259] Suitable diseases and expression products are described in detail below.

[0260] Suitably, the synthetic muscle-specific expression cassette comprises a sequence providing 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.

[0261] Activity of the muscle-specific promoter and expression cassette

[0262] heart

[0263] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% of the activity of the control promoter CK7 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 10,000%, 15,000%, 20,000%, preferably 30,000%, 31,000%, 32,000%, 33,000%, more preferably at least 35,000% of the activity of the control promoter CK7 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in cardiac cells or cardiac tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times higher, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher than the control promoter CK7. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in cardiac cells or cardiac tissue that is at least 50, 100, 150, 200 times, preferably at least 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 times, more preferably at least 310, 320, 330, 340 or 350 times higher than the control promoter CK7.

[0264] like Figure 2 and 3 As shown in Figure 2, SP0525 (SEQ ID NO: 1) showed approximately 1500% of the CK7 activity in the heart (15 times higher than the CK7 activity in the heart). Figure 2 and 4As shown in FIG, SP0526 (SEQ ID NO: 2) exhibited approximately 800% of the activity of CK7 in the heart (8 times higher than the activity of CK7 in the heart). Figure 2 and 5 As shown in FIG, SP0527 (SEQ ID NO: 3) exhibited approximately 3600% of the CK7 activity in the heart (36 times higher than the CK7 activity in the heart). Figure 2 and 6 As shown in FIG, SP0528 (SEQ ID NO: 4) exhibited approximately 1400% of the CK7 activity in the heart (14 times higher than the CK7 activity in the heart). Figure 2 and 7 As shown, expression cassette 529 showed approximately 35,000% of the CK7 activity in the heart (350 times higher than the CK7 activity in the heart). Figure 2 and 8 As shown in Figure 2, SP0530 (SEQ ID NO: 27) showed approximately 23,000% of the activity of CK7 in the heart (230 times higher than the activity of CK7 in the heart). Figure 2 and 9 As shown, SP0531 (SEQ ID NO: 28) exhibited approximately 300% of the activity of CK7 in the heart (3 times higher than the activity of CK7 in the heart).

[0265] In some preferred embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 300% of the activity of the control promoter CK7 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 3 times higher activity in cardiac cells or cardiac tissue than the control promoter CK7.

[0266] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% of the activity of the control promoter CK8 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in cardiac cells or cardiac tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times higher, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher than the control promoter CK8.

[0267] like Figure 1 and 7 As shown, expression cassette 529 showed approximately 250% of the CK8 activity in the heart (2-fold higher than the CK8 activity in the heart). Figure 1 and 8 As shown, SP0530 (SEQ ID NO: 27) exhibited approximately 160% of the CK8 activity in the heart.

[0268] In some preferred embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 200% of the activity of the control promoter CK8 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 2-fold higher activity than the control promoter CK8 in cardiac cells or cardiac tissue.

[0269] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 110%, 120%, 130%, 140%, 150%, 160%, preferably at least 170%, 180%, 190%, 200%, most preferably at least 210%, 220%, 230%, 240%, 250% of the activity of SP0524 in cardiac cells or cardiac tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 2-fold greater activity than SP0524 in cardiac cells or cardiac tissue.

[0270] like Figure 8 and application PCT / GB2022 / 051611 (particularly page 9, lines 7-11 and Figure 5 、 6 As shown in Figures 1 and 18), SP0530 showed approximately 230% of the activity of SP0524 in the heart (2-fold higher activity than SP0524 in the heart). Thus, addition of the tMCK SA / SD intron to SP0524 resulted in a promoter (SP0530) with higher activity in the heart.

[0271] diaphragm

[0272] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% of the activity of the control promoter CK7 in diaphragm cells or diaphragm tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in diaphragm cells or diaphragm tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times higher, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher than the control promoter CK7.

[0273] like Figure 2 and 4 As shown in FIG, SP0526 (SEQ ID NO: 2) showed approximately 1500% of the CK7 activity in diaphragm muscle (15-fold higher than the CK7 activity in diaphragm muscle). Figure 2 and 5 As shown in FIG, SP0527 (SEQ ID NO: 3) showed approximately 1100% of the CK7 activity in diaphragm muscle (11-fold higher than the CK7 activity in diaphragm muscle). Figure 2 and 6 As shown in FIG, SP0528 (SEQ ID NO: 4) showed approximately 300% of the CK7 activity in diaphragm muscle (3 times higher than the CK7 activity in diaphragm muscle). Figure 2 and 7 As shown, expression cassette 529 showed approximately 1800% of the CK7 activity in diaphragm muscle (18-fold higher than the CK7 activity in diaphragm muscle). Figure 2 and 8 As shown in FIG, SP0530 (SEQ ID NO: 27) showed approximately 2400% of the CK7 activity in diaphragm muscle (24 times higher than the CK7 activity in diaphragm muscle). Figure 2 and 9 As shown, SP0531 (SEQ ID NO: 28) showed approximately 2800% of the CK7 activity in diaphragm muscle (28-fold higher than the CK7 activity in diaphragm muscle).

[0274] In some preferred embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 300% of the activity of a control promoter CK7 in diaphragm cells or diaphragm muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 3 times greater activity in diaphragm cells or diaphragm muscle tissue than the control promoter CK7.

[0275] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% of the activity of the control promoter CK8 in diaphragm cells or diaphragm tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in diaphragm cells or diaphragm tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher than that of the control promoter CK8, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher.

[0276] like Figure 1 and 4 As shown in Figure 2, SP0526 (SEQ ID NO: 2) showed approximately 180% of the CK8 activity in diaphragm muscle. Figure 1 and 5 As shown in Figure 2, SP0527 (SEQ ID NO: 3) showed approximately 130% of the CK8 activity in diaphragm muscle. Figure 1 and 7 As shown, expression cassette 529 showed approximately 200% of the CK8 activity in diaphragm muscle (2-fold higher than the CK8 activity in diaphragm muscle). Figure 1 and 8 As shown in FIG, SP0530 (SEQ ID NO: 27) showed approximately 280% of the CK8 activity in diaphragm muscle (2-fold higher than the CK8 activity in diaphragm muscle). Figure 1 and 9 As shown, SP0531 (SEQ ID NO: 28) showed approximately 300% of the CK8 activity in diaphragm muscle (3-fold higher than the CK8 activity in diaphragm muscle).

[0277] gastrocnemius muscle

[0278] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% of the activity of the control promoter CK7 in gastrocnemius muscle cells or gastrocnemius muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in gastrocnemius muscle cells or gastrocnemius muscle tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times higher, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher than the control promoter CK7.

[0279] like Figure 2 and 4 As shown in FIG, SP0526 (SEQ ID NO: 2) showed approximately 400% of the CK7 activity in the gastrocnemius muscle (4 times higher than the CK7 activity in the gastrocnemius muscle). Figure 2 and 5 As shown in FIG, SP0527 (SEQ ID NO: 3) showed approximately 260% of the CK7 activity in the gastrocnemius muscle (2-fold higher than the CK7 activity in the gastrocnemius muscle). Figure 2 and 9 As shown, SP0531 (SEQ ID NO: 28) showed approximately 200% of the CK7 activity in gastrocnemius muscle (2-fold higher than the CK7 activity in gastrocnemius muscle).

[0280] In some preferred embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 200% of the activity of the control promoter CK7 in gastrocnemius muscle cells or gastrocnemius muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 2-fold higher activity than the control promoter CK7 in gastrocnemius muscle cells or gastrocnemius muscle tissue.

[0281] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has about 100% or less than 100% of the activity of the control promoter CK8 in gastrocnemius muscle cells or gastrocnemius muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has similar activity or less than the control promoter CK8 in gastrocnemius muscle cells or gastrocnemius muscle tissue.

[0282] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 110%, preferably 120%, or most preferably 130% of the activity of SP0524 in gastrocnemius muscle cells or tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 150%, 160%, 170%, 180%, 190%, 200%, preferably 210%, 250%, 300%, 350%, 400%, 450%, most preferably 500% or 550% of the activity of SP0524 in gastrocnemius muscle cells or tissue.

[0283] like Figure 8 and application PCT / GB2022 / 051611 (particularly page 9, lines 7-11 and Figure 5 、 6 As shown in Figures 1 and 18), SP0530 showed approximately 130% of the activity of SP0524 in gastrocnemius muscle. Thus, addition of the tMCK SA / SD intron to SP0524 resulted in a promoter (SP0530) with higher activity in gastrocnemius muscle.

[0284] like Figure 9 and application PCT / GB2022 / 051611 (particularly page 9, lines 7-11 and Figure 5 、 6 As shown in Figures 1 and 18), SP0531 exhibited approximately 550% of the activity of SP0524 in gastrocnemius muscle. Thus, addition of the MVM truncated intron to SP0524 resulted in a promoter (SP0531) with higher activity in gastrocnemius muscle. Furthermore, addition of the MVM truncated intron to SP0524 resulted in a promoter (SP0531) with lower activity in diaphragm, heart, soleus, and tibialis anterior muscles.

[0285] soleus muscle

[0286] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less than 100% of the activity of the control promoter CK7 in soleus muscle cells or soleus muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has lower activity than the control promoter CK7 in soleus muscle cells or soleus muscle tissue.

[0287] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less than 100% of the activity of the control promoter CK8 in a soleus muscle cell or soleus muscle tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less activity than the control promoter CK8 in a soleus muscle cell or soleus muscle tissue.

[0288] Tibialis anterior muscle

[0289] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has at least 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, preferably at least 1100%, 1200%, 1300%, 1400%, 1600%, 1700%, 1800%, 1900%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600%, 3700%, 3800%, 3900%, 4000%, 4100%, 4200%, 4300%, 4400%, 4500%, 4600%, 4700%, 4800%, 4900%, 5000%, 5100%, 5200%, 5300%, 5400%, 5500%, 5600%, 5700%, 5800%, 5900%, 6000%, 6100%, 6100%, 6200%, 6300%, 6400%, 6500%, 6600%, 6700%, 6800%, 6900%, 7000%, 7100%, 7200%, 7300%, 7400%, 7500%, 7600%, 7700%, 7800%, 7900%, 8 0%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000% and most preferably at least 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3100%, 3200%, 3300%, 3400%, 3500%, 3600% activity. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in tibialis anterior muscle cells or tibialis anterior muscle tissue that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher than that of the control promoter CK7, preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, more preferably at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 times higher.

[0290] like Figure 2 and 4As shown in FIG, SP0526 (SEQ ID NO: 2) showed approximately 240% of the CK7 activity in the tibialis anterior muscle (2-fold higher than the CK7 activity in the tibialis anterior muscle). Figure 2 and 5 As shown, SP0527 (SEQ ID NO: 3) exhibited approximately 140% of the CK7 activity in the tibialis anterior muscle.

[0291] liver

[0292] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less than 100% of the activity of the control promoter CK7 or CK8 in liver cells or liver tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has lower activity in liver cells or liver tissue than the control promoter CK7 or CK8.

[0293] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less than 100% of the activity of SP0524 in liver cells or liver tissue. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention has less activity than SP0524 in liver cells or liver tissue.

[0294] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention is expressed in liver cells or liver tissue at a concentration above 1.2 e 12 In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the present invention or the expression cassette according to the ninth or tenth aspect of the present invention is effective in liver cells or liver tissue at a concentration of more than 1.2 e 12 It has lower activity than SP0524 at a dose of 100 vg / 200 μl.

[0295] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in the liver that is about 2, 3, 4, 5 times lower, preferably 6, 7, 8, 9, 10 times, most preferably 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times lower than in the muscle tissue with the lowest activity. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in the liver that is at least 2, 3, 4, 5 times lower, preferably 6, 7, 8, 9, 10 times, most preferably 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times lower than in the muscle tissue with the lowest activity. The muscle tissue with the lowest activity is the muscle tissue in which the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention is the least active (i.e., the lowest average activity).

[0296] In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in the liver that is about 100, 200, 300, 400, 500-fold, preferably about 600, 700, 800, 900-fold, most preferably about 1000, 1100, 1200, 1300, 1400, or 1500-fold lower than that in the muscle tissue with the highest activity. In some embodiments, the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention has an activity in the liver that is at least 100, 200, 300, 400, 500-fold, preferably at least 600, 700, 800, 900-fold, most preferably 1000, 1100, 1200, 1300, 1400, or 1500-fold lower than that in the muscle tissue with the highest activity. The muscle tissue with the highest activity is the muscle tissue in which the synthetic muscle-specific promoter according to any one of the first to fifth aspects of the invention or the expression cassette according to the ninth or tenth aspect of the invention is most active (ie the average activity is the highest).

[0297] Vectors and viral particles

[0298] Disclosed herein are various vectors comprising a synthetic muscle-specific promoter according to any aspect of the invention or an expression cassette according to the invention.

[0299] In some embodiments of the present invention, the vector is a plasmid. Such a plasmid may include a variety of other functional nucleic acid sequences, such as one or more selection markers, one or more replication origins, multiple cloning sites, etc. In some embodiments of the present invention, the vector is a viral vector.

[0300] In some embodiments of the present 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, and pCMV-EGFP available from Clontech. Many other vectors are well-known and commercially available. For mammalian adenovirus vectors, the pSV 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 plasmids (Yip) and yeast replicating plasmids (Yrp). For plants, the Ti plasmid of Agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0301] In some preferred embodiments, the vector is a gene therapy vector. Various gene therapy vectors are known in the art, and AAV vectors, adenoviral vectors, retroviral vectors and lentiviral vectors can be mentioned. When the vector is a gene therapy vector, the vector preferably comprises a nucleic acid sequence operably linked to a synthetic muscle-specific promoter of the present invention, and the nucleic acid sequence encodes a therapeutic product, suitably a therapeutic protein. The therapeutic protein can be a secretable protein. Non-limiting examples of secretable therapeutic proteins include coagulation factors, such as factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, toxic proteins, etc.

[0302] In some embodiments of the present invention, the vector is a viral vector, such as a retrovirus, a lentivirus, an adenovirus or an adeno-associated virus (AAV) vector. In some preferred embodiments, the vector is an AAV vector. In some preferred embodiments, AAV has a serotype suitable for muscle transduction. In some embodiments, AAV is selected from the group consisting of: AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8 and AAV2G9, or derivatives thereof. The AAV vector is preferably used as a self-complementary double-stranded AAV vector (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., single-stranded to double-stranded AAV conversion), although the use of single-stranded AAV vectors (ssAAV) is also included in this article. In some embodiments of the present invention, the AAV vector is chimeric, meaning that it comprises components from at least two AAV serotypes, such as the ITR of AAV2 and the capsid protein of AAV5. It is known that AAV9 can effectively transduce skeletal muscle and cardiac muscle, especially effectively, so AAV9 and its derivatives are particularly meaningful for targeting skeletal muscle and cardiac muscle. It is also known that AAV1, AAV6, AAV7 and AAV8 target skeletal muscle, so these AAV serotypes and their derivatives are also particularly meaningful for targeting skeletal muscle. It is also known that AAV1 and AAV8 target cardiac muscle, so these AAV serotypes and their derivatives are also particularly meaningful for targeting cardiac muscle. In some embodiments, the rAAV vector is an AAV3b serotype, including but not limited to AAV3b265D virions, AAV3b265D549A virions, AAV3b549A virions, AAV3bQ263Y virions or AAV3bSASTG virions (i.e., virions comprising an AAV3b capsid containing Q263A / T265 mutations). In some embodiments, the virion can be a reasonable haploid or chimera or any mutant, such as a capsid that can be customized to increase turnover at a desired location, such as the heart. Other capsids can include capsids from any known AAV serotype, including AAV1, AAV3, AAV4, AAV5, AAV7, AAV10, etc. In some embodiments, the vector is AAV9. In some embodiments, the vector is AAVMYO.AAVMYO capsid has been shown to have high transduction in muscle and is described in Weinmann, J., Weis, S., Sippel, J. et al. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat Commun 11, 5432 (2020). https: / / doi.org / 10.1038 / s41467-020-19230-w, which is incorporated herein by reference. In some embodiments, the vector is a MyoAAV vector. MyoAAV capsids have been shown to have high transduction in muscle and are reported in Mohammadsharif Tabebordbar, Kim A. Lagerborg, Alexandra Stanton, Emily M. King, Simon Ye, Liana Tellez, Allison Krunnfusz, Sahar Tavakoli, Jeffrey J. Widrick, Kathleen A. Messemer, Emily C. Troiano, Behzad Moghadaszadeh, Bryan L. Peacker, Krystynne A. Leacock, Naftali Horwitz, Alan H. Beggs, Amy J. Wagers, Pardis C. Sabeti, Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species, Cell, Volume 184, Issue 19, 2021, Pages 4919-4938.e22, ISSN 0092-8674, https: / / doi.org / 10.1016 / j.cell.2021.08.028, which is incorporated herein by reference.

[0303] The present invention further provides a recombinant virion (viral particle) comprising the above vector.

[0304] Pharmaceutical composition

[0305] The synthetic muscle-specific promoter, expression cassette, vector, or virion of the present invention can be formulated into a pharmaceutical composition together with one or more pharmaceutically acceptable excipients, i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition can be provided in the form of a kit. Pharmaceutical compositions and delivery systems suitable for AAV vectors, and methods and uses thereof are known in the art.

[0306] Treatment and other methods and uses

[0307] The synthetic muscle-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to various aspects of the present invention can be used to treat a disease, preferably a disease associated with aberrant gene expression, optionally in muscle (eg, muscle disease).

[0308] In one embodiment, the present invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the present invention for use in treating skeletal muscle diseases. In one embodiment, the present invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the present invention for use in treating myocardial diseases. In one embodiment, the present invention provides a synthetic muscle-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to various aspects of the present invention for use in treating skeletal and myocardial muscle diseases.

[0309] Related diseases and expression products are discussed below.

[0310] The synthetic muscle-specific promoter, expression cassette, vector or virion according to various aspects of the present invention may be used to prepare a pharmaceutical composition for treating any of the conditions or diseases mentioned herein.

[0311] cell

[0312] The present invention further provides cells comprising a synthetic muscle-specific promoter, expression cassette, vector or virion according to various aspects of the present invention. Suitably, the cell is a eukaryotic cell. Suitably, the eukaryotic cell can 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 can be a prokaryotic cell. In some embodiments of the present invention, the cell is isolated, e.g., in cell culture. In other embodiments of the present invention, the cell can be part of a tissue or a multicellular organism.

[0313] In a preferred embodiment, the cell is a muscle cell (myoblast), which can be isolated or in vivo. In a preferred embodiment, the cell is a cardiomyocyte, which can be isolated or in vivo. In an alternative preferred embodiment, the cell is a skeletal muscle cell, which can be isolated or in vivo. The muscle cell can be a primary muscle cell or a cell of a myogenic cell line, such as an immortalized cell line. The cell can be present in a muscle tissue environment (e.g., in the muscle of an animal) or can be isolated from muscle tissue, such as it can be in cell culture. Suitably, the cell is a human cell. Suitably, the cell is a mammalian cell excluding humans.

[0314] Skeletal muscle cells can be from fast-twitch muscles or slow-twitch muscles. Skeletal muscle cells can be selected from moderate-twitch cells, myocytes, myotubes, myoblasts, and satellite cells.

[0315] The cardiomyocytes may be selected from ventricular cardiomyocytes, atrial cardiomyocytes, pericytes, cardiac smooth muscle cells, cardiac fibroblasts or endothelial cells (EC) in the heart, as well as perivascular cells and pacemaker cells.

[0316] The synthetic muscle-specific promoter, expression cassette or vector according to the invention may be inserted into the genome of a cell, or it may be episomal (eg present in an episomal vector).

[0317] Methods for producing expression products

[0318] Also disclosed herein is a method for producing an expression product, the method comprising providing a muscle-specific expression cassette (preferably in a vector as described above) according to the synthesis of the present invention in a cell, preferably a muscle cell, and expressing the expression product present in the synthetic muscle-specific expression cassette, suitably a gene. Suitably, the method comprises maintaining the muscle cell under suitable conditions for expression of the expression product (suitably a gene). In culture, this can include incubating the cell or tissue comprising the cell under suitable culture conditions. Suitably, the culture conditions can be incubated at 37° C., 5% CO 2 . Expression can certainly be in vivo, for example, in one or more cells of a subject's muscle. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. Skeletal muscle and cardiomyocyte cell types are described above.

[0319] Suitably, the method comprises the step of introducing the synthetic muscle-specific expression cassette into muscle cells. A wide range of methods for transfecting muscle cells are well known in the art. A preferred method for transfecting muscle cells is to transduce the cells with a viral vector (e.g., an AAV vector) containing the synthetic muscle-specific expression cassette.

[0320] A wide range of methods for transfecting cells are well known in the art, such as viral-mediated transfection, such as transfection using viral vectors; chemical-based transfection, such as lipofection, calcium phosphate transfection, cationic polymers of Fugene reagent; non-chemical-based transfection, such as electroporation; microinjection; Agrobacterium-mediated transfer; gene gun; impalefection; hydrostatic pressure; direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment.

[0321] Methods for expressing expression products

[0322] Also disclosed herein is a method for expressing an expression product (suitably a therapeutic transgene) in a muscle cell, the method comprising introducing an expression cassette or vector according to the present invention into the muscle cell. Suitably, introducing the expression cassette or vector may comprise transfecting the muscle cell with the expression cassette or vector. A wide range of methods for transfecting muscle cells are well known in the art. A preferred method for transfecting muscle cells is to transduce the cells using a viral vector (e.g., an AAV vector) comprising a synthetic muscle-specific expression cassette. The muscle cell may be in vivo or ex vivo. In one embodiment, the muscle cell is a cardiomyocyte. In one embodiment, the muscle cell is a skeletal muscle cell. Skeletal muscle and cardiomyocyte cell types are as described above.

[0323] Gene therapy methods

[0324] It is obvious to the skilled person that the synthetic muscle-specific promoter, expression cassette, vector, pharmaceutical composition or virosome according to various aspects of the present invention can be used for gene therapy. Therefore, the use of such nucleic acid constructs in gene therapy forms part of the present invention.

[0325] The expression cassette, vector, pharmaceutical composition or virion according to the present invention can be used for gene therapy in a subject, preferably gene therapy performed by muscle-specific expression of an expression product (suitably a therapeutic gene). Suitably, the expression cassette, vector, pharmaceutical composition or virion according to the present invention can be used for gene therapy performed by myocardial-specific (or cardiac-selective) expression and / or skeletal muscle-specific (or bone-selective) expression of an expression product (suitably a therapeutic gene). The therapy may involve treating a disease by secreting an expression product (suitably a gene, therapeutic transgene or therapeutic product) from muscle cells, suitably in diseases involving abnormal gene expression in muscle. Suitable diseases are discussed below.

[0326] Also provided herein is a method of gene therapy for a subject, preferably a human, in need thereof, comprising:

[0327] - administering to a subject (suitably introducing into the subject's muscle) a synthetic muscle-specific expression cassette, vector, virion or pharmaceutical composition of the invention comprising an expression product, suitably a gene encoding a therapeutic expression product. Suitable expression products are described in detail below.

[0328] In one embodiment, the muscle is cardiac muscle. In one embodiment, the muscle is skeletal muscle.

[0329] Suitably, the method comprises expressing a therapeutic amount of an expression product (suitably a therapeutic expression product) from the gene in muscle of the subject. Various conditions and diseases that may be treated, and suitable diseases, are discussed below.

[0330] In some embodiments, the method comprises administering to a subject a vector or virosome according to the invention. Suitably, the vector is a viral gene therapy vector, such as an AAV vector.

[0331] In some embodiments, the method includes systemic administration of the viral gene therapy 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.

[0332] In some embodiments, the viral gene therapy vector may be administered simultaneously 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.

[0333] When the vector is an AAV vector, the dose of the vector can be from 1x10 10 gc / kg to 1x10 15 gc / kg or more, suitably from 1x10 12 gc / kg to 1x10 14 gc / kg, suitably from 5x10 12 gc / kg to 5x10 13 gc / kg (gc / kg stands for genome copies per kilogram). In some preferred embodiments, the AAV dose is 1e 14 gc / kg or about 1e 14 gc / kg.

[0334] In general, the subject in need is a mammal, preferably a primate, more preferably a human. In some embodiments, the subject is non-human. Typically, the subject in need will exhibit characteristic symptoms of the disease. The method typically comprises improving the symptoms exhibited by the subject in need by expressing a therapeutic amount of an expression product (suitably a therapeutic expression product).

[0335] Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well known in the art and will not be discussed in detail herein. In short, they include intramuscular injection, interstitial injection, airway instillation, application to endothelium, intrahepatic parenchyma, and intravenous or intraarterial administration (e.g., intrahepatic artery, intrahepatic vein) of plasmid DNA vectors (naked or in liposomes) or viral vectors. Various devices have been developed to improve the availability of DNA to target cells. Although a simple method is to physically contact the target cells with a catheter or implantable material containing the relevant vector, a more complex method can use a jet injection device, etc. In vitro and in vivo procedures have been used to transfer genes into mammalian muscle cells. In vitro methods generally require harvesting muscle cells, performing in vitro transduction with a suitable expression vector, and then reintroducing the transduced muscle cells into the muscle. In vivo gene transfer is achieved by injecting DNA or viral vectors into the muscle. In some preferred embodiments, the preferred route of administration is intravenous. Intravenous delivery is particularly preferred for gene therapy for muscle. In some preferred embodiments, the preferred route of administration is intracoronary. Intracoronary delivery is particularly preferred for gene therapy for myocardium.

[0336] According to some preferred embodiments, the above methods can be used to treat a subject suffering from the diseases discussed above, such as muscular dystrophy or congestive heart failure.

[0337] disease

[0338] The disease can be any disease. Suitably, the patient's condition or disease are associated with abnormal gene expression, optionally associated with abnormal gene expression in muscle cells (myoblasts) or tissues. Suitably, the patient's condition or disease are associated with abnormal gene expression, optionally associated with abnormal gene expression in cardiac muscle and / or skeletal muscle cells. In a preferred embodiment, the disease is a disease that can be alleviated by muscle-specific expression of a suitable expression product.

[0339] In some embodiments, the disease is vascular disease, muscular dystrophy, cardiomyopathy, myotonia, muscle atrophy, myoclonic dystonia (affected gene: SGCE), mitochondrial myopathy, rhabdomyolysis, fibromyalgia, and / or myofascial pain syndrome.

[0340] In one embodiment, the disease may be a cardiovascular condition, a heart disease, or a cardiac disorder. In one embodiment, the disease may be heart failure, such as congestive heart failure. In one embodiment, the disease may be selected from the group consisting of ischemia, arrhythmia, myocardial infarction (MI), abnormal cardiac contractility, non-ischemic cardiomyopathy, peripheral arterial occlusive disease, and abnormal Ca 2+Metabolism and combinations thereof. In some embodiments, the disease can be selected from the group consisting of congestive heart failure, cardiomyopathy, myocardial infarction, tissue ischemia, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, conduction system dysfunction, coronary artery dysfunction, and cor pulmonale. In some embodiments, the disease can be selected from the group consisting of congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, arrhythmia, muscular dystrophy, muscle mass abnormalities, muscle degeneration, infectious myocarditis, drug- or toxin-induced muscle abnormalities, allergic myocarditis, autoimmune endocarditis, and congenital heart disease.

[0341] In some embodiments, the disease is cardiomyopathy. In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy, restrictive cardiomyopathy, left ventricular noncompaction, Takotsubo cardiomyopathy, myocarditis, eosinophilic myocarditis and ischemic cardiomyopathy. Preferably, the arrhythmogenic right ventricular dysplasia is ARVD1 (gene: TGFB3), ARVD2 (gene: RYR2), ARVD3, ARVD4, ARVD5 (gene: TMEM43), ARVD6, ARVD7 (gene: DES), ARVD8 (gene: DSP), ARVD9 (gene: PKP2), ARVD10 (gene: DSG2), ARVD11 (gene: DSC2) and / or ARVD12 (gene: JUP). In some embodiments, the disease is hypertrophic cardiomyopathy. Preferably, the hypertrophic cardiomyopathy is CMH1 (gene: MYH7), CMH2 (gene: TNNT2), CMH3 (gene: TPM1), CMH4 (gene: MYBPC3), CMH5, CMH6 (gene: PRKAG2), CMH7 (gene: TNNI3), CMH8 (gene: MYL3), CMH9 (gene: TTN), CMH10 (gene: MYL2), CMH11 (gene: ACTC1) or CMH12 (gene: CSRP3).

[0342] In some embodiments, the disease is a vascular disease. The vascular disease can be coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, or aortic aneurysm.

[0343] In some embodiments, the disease can be a cardiomyopathy. The cardiomyopathy can be hypertensive heart disease, heart failure (such as congestive heart failure), cor pulmonale, arrhythmia, inflammatory heart disease (such as endocarditis, inflammatory cardiac hypertrophy, myocarditis), valvular heart disease, congenital heart disease and rheumatic heart disease.

[0344] In some embodiments, the disease is a muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (affected gene: DMD), Becker muscular dystrophy (affected gene: DMD), limb-girdle muscular dystrophy (subtype 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: P OMT1), LGMD2L (gene: AN05), LGMD2M (gene: FKTN), LGMD2N (gene: POMT2), LGMD20 (gene: POMGNT1), LGMD2Q (gene: PLEC1)), congenital muscular dystrophy, distal muscular dystrophy (subtypes and affected genes: Miyoshi myopathy (gene: DYSF), distal tibial anterior myopathy (gene: DYSF), Welander distal myopathy (gene: TIA1), Gowe The disease is characterized by rs-Laing distal myopathy (gene: MYH7), Nonaka distal myopathy, hereditary inclusion body myositis type 1, distal myopathy with vocal cord and pharyngeal weakness, ZASP-related 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)). In some preferred embodiments, the disease is limb-girdle muscular dystrophy type 2i (LGMD2I; affected gene: FKRP).

[0345] In some embodiments, the disease is myotonia. In some embodiments, the myotonia is myotonia congenita (affected gene: CLCN1; subtypes: Thomsen type, Beck type) and / or myotonia congenita (affected gene: SCN4A).

[0346] In some embodiments, the disease is Duchenne muscular dystrophy (gene: DMD), myotubular myopathy (gene: MTM1), spinal muscular atrophy (gene: SMA), glycogen storage disease type II (Pompe disease, gene: GAA) or cardiomyopathy. In some preferred embodiments, the disease is Duchenne muscular dystrophy (affected gene: DMD).

[0347] Other exemplary diseases include, but are not limited to, acid maltase deficiency (AMD), alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis (ALS), Andersen-Tawil syndrome, Becker muscular dystrophy (BMD), Becker myotonia congenita, Bethlem myopathy, carnitine deficiency, carnitine palmitoyltransferase deficiency (CPT deficiency), Central Core Disease,CCD), Centronuclear Myopathy, Charcot-Marie-Tooth Disease (CMT), Congenital Myasthenic Syndrome (CMS), Congenital Myotonic Dystrophy, Cori Disease (Debranching Enzyme Deficiency), Debranching Enzyme Deficiency, Dejerine-Sottas Disease (DSD), Dermatomyositis (DM), Endocrine Myopathies, Eulenberg Disease (Myotonia Congenita), 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, Gowers-Laing Distal Myopathy, Hauptmann-Thanheuser 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), hereditary myopathies, integrin-deficient congenital muscular dystrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome (LEMS), McArdle disease (phosphatase deficiency), muscle metabolic diseases, mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease, muscle-eye-brain disease, myasthenia gravis (MG), myoadenylate deaminase deficiency, myofibrillar myopathy, muscle phosphorylase deficiency, myotonia congenita (MC), myotonic dystrophy (MMD), myotubular myopathy (MTM or MM), nematode myopathy, Nonaka distal myopathy, oculopharyngeal myopathy dystrophies (OPMD), myotonia congenita, Pearson syndrome, periodic paralysis, Charcot-Marie-Tooth disease, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, phosphorylase deficiency, polymyositis (PM), Pompe disease (acid maltase deficiency), progressive external ophthalmoplegia (PEO), rod body disease (nematode myopathy), spinal muscular atrophy (SMA), spinal bulbar muscular atrophy (SBMA), Steinert disease (myotonic dystrophy), Tarui disease (phosphofructokinase deficiency), Thomsen disease (myotonia congenita), Ullrich congenital muscular dystrophy, Walker-Warburg syndrome (congenital muscular dystrophy), Welander distal myopathy, and ZASP-related myopathy.

[0348] In some preferred embodiments, the disease is a myocardial disease. In some preferred embodiments, the disease is congestive heart failure. In some preferred embodiments, the disease is congenital heart failure.

[0349] In some preferred embodiments, the disease is a cardiac and skeletal muscle disease. In some embodiments, the disease is Duchenne muscular dystrophy (gene: DMD).

[0350] In some preferred embodiments, the disease is Danon disease. Danon disease is an X-linked dominant genetic disorder associated with hypertrophic cardiomyopathy, skeletal muscle weakness, and intellectual disability.

[0351] Promoters according to the present invention are particularly useful in diseases where expression of a large expression product (e.g., a large transgene) is desired due to their small size (e.g., less than 390 bp). Promoters according to the present invention are particularly useful in diseases where expression of the expression product in non-muscle tissues and cells (e.g., liver) is desired to be reduced. Promoters according to the present invention are particularly useful in diseases where expression of a large expression product is desired and where hepatic expression of the expression product is desired to be reduced.

[0352] Expression product

[0353] The expression product can be any product desired to be expressed. Expression in skeletal muscle and / or cardiac muscle may be desired. The expression product can be a gene. The expression product can be a gene encoding a desired gene expression product (e.g., a product of interest), such as a polypeptide (protein) or RNA. The expression product can be a transgene. The expression product can be a protein or polypeptide. The expression product can be a nucleic acid sequence. The expression product can be a therapeutic expression product.

[0354] In some preferred embodiments, the expression product is lysosomal associated membrane protein type 2 (LAMP2). Mutations in LAMP2 cause Danon disease, and providing a non-disease copy of LAMP2 may be desirable for treating Danon disease.

[0355] In some preferred embodiments, the expression product is fukutin-related protein (FKRP). Mutations in FKRP cause limb-girdle muscular dystrophy-dystrophinopathy (LGMD), and providing a non-disease copy of FKRP may be desirable for treating LGMD.

[0356] In some embodiments, the expression product, suitably a gene, encodes a non-disease-mediated variant, for example a wild-type variant of at least one human gene selected from the group consisting of DMD, GALGT2, SMA, GAA, MTM1, TTID, LMNA, CAV3, DNAJB6, DES, TNP03, HNRPDL, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, FKRP, TTN, POMT1, ANOVA. 5, FKTN, POMT2, PFEC1, DYSF, TIA1, MYH7, DUX4, SMCHD, PABPN1, DMPK, ZNF9, CFCN1, SCN4A, MYH7, TNNT2, TPM1, MYBPC3, PRKAG2, TNNI3, MYF3, TTN, MYF2, ACTC1, CSRP3, TGFB3, RYR2, TMEM43, DES, DSP, PKP2, DSG2, DSC2, JUP and HYPP. In some preferred embodiments, the gene is DMD.

[0357] In some embodiments, the expression products include dystrophin (including micro-dystrophin), β1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthetase I, alpha-1 antitrypsin, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, porphobilinogen deaminase, cystathionine β-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, and cystic fibrosis transmembrane regulator (CFTR).

[0358] In some embodiments, the expression product can be dystrophin. The mutation of the DMD gene that damages the function of dystrophin leads to Becker muscular dystrophy or Duchenne muscular dystrophy, which are X-linked recessive muscular dystrophies characterized by muscle weakness. Providing a non-disease copy of dystrophin may be desirable for treating Becker muscular dystrophy and / or Duchenne muscular dystrophy. The DMD gene is the largest gene known in humans (approximately 2.4 million base pairs). Therefore, the full-length DMD gene is too large to be packaged into some viral vectors with limited capacity (payload), such as AAV vectors. A shorter version of the DMD gene (called mini-dystrophin) is being used to solve this problem. Nevertheless, even mini-dystrophin is quite large (e.g., 3.5-4kB), which still makes AAV packaging difficult. Thus, short length synthetic muscle-specific promoters (e.g., less than 400 nucleotides in length, less than 350 nucleotides in length, preferably less than 300 nucleotides in length, still more preferably less than 290 nucleotides in length, 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 expression cassettes where the sequence encoding the expression product is a DMD gene or a miniature version of the DMD gene (mini-dystrophin). In some preferred embodiments, the expression product is dystrophin, preferably mini-dystrophin.

[0359] In some preferred embodiments, the expression product is a smaller version of utrophin (eg, mini-utrophin or micro-utrophin). In some preferred embodiments, the expression product is a smaller version of dystrophin (eg, mini-dystrophin or micro-dystrophin). In some embodiments, the expression product can be disclosed in Song Y, Morales L, Malik AS, Mead AF, Greer CD, Mitchell MA, Petrov MT, Su LT, Choi ME, Rosenblum ST, Lu X, Van Belzen DJ, Krishnankutty RK, Balzer FJ, Loro E, French R, Propert KJ, Zhou S, Kozyak BW, Nghiem PP, Khurana TS, Kornegay JN, Stedman HH. Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models. Nat Med. 2019 Oct; 25(10): 1505-1511. doi: 10.1038 / s41591-019-0594-0. Epub 2019 Oct 7.PMID:31591596;PMCID:PMC7274039 (and especially the extended data Figure 1, which is incorporated herein by reference). In some embodiments, the expression product may be the micro-dystrophin disclosed in Duan D.Micro-Dystrophin Gene Therapy Goes SystemicinDuchenne Muscular Dystrophy Patients. Hum Gene Ther. 2018 Jul; 29(7): 733-736. doi: 10.1089 / hum.2018.012. Epub 2018 Apr 5. PMID: 29463117; PMCID: PMC6066190 (which is incorporated herein by reference). In some embodiments, the expression product may be disclosed in Duan D.Micro-utrophin Therapy for Duchenne Muscular Dystrophy.MolTher.2019Nov6; 27(11):1872-1874.doi:10.1016 / j.ymthe.2019.10.011.Epub 2019Oct22.PMID:31653398; PMCID:PMC6838911 (and particularly Figure 1 , which is incorporated herein by reference). In some embodiments, the expression product may be a microutrophin disclosed in Starikova, AV, Skopenkova, VV, Polikarpova, AV et al. Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression. Sci Rep 12, 848 (2022). https: / / doi.org / 10.1038 / s41598-022-04892-x (and in particular Figure 1, which is incorporated herein by reference). In some embodiments, the expression product may be the micro-utrophin disclosed in Kennedy TL, Guiraud S, Edwards B, Squire S, Moir L, Babbs A, Odom G, Golebiowski D, Schneider J, Chamberlain JS, Davies KE. Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2 / mdx Mice. Mol Ther Methods Clin Dev. 2018 Oct 16; 11: 92-105. doi: 10.1016 / j.omtm.2018.10.005. PMID: 30417024; PMCID: PMC6216100 (which is incorporated herein by reference). In some embodiments, the expression product may be disclosed in Banks GB, Chamberlain JS, Odom GL.Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects.PLoS Genet.2020Nov11;16(11):e1009179.doi:10.1371 / journal.pgen.1009179.PMID:33175853;PMCID:PMC7682874 (and particularly Figure 1, which is incorporated herein by reference). In some embodiments, the expression product may be a micro-dystrophin disclosed in Howard ZM, Dorn LE, Lowe J, Gertzen MD, Ciccone P, Rastogi N, Odom GL, Accornero F, Chamberlain JS, Rafael-Fortney JA.Micro-dystrophin gene therapy prevents heart failure in an improved Duchennemuscular dystrophy cardiomyopathy mouse model. JCI Insight. 2021 Apr 8; 6(7): e146511. doi: 10.1172 / jci.insight.146511. PMID: 33651713; PMCID: PMC8119181 (and in particular the methods, which are incorporated herein by reference). In some embodiments, the expression product may be the micro-dystrophin disclosed in Mendell JR, Sahenk Z, Lehman K et al. Assessment of Systemic Delivery of rAAVrh74.MHCK7.micro-dystrophin in Children With Duchenne Muscular Dystrophy: A Nonrandomized Controlled Trial. JAMA Neurol. 2020; 77(9): 1122–1131. doi: 10.1001 / jamaneurol.2020.1484 (and in particular the introduction, which is incorporated herein by reference). In some embodiments, the expression product may be the micro-dystrophin disclosed in https: / / www.clinicaltrials.gov / ct2 / show / NCT03368742 (which study is incorporated herein by reference).

[0360] Other expression products include enzymes for enzyme replacement therapy, and enzymes for various conditions caused by insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate can be used for the treatment of lysosomal storage diseases (e.g., suitable genes include genes encoding β-glucuronidase (GUSB)).

[0361] In some embodiments, exemplary polypeptide expression products include neuroprotective polypeptides and anti-angiogenic polypeptides. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), urturing, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-β), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelium-derived factor (PEDF), Wnt polypeptides, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibodies, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and hedgehog family members (sonic hedgehog, Indian hedgehog, and desert hedgehog, etc.).

[0362] In some embodiments, the expression products include hormones and growth and differentiation factors, including but not limited to insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, 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 factors I and II (IGF-I and IGF-II), transforming growth factor alpha superfamily (including TGFa, activin, inhibin, or any bone morphogenetic protein (BMP) BMP 1-15), any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0363] In some embodiments, the expression products include proteins that regulate the immune system, including but not limited to cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-25 (including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon (α, β and γ), stem cell factor, flk-2 / flt3 ligand. Gene products produced by the immune system can also be used in the present invention. In some embodiments, the expression products include immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. In some embodiments, the expression product further comprises complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

[0364] In some embodiments, the expression product includes any one of a receptor for a hormone, growth factor, cytokine, lymphokine, regulatory protein, and immune system protein. Useful heterologous nucleic acid sequences also include cholesterol-regulated and / or lipid-regulated receptors, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. The present invention also includes 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). In addition, 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 protein, 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.

[0365] In some embodiments, the expression products include those 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; U.S. Pat. No. 6,200,560 and U.S. Pat. No. 6,221,349).

[0366] In some embodiments, the expression product can be a modulator of phosphatase activity, such as type 1 phosphatase activity. The modulator can be a protein that inhibits phosphatase activity, such as type 1 phosphatase activity. The modulator can be a nucleic acid that increases expression of an endogenous nucleic acid that encodes a protein that inhibits phosphatase activity, such as a transcription factor. The modulator can be a regulatory sequence integrated into or near an endogenous nucleic acid that encodes a protein that inhibits phosphatase activity. The modulator can be a nucleic acid that provides a nucleic acid modulator of gene expression, such as an siRNA.

[0367] In some embodiments, the expression product can be an inhibitor of protein phosphatase 1 (PP1), such as an I-1 polypeptide. Phosphatase inhibitor-1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatase. Increasing I-1 levels or activity can restore beta-adrenergic responsiveness to failing human cardiomyocytes. Suitably, the I-1 protein can be constitutively active, such as an I-1 protein in which threonine 35 is replaced by glutamic acid instead of aspartic acid. The expression product can be any one or more inhibitors selected from the group consisting of: phosphatase inhibitor 2 (PP2); okadaic acid or caliculin; and nippl (which is an endogenous nuclear inhibitor of protein phosphatase 1).

[0368] In some embodiments, the expression product can be any protein that regulates cardiac activity, such as a type 1 phosphatase inhibitor, e.g., I-1 or sarcoplasmic reticulum Ca 2+ ATPase (SERCA), such as SERCA1 (eg, 1a or 1b), SERCA2 (eg, 2a or 2b), or SERCA3.

[0369] In some embodiments, the expression product can be a nucleic acid sequence encoding a mutant form of a phosphatase inhibitor-1 protein, wherein the mutant form comprises at least one amino acid at the position of a PKC-α phosphorylation site in the wild type, wherein at least one amino acid is constitutively non-phosphorylated or mimics a non-phosphorylated state in the mutant form. The expression product can be adenylate cyclase 6 (AC6, also known as adenylate cyclase VI), S100A1, beta-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 anthonogene-3 (BAG3).

[0370] In some embodiments, the expression product can be an inhibitor of a cytokine, such as an IL-18 inhibitor. The expression product can encode a β-adrenergic signaling protein (β-ASP) (including β-adrenergic receptor (β-Ar), G-protein receptor kinase inhibitor (GRK inhibitor) and adenylate cyclase (Ac)) to enhance cardiac function.

[0371] In some embodiments, the expression product can be an angiogenic protein. Angiogenic proteins promote the development and differentiation of blood vessels. Examples of angiogenic proteins include members of the fibroblast growth factor (FGF) family, 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.

[0372] In some embodiments, the 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.

[0373] Other suitable expression products include microRNA (miRNA), interfering RNA, antisense RNA, ribozymes, and aptamers.

[0374] In some preferred embodiments, the expression product is a protein phosphatase 1 (PP1) inhibitor.

[0375] In some embodiments of the present invention, the expression product can be used for gene editing, for example, a gene encoding a site-specific nuclease, such as a homing endonuclease (meganuclease), a zinc finger nuclease (ZFN), a nuclease based on a transcription activator-like effector (TALEN) or a clustered regularly interspaced short palindromic repeat system (CRISPR-Cas). Suitably, the site-specific nuclease is suitable for editing the desired target genomic site by cutting (usually a site-specific double-strand break), and then repairing the cutting by non-homologous end joining (NHEJ) or homology-dependent repair (HDR), producing the desired editing. The editing can be to partially or completely repair a dysfunctional gene, or to knock down or knock out a functional gene. Alternatively, a suitable system known in the art can be used for editing by basic editing or major editing.

[0376] The expression product can be a gene. The gene usually encodes the desired gene expression product, such as a polypeptide (protein) or RNA. The gene can be a full-length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof having at least some desired biological activity.

[0377] When the gene encodes a protein, it can be essentially any type of protein. As non-limiting examples, 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).

[0378] In some preferred embodiments, the gene encodes a therapeutic expression product, preferably a therapeutic polypeptide useful for treating a disease or condition associated with aberrant gene expression, optionally in muscle, optionally in cardiac and / or skeletal muscle.

[0379] The expression product may be a therapeutic expression product. The therapeutic expression product may be used to treat cardiovascular conditions or heart diseases and disorders, such as heart failure or CHF. In some preferred embodiments, the therapeutic expression product may be used to treat DMD. In some preferred embodiments, the therapeutic expression product may be used to treat Danon's disease. The expression product may be a LAMP2 protein. The therapeutic expression product may be any protein that regulates cardiac activity, such as a type 1 phosphatase inhibitor, for example, I-1 or sarcoplasmic reticulum Ca 2+. 2+ ATPase (SERCA), such as SERCA1 (eg, 1a or 1b), SERCA2 (eg, 2a or 2b), or SERCA3.

[0380] The therapeutic expression product can be a modulator of phosphatase activity, such as type 1 phosphatase activity. The modulator can be a protein that inhibits phosphatase activity, such as type 1 phosphatase activity. The modulator can be a nucleic acid that increases the expression of an endogenous nucleic acid encoding a protein that inhibits phosphatase activity, such as a transcription factor. The modulator can be a regulatory sequence integrated into or near an endogenous nucleic acid encoding a protein that inhibits phosphatase activity. The modulator can be a nucleic acid that provides a nucleic acid modulator of gene expression, such as an siRNA.

[0381] The therapeutic expression product can be an inhibitor of protein phosphatase 1 (PP1), such as an I-1 polypeptide. Phosphatase inhibitor-1 (or "I-1") protein is an endogenous inhibitor of type 1 phosphatases. Increasing I-1 levels or activity can restore beta-adrenergic responsiveness in failing human cardiomyocytes. Suitably, the I-1 protein can be constitutively active, such as an I-1 protein in which threonine 35 is replaced by glutamate instead of aspartate. The therapeutic expression product can be any one or more inhibitors selected from the group consisting of: phosphatase inhibitor 2 (PP2); okadaic acid or caliculin; and nippl (which is an endogenous nuclear inhibitor of protein phosphatase 1). In some preferred embodiments, the expression cassette comprises a muscle-specific promoter operably linked to a protein phosphatase 1 (PP1) inhibitor. Type 1 phosphatases include, but are not limited to, PP1cα, PP1cβ, PP1cδ, and PP1cγ.

[0382] The therapeutic expression product can be a nucleic acid sequence encoding a mutant form of a phosphatase inhibitor-1 protein, wherein the mutant form comprises at least one amino acid at the position of a PKC-α phosphorylation site in the wild type, wherein at least one amino acid is constitutively non-phosphorylated or mimics a non-phosphorylated state in the mutant form. The therapeutic expression product can be adenylate cyclase 6 (AC6, also known as adenylate cyclase VI), S100A1, beta-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 anthonogene-3 (BAG3).

[0383] The therapeutic expression product can be an inhibitor of a cytokine, such as an IL-18 inhibitor. The therapeutic expression product can encode a β-adrenergic signaling protein (β-ASP) (including β-adrenergic receptor (β-Ar), G-protein receptor kinase inhibitor (GRK inhibitor) and adenylate cyclase (Ac)) to enhance cardiac function.

[0384] The therapeutic expression product can be an angiogenic protein. Angiogenic proteins promote the development and differentiation of blood vessels. Examples of angiogenic proteins include members of the fibroblast growth factor (FGF) family, 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.

[0385] Definitions and general points

[0386] Although the preparation and use of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in various variations in a specific context. The specific embodiments discussed herein are only intended to illustrate specific ways to prepare and use the present invention and are not intended to limit the scope of the invention.

[0387] This discussion of the background to the invention is included to explain the context of the invention. This should not be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country before the priority date of any claim.

[0388] Throughout the disclosure, various publications, patents, and published patent specifications are cited by identifying references. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or portions of such documents specifically mentioned herein are incorporated by reference.

[0389] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art and are fully explained in the literature. For example, see Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and Son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (edited by Harries and Higgins 1984); Transcription and Translation (edited by Hames and Higgins 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRLPress, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154-155 (Wu et al. ed.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, editors, Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, editors, 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0390] To facilitate understanding of the present invention, various terms are defined or explained below. The terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention relates. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but rather to encompass general categories that can be illustrated using specific examples. The terms herein are used to describe specific embodiments of the present invention, but their use does not limit the invention unless outlined in the claims.

[0391] The term "muscle" is well known to those skilled in the art. Preferably, the muscle is skeletal muscle (including diaphragm) or cardiac muscle. Preferably, the muscle is vertebrate muscle, more preferably mammalian muscle, and even more preferably human muscle. Preferably, the muscle is striated muscle. The promoter of the present invention can be active in skeletal muscle and / or cardiac muscle.

[0392] The term "muscle cell" or "myoblast" refers to cells found in muscle (muscle tissue) or derived from muscle tissue. Muscle cells can be primary cells or cell lines (such as C2C12 or H2K cells (skeletal muscle cell lines) or H9C2 cells (cardiac muscle cell lines)). Muscle cells can be in vivo (e.g., in muscle tissue) or in vitro (e.g., in cell culture). The myocytes found in muscle tissue are typically long tubular cells that develop into muscle from myoblasts, a process known as myogenesis. As used herein, the term muscle cell or myocyte includes myocytes from skeletal muscle and from cardiac muscle (cardiac myocytes). The synthetic promoter of the present invention can be active in skeletal muscle cells and / or cardiac myocytes.

[0393] The term "cis-regulatory element" or "CRE" is a term well known to technicians and refers to a nucleic acid sequence that can regulate or modulate the transcription of adjacent genes (i.e., cis), such as enhancers, promoters, insulators or silencers. CREs are present near the genes they regulate. CREs typically regulate gene transcription by binding to TFs, i.e., they include TFBSs. A single TF can bind to many CREs, thereby controlling the expression of many genes (pleiotropy). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the genes they regulate. "Enhancers" in this context are CREs that enhance (i.e., upregulate) the transcription of genes that are operably associated with them, and can be present upstream, downstream, or even in introns of the genes they regulate. Multiple enhancers can work together to regulate the transcription of a gene. In this article, "silencers" relate to CREs that are bound to TFs called repressors, which act to prevent or downregulate gene transcription. The term "silencer" can also refer to a region in the 3' untranslated region of a messenger RNA that binds to a protein that inhibits translation of the mRNA molecule, but this usage is different from its use in describing CRE. Typically, the CRE of the present invention is a muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) enhancer element (commonly referred to as muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) CRE, or muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) CRE enhancer, etc.). In some embodiments, CRE0145 (SEQ ID NO: 10) and DES_MT_enhancer_48bp (SEQ ID NO: 11) are muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) enhancer elements. In this article, it is preferred that CRE is 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, suitably 1000, 750, 500, 250, 200, 150 or 100 nucleotides or less from the TSS. The CRE of the present invention is preferably shorter in length, preferably 500 nucleotides or less in length, for example, their length can be 400, 300, 200, 175, 150, 90, 80, 70, 60 or 50 nucleotides or less. The CRE of the present invention is generally provided in combination with an operably linked promoter element, which can be a minimal promoter or a proximal promoter; the CRE of the present invention enhances the muscle-specific, myocardial-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) activity of the promoter element.In any combination of CRE or its functional variant disclosed herein, suitably some or all of the CRE and promoter elements can be placed adjacent to each other in the promoter (i.e., without any intervening CRE or other regulatory elements). The CRE can be continuous or discontinuous (i.e., they can be placed next to each other, or they can be separated by spacers or other sequences). The CRE can be arranged in any order. In some preferred embodiments, the CRE or its functional variant is provided in the order listed and is adjacent to each other. For example, a synthetic muscle-specific synthetic promoter can include CRE0145 immediately upstream of DES_MT_enhancer_48bp, etc. In some embodiments, preferably some or all of the CRE are continuous.

[0394] The term "cis-regulatory module" or "CRM" refers to a functional regulatory nucleic acid module, which generally comprises two or more CREs or other regulatory elements; in the present invention, CREs are generally muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) enhancers, and thus CRMs are synthetic muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) regulatory nucleic acids. Therefore, in the present application, a CRM generally comprises multiple muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) CREs, and optionally other regulatory elements. Typically, the multiple CREs in a CRM act together (e.g., additively or synergistically) to enhance transcription of a gene operably associated with a synthetic promoter comprising the CRM. There is considerable scope for shuffling (i.e., reordering) the CREs, reversing (i.e., reversing the direction) and changing the spacing of the CREs in a CRM. Therefore, functional variants of the CRMs of the present invention particularly include variants of the referenced CRMs in which the CREs have been shuffled and / or reversed, and / or the spacing between the CREs has been altered. A CRM may include other regulatory elements, such as inducible or repressible elements, intrinsic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements and splicing elements, etc., as long as they do not render the CRM essentially non-functional.

[0395] As used herein, the phrase "promoter" refers to a region of DNA, typically located upstream of a nucleic acid sequence to be transcribed, that is required for transcription to occur, i.e., for its initiation. A promoter allows for the proper activation or repression of transcription of the coding sequence under its control. Promoters typically contain specific sequences that are recognized and bound by multiple TFs. TFs bind to the promoter sequence and lead to 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.

[0396] In some cases, the terms "promoter" or "composite promoter" as used herein refer to a combination of a promoter and one or more additional regulatory elements. In some cases, the promoter has a desired expression profile, and adding one or more additional regulatory elements can regulate the expression profile. Regulating refers to increasing, decreasing, or maintaining the level of activity in any particular cell type or tissue. In some cases, additional regulatory elements may be immediately downstream of the transcription start site (TSS), such as introns. Such sequences downstream of the TSS may help regulate expression at the transcription and / or translation stage. In some cases, additional regulatory elements may be located between the CRE and promoter elements, such as detargeting elements or liver detargeting elements. Such sequences located between the CRE and promoter elements may result in lower expression in specific tissues or cell types, such as in the liver or hepatocytes.

[0397] As used herein, the term "synthetic promoter" refers to a promoter that does not occur in nature. In this article, it generally comprises a CRE and / or CRM of the present invention that is operably linked to a promoter element such as a minimal (or core) promoter or a muscle-specific (or cardiac-selective), cardiac-specific (or cardiac-selective) or skeletal-muscle-specific (or bone-selective) proximal promoter. The CRE and / or CRM of the present invention are used to enhance muscle-specific, cardiac-specific (or cardiac-selective) or skeletal-muscle-specific (or bone-selective) transcription of a gene that is operably linked to the synthetic promoter. Partial synthetic promoters can be naturally occurring (e.g., a minimal promoter or one or more CREs in a promoter), but synthetic promoters as an entity do not occur naturally.

[0398] As used herein, a "minimal promoter" (also referred to as a "core promoter") refers to a generally short DNA fragment that is inactive or mostly 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 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 a transcription start site (TSS) and elements directly upstream, a binding site for RNA polymerase II, and a general transcription factor binding site (usually a TATA box). Minimal promoters may also include some elements downstream of the TSS, but these elements typically have little function in the absence of additional regulatory elements.

[0399] As used herein, a "proximal promoter" refers to a minimal promoter plus at least some additional regulatory sequences, typically a proximal sequence upstream of a gene that tends to contain primary regulatory elements. It typically extends approximately 250 base pairs upstream of the TSS and includes a specific TFBS. The proximal promoter may also include one or more regulatory elements downstream of the TSS, such as UTRs or introns. In the present case, suitably, the proximal promoter may be a naturally occurring muscle-specific, myocardial-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) proximal promoter, which may be combined with one or more CREs or CRMs of the present invention. However, the proximal promoter may be synthetic.

[0400] As used herein, "promoter element" refers to a minimal promoter or proximal promoter as defined above. In the context of the present invention, a promoter element is typically combined with a CRM or one or more CREs and optionally one or more additional regulatory elements to provide a synthetic muscle-specific, cardiac muscle-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) promoter of the present invention.

[0401] In the context of the present invention, a "functional variant" of a CRE, CRM, promoter element, promoter, or other regulatory nucleic acid is a variant of a reference sequence that retains the ability to function in the same manner as the reference sequence (e.g., as a muscle-specific, cardiac-specific (or cardiac-selective), skeletal muscle-specific (or skeletal-selective) CRE, a muscle-specific, cardiac-specific (or cardiac-selective), skeletal muscle-specific (or skeletal-selective) CRM, a muscle-specific, cardiac-specific (or cardiac-selective), skeletal muscle-specific (or skeletal-selective) promoter element, or a muscle-specific, cardiac-specific (or cardiac-selective), skeletal muscle-specific (or skeletal-selective) promoter. A "functional variant" of an additional regulatory element, detargeting element, liver detargeting element, or intron is a variant of a reference sequence that retains the ability to function in the same manner as the reference sequence (e.g., as an additional regulatory element, detargeting element, liver detargeting element, or intron). Alternative terms for such functional variants include "bioequivalent" or "equivalent."

[0402] It will be understood that the ability of a given CRE, CRM, promoter, or other regulatory sequence to function as a muscle-specific, cardiac-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) enhancer depends significantly on the ability of the sequence to bind to the same muscle-specific, cardiac-specific (or cardiac-selective), or skeletal muscle-specific (or skeletal-selective) TF that the reference sequence binds to. Thus, in most cases, a functional variant of a CRE, CRM, promoter, or other regulatory sequence will contain most or all of the same TFs as the reference CRE, CRM, promoter, or other regulatory sequence. Preferably, but not necessarily, the TFBS of the functional variant is in the same relative position (i.e., sequence and general position) as the reference CRE, CRM, promoter, or other regulatory sequence. It is also preferred, but not necessarily, that the TFBS of the functional variant is in the same orientation as the reference sequence (it should be noted that the TFBS can in some cases be present in reverse, for example, as a reverse complement vis-à-vis sequence in the reference sequence). It is also preferred, but not necessarily, that the TFBS of the functional variant is on the same chain as the reference sequence. Thus, in a preferred embodiment, the functional variant contains the same TF, in the same order, in the same position, in the same orientation, and on the same chain as the reference sequence. It will also be understood that the sequences located between the TFBS (in some cases referred to as spacer sequences, etc.) have less effect on the function of the CRE, CRM, promoter or other regulatory sequences. Such sequences can generally vary widely, and their lengths can vary. However, in preferred embodiments, the spacing in the functional variant (i.e., the distance between adjacent TFBSs) is substantially the same as the spacing in the reference sequence (e.g., it varies by no more than 20%, preferably no more than 10%, and more preferably is approximately the same). Obviously, in some cases, the functional variant of a CRE, CRM, promoter or other regulatory sequence can exist in the opposite orientation, for example, it can be the reverse complement of the above-mentioned CRE, CRM, promoter or other regulatory sequence, or a variant thereof.

[0403] The level of sequence identity between a functional variant and a reference sequence can also be an indicator of retained function. A high level of sequence identity within a CRE, CRM, or promoter TFBS is generally more important than sequence identity within a spacer sequence (where little or no sequence conservation is required). However, it should be understood that even within a TFBS, a considerable degree of sequence variation can be accommodated, as the sequence of a functional TFBS need not completely match the consensus sequence.

[0404] The ability of one or more TFs to bind to the TFBS in a given functional variant can be determined by any relevant means known in the art, including but not limited to electromobility shift assays (EMSAs), 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. Sambrook et al. (cited above) describe suitable methods. Many relevant articles describing this process are available, for example, Hellman and Fried, Nat Protoc. 2007; 2(8): 1849–1861.

[0405] "Muscle specificity" or "muscle-specific expression" refers to the ability of a cis-regulatory element, a cis-regulatory module, a promoter element or a promoter to enhance or drive gene expression in muscle cells (or muscle-derived cells) in a preferential or predominant manner 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, the expression in muscle cells is at least 75%, 80%, 85%, 90% or 95% compared to other cells. "Myocardial specificity," "cardiac selectivity" or "myocardial-specific expression" refers to the ability of a cis-regulatory element, a cis-regulatory module, a promoter element or a promoter to enhance or drive gene expression in myocardium in a preferential or predominant manner compared to other tissues (e.g., spleen, liver, lung and brain) and compared to skeletal muscle tissue. "Skeletal muscle specificity," "skeletal selectivity," 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 gene expression in skeletal muscle in a preferential or predominant manner compared to other tissues (e.g., spleen, liver, lung, and brain) and compared to cardiac muscle tissue. There may be situations where a lower degree of specificity is desired and is part of the present invention.

[0406] "CNS-specific" or "CNS-specific expression" refers to the ability of a promoter to enhance or drive the expression of a gene in cells of the central nervous system (CNS) (or cells of CNS origin) in a preferential or predominant manner compared to other tissues (e.g., liver, kidney, spleen, heart, lung, muscle, and brain). The expression of a gene can be in the form of mRNA or protein. In a preferred embodiment, CNS-specific expression is little expression in other (i.e., non-CNS) tissues or cells, i.e., the expression is highly CNS-specific. For example, expression in CNS cells is at least 75%, 80%, 85%, 90%, or 95% compared to other cells. There may be situations where a lower degree of specificity is desired and is part of the present invention.

[0407] "Kidney-specific" or "kidney-specific expression" refers to the ability of a promoter to enhance or drive the expression of a gene in kidney cells (or cells of kidney origin) in a preferred or predominant manner compared to other tissues (e.g., liver, CNS, spleen, heart, lung, muscle, and brain). The expression of the gene can be in the form of mRNA or protein. In a preferred embodiment, kidney-specific expression is little expression in other (i.e., non-kidney) tissues or cells, i.e., the expression is highly kidney-specific. For example, expression in kidney cells is at least 75%, 80%, 85%, 90%, or 95% compared to other cells. There may be situations where a lower degree of specificity is desired and is part of the present invention.

[0408] "Lung-specific" or "lung-specific expression" refers to the ability of a promoter to enhance or drive expression of a gene in lung cells (or cells of lung origin) in a preferential or predominant manner compared to other tissues (e.g., liver, CNS, spleen, heart, muscle, and brain). The expression of the gene can be in the form of mRNA or protein. In preferred embodiments, lung-specific expression is little expression in other (i.e., non-lung) tissues or cells, i.e., the expression is highly lung-specific. For example, expression in lung cells is at least 75%, 80%, 85%, 90%, or 95% compared to other cells. There may be situations where a lower degree of specificity is desired and is part of the present invention.

[0409] The skilled person can easily assess the ability of a CRE, CRM or promoter to function as muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) CRE, CRM or promoter. Therefore, the skilled person can easily determine whether any variant of the above-described specific CRE, CRM or promoter retains function (that is, it is a functional variant as defined above). For example, any given CRE or CRM to be assessed can be operably linked to a minimal promoter (e.g., located upstream of CMV-MP), and the ability of the CRE or CRM to drive the expression of muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) gene (typically a reporter gene) can be measured. Alternatively, the variant of a CRE or CRM can be substituted into a synthetic muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) promoter in place of a reference CRE or CRM, and the impact of the muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or skeletal-selective) expression driven by the modified promoter can be determined, and compared with an unmodified form. Similarly, the technician can easily assess the ability (for example, as described in the following examples) that promoter drives muscle-specific, cardiac muscle-specific (or heart selectivity) or skeletal muscle-specific (or bone selectivity) expression.The expression level of the gene driven by the variant of the reference promoter can be compared with the expression level driven by the reference promoter.In some embodiments, the muscle-specific, cardiac muscle-specific (or heart selectivity) or skeletal muscle-specific (or bone selectivity) expression level driven by the variant promoter is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the expression level driven by the reference promoter, so to speak, the variant retains function. Suitable nucleic acid constructs and reporter gene assays for assessing muscle-specific, cardiac muscle-specific (or heart selectivity) or skeletal muscle-specific (or bone selectivity) expression enhancement can be easily constructed, and the embodiments listed below provide suitable methodology.

[0410] Muscle-specific, myocardial-specific (or cardiac selectivity) or skeletal muscle-specific (or skeletal selectivity) can be identified, wherein the expression of gene (such as therapeutic or reporter gene) preferentially or predominantly occurs in cells (or muscle tissue) of muscle origin, cells (or cardiac tissue) of myocardial origin or cells (or skeletal tissue) of skeletal muscle origin.For example, preferential or predominant expression can be defined, wherein the expression level in the cell of muscle origin, myocardial origin or skeletal muscle origin is higher than, preferably significantly higher than other types of cells (i.e., cells of non-muscle origin, cells of non-myocardial origin or cells of non-skeletal muscle origin).For example, suitably, the expression in the cell of muscle origin, myocardial origin or skeletal muscle origin is higher than the expression in non-muscle cell, non-myocardial cell or non-skeletal muscle cell by at least 1.2 times, 1.5 times, 2 times, 4 times or 5 times, preferably at least higher than non-muscle cell, non-myocardial cell or non-skeletal muscle cell by at least 10 times, and in some cases may be higher by 50 times or more. For convenience, muscle-specific expression can be demonstrated by comparing the expression level in a muscle cell line (e.g., a muscle-derived cell line such as C2C12 or H2K cells (skeletal muscle) or H9C2 cells (heart)) with the expression level in a liver-derived cell line (e.g., Huh7 or HepG2), a kidney-derived cell line (e.g., HEK-293), a cervical tissue-derived cell line (e.g., HeLa), and / or a lung-derived cell line (e.g., A549). Cardiac-specific or cardiac-selective expression can be demonstrated by comparing the expression level in a cardiomyocyte cell line (e.g., a cardiomyocyte-derived cell line such as H9C2) or primary cardiomyocytes with the expression level 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 line (e.g., C2C12 or H2K). Suitably, skeletal muscle-specific or bone-selective expression can be demonstrated by comparing 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).

[0411] Preferably, the synthetic muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) promoter of the present invention shows reduced expression in cells of non-muscle origin (suitably in Huh7, HEK-293, HeLa and / or A549 cells) when compared with non-tissue-specific promoters such as CMV-IE. Preferably, the synthetic muscle-specific, cardiac-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) promoter of the present invention has 50% or less activity of CMV-IE promoter in cells of non-muscle origin (suitably in Huh7, HEK-293, HeLa and / or A549 cells), suitably 25% or less, 20% or less, 15% or less, 10% or less, 5% or less or 1% or less activity. Typically, it is preferred that the expression in cells of non-muscle origin be minimized, but in some cases, this may not be necessary. Even if a synthetic promoter of the invention has higher expression in, for example, one or two non-muscle cells, it can still be a muscle-specific promoter as long as it generally has higher expression overall in a range of muscle cells relative to non-muscle cells. In some embodiments, a muscle-specific promoter expresses a gene at least 25%, or at least 35%, or at least 45%, or at least 55%, or at least 65%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or any integer between 25% and 95% higher in muscle cells than in non-muscle cells.

[0412] The synthetic muscle-specific promoters of the present invention are preferably suitable for promoting expression in the muscle of a subject, e.g., driving muscle-specific expression of a transgene, preferably muscle-specific expression of a therapeutic transgene. The synthetic cardiac muscle-specific or cardiac-selective promoters of the present invention are preferably suitable for promoting expression in the heart of a subject, e.g., driving cardiac muscle-specific or cardiac-selective expression of a transgene, preferably cardiac muscle-specific expression of a therapeutic transgene. The synthetic skeletal muscle-specific or skeletal-selective promoters of the present invention are preferably suitable for promoting expression in the skeletal muscle of a subject, e.g., driving skeletal muscle-specific, skeletal-selective expression of a transgene, preferably skeletal muscle-specific expression of a therapeutic transgene. Preferred synthetic muscle-specific promoters of the present invention are suitable for promoting muscle-specific transgene expression and have an activity in muscle cells of at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 105%, 110%, 115%, 120%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity of the CBA, spc5-1, CK7, or CK8 promoters. In some embodiments, the synthetic muscle-specific promoters of the present invention are suitable for promoting muscle-specific transgene expression at a level of at least 100% of the activity of the CBA, spc5-1, CK7, or CK8 promoters, preferably 150%, 200%, 300%, or 500% of the activity of the CBA, spc5-12, CK7, or CK8 promoters. In some embodiments, the synthetic cardiac-specific or cardiac-selective promoters of the invention are suitable for promoting cardiac-specific or cardiac-selective transgene expression at a level of at least 100% of the activity of the Tnnt2 or My12 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the Tnnt2 or My12 promoter. In some embodiments, the synthetic skeletal muscle-specific or bone-selective promoters of the invention are suitable for promoting skeletal muscle-specific or bone-selective transgene expression at a level of at least 100% of the activity of the Tnnt2 or My12 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the spc5-12 promoter. Suitably, such muscle-specific expression is determined in cells of muscle origin, such as C2C12 or H2K cells (skeletal muscle) or H9C2 cells (heart), or primary muscle cells, suitably primary human myocytes.Himeda, CL, Chen, https: / / doi.org / 10.1007 / 978-1-61737-982-6_1 The CK8 promoter is disclosed in .

[0413] The synthetic muscle-specific, cardiac muscle-specific (or cardiac-selective) or skeletal muscle-specific (or bone-selective) promoters of the present invention are also capable of promoting muscle-specific, cardiac muscle-specific (or cardiac-specific) or skeletal muscle-specific (or bone-selective) expression of genes in muscle-derived cells (e.g., C2C12 or H2K cells (skeletal muscle) or H9C2 cells (heart)) at a level of at least 50%, 100%, 150% or 200% compared to CMV-IE.

[0414] As used herein, the term "nucleic acid" generally refers to an oligomer or polymer (preferably a linear polymer) of any length that is essentially composed of nucleotides. Nucleotide units generally include a heterocyclic base, a sugar group and at least one (e.g., one, two, or three) phosphate group, including modified or substituted phosphate groups. Heterocyclic bases can especially include 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), and chemically or biochemically modified (e.g., methylated), non-natural or derived bases. Sugar groups can especially include pentose (pentofuranosyl) groups, such as ribose and / or 2-deoxyribose, or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as referred to herein can include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. The modified nucleotides can include modified heterocyclic bases, modified sugar moieties, modified phosphate groups or combinations thereof. Modifications of phosphate groups or sugars can be introduced to improve stability, resistance to enzymatic degradation or some other useful properties. The term "nucleic acid" further preferably includes DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides and synthetic (e.g., chemically synthesized) DNA, RNA or DNA RNA hybrids. Nucleic acids can be naturally occurring, for example, present in nature or isolated from nature; or they can be non-naturally occurring, for example, recombinant, i.e., produced by recombinant DNA technology, and / or partially or completely chemically or biochemically synthesized. "Nucleic acid" can be double-stranded, partially double-stranded or single-stranded. In the case of single-stranded, nucleic acid can be a sense strand or an antisense strand. In addition, nucleic acid can be circular or linear.

[0415] The term "isolated" when referring to a nucleic acid refers to a nucleic acid molecule that is completely or partially lacking sequences with which it is normally associated in nature; or a sequence that, while present in nature, has heterologous sequences associated with it; or a molecule that is separated from a chromosome.

[0416] The terms "identity" and "identical" and the like refer to the sequence similarity between two polymeric molecules, for example between two nucleic acid molecules, such as between two DNA molecules. Sequence alignments and determination of sequence identity can be performed, for example, using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215:403-10), for example the "Blast 2 Sequence" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250).

[0417] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol.Biol.24:307-31; Tatiana et al. (1999) FEMS Microbiol.Lett.174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol.Biol.215:403-10.

[0418] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST TM ; Altschul et al. (1990)) are available from a number of sources, including the National Center for Biotechnology Information (Bethesda, MD) and the Internet for use with a number of sequence analysis programs. BLAST on the Internet TM Instructions on how to use this program to determine sequence identity are provided under the Help section of the program. For comparison of nucleic acid sequences, BLAST can be used. TMThe "Blast 2 Sequences" function of the (Blastn) program uses default parameters. When evaluated by this method, nucleic acid sequences with greater similarity to the reference sequence will show a higher percent identity. Typically, the sequence identity percentage is calculated over the entire sequence length.

[0419] For example, suitably the global optimal alignment is found by the Needleman-Wunsch algorithm using the following scoring parameters: match score: +2, mismatch score: -3; gap penalty: gap open 5, gap extension 2. Suitably the percent identity of the resulting best global alignment is calculated as the ratio of the number of bases in the alignment to the total length of the alignment, wherein the length of the alignment includes matches and mismatches, multiplied by 100.

[0420] The term "hybridization" refers to the annealing of two at least partially complementary nucleotide sequences during the hybridization process. In order for hybridization to occur, complementary nucleic acid molecules are typically denatured thermally or chemically to melt the double strand into two single strands and / or to remove hairpins or other secondary structures from the single stranded nucleic acid. The stringency of hybridization is affected 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, 3 rd

[0014] The present invention is described in

[0015] Cold Spring Harbor Laboratory Press, CSH, New York, but the skilled artisan will appreciate that many different hybridization conditions can be designed based on the homology and / or length of known or anticipated nucleic acid sequences. High stringency conditions for hybridization include high temperature and / or low sodium / salt concentration (salts include sodium, such as in sodium chloride and sodium citrate) and / or the inclusion of formamide in the hybridization buffer and / or reducing the concentration of compounds such as SDS (sodium dodecyl sulfate detergent) in the hybridization buffer and / or excluding compounds such as dextran sulfate or polyethylene glycol (which promote molecular aggregation) from the hybridization buffer. As a non-limiting example, representative salt and temperature conditions for stringent hybridization are: 1x SSC, 0.5% SDS, 65°C. The abbreviation SSC refers to the buffer used in nucleic acid hybridization solutions. One liter of 20X (20 times concentrated) stock SSC buffer (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate. A representative time period for achieving hybridization is 12 hours.

[0421] The term "transcription factor binding site" (TFBS) is well known in the art. It will be apparent to the skilled artisan that alternative TFBS sequences may be used, as long as they are bound by the intended TF. The consensus sequences of various TFBS are known in the art, and the skilled artisan can readily use this information to determine alternative TFBSs. Furthermore, the ability of a TF to bind to a given putative sequence can be readily determined experimentally by the skilled artisan (e.g., by EMSA and other methods well known in the art and discussed herein).

[0422] The meaning of "consensus sequence" is well known in the art. In this application, unless the context indicates otherwise, the following symbols are used for consensus sequences. Consider the following exemplary DNA sequence:

[0423] A[CT]N{A}YR

[0424] A means A is always found at that position; [CT] means C or T at that position; N means any base at that position; and {A} means any base is found at that position except A. Y represents any pyrimidine, and R represents any purine.

[0425] "Synthetic" in this application refers to nucleic acid molecules that do not exist in nature. The synthetic nucleic acids of the present invention are artificially produced, usually by recombinant technology or de novo synthesis. Such synthetic nucleic acids can contain naturally occurring sequences (e.g., promoters, enhancers, introns and other such regulatory sequences), but these are present in non-naturally occurring environments. For example, a synthetic gene (or a portion of a gene) typically contains one or more nucleic acid sequences that are discontinuous in nature (chimeric sequences), and / or can encompass substitutions, insertions and deletions and combinations thereof.

[0426] As used herein, "complementarity" or "complementarity" refers to the 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," where only some bases bind to their complements, or it can be complete, where every base in the sequence binds to its complementary base. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands.

[0427] As used herein, "transfection" refers broadly to any process by which nucleic acid is intentionally introduced into a cell, including introduction by viral and non-viral vectors, and includes or is equivalent to terms and processes such as transformation and transduction. 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 microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0428] 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 pharmaceutically useful compound, or a gene that encodes a desired trait. In another example, the transgene encodes a useful nucleic acid, such as an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. The transgene preferably encodes a therapeutic product, such as a protein.

[0429] The term "vector" is well known in the art and, as used herein, refers to a nucleic acid molecule, such as double-stranded DNA, which may have been inserted into a nucleic acid sequence according to the present invention. The vector is suitable for transporting the inserted nucleic acid molecule into a suitable host cell. The vector generally contains all the necessary elements that allow the inserted nucleic acid molecule to be transcribed and preferably the transcript to be translated into a polypeptide. The vector generally contains all the necessary elements so that once the vector enters the host cell, the vector can replicate independently of the host chromosomal DNA, or replicate simultaneously with the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule can be produced. The vector of the present invention can be an episomal vector (i.e., not integrated into the host cell genome), or it can be a vector that is integrated into the host cell genome. This definition includes 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 that do not contain bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC) or humans (HAC)) can be used to accommodate larger inserts. Viral vectors are derived from viruses, including but not limited to retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis virus vectors, etc. Typically, but not necessarily, viral vectors are replication-defective because they have lost the ability to reproduce in a given cell because the viral genes necessary for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as a cancer cell, and are typically used to induce (cancer) cell-specific (tumor) lysis. Virosomes are non-limiting examples of vectors comprising viral and non-viral elements, particularly they combine liposomes with inactivated HIV or influenza viruses (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0430] As used herein, the term "operably connected" or equivalent expression refers to the arrangement of various nucleic acid elements relative to each other so that the elements are functionally connected and can interact with each other in an expected manner. Such elements can include, but are not limited to, promoters, CREs (such as enhancers or other regulatory elements), detargeting elements (such as liver detargeting elements), promoter elements, polyadenylation sequences, one or more introns and / or exons, one or more UTRs, and the coding sequence of the gene of interest to be expressed. When correctly oriented or operably connected, nucleic acid sequence elements act together to regulate each other's activity and may ultimately affect the expression level of the expression product. Regulating refers to increasing, reducing, or maintaining the activity level of a particular element. The position of each element relative to other elements can be represented by the 5' end and 3' end of each element or their position upstream or downstream of another element or position (such as TSS or promoter element), and the distance between any particular element can be referenced by the number of inserted nucleotides or base pairs between the elements. As understood by the skilled artisan, being operably connected means functional activity and is not necessarily related to being connected in natural position. In practice, when used in a nucleic acid expression cassette, a CRE will typically be located immediately upstream of a promoter element (although this is often the case, it should never be interpreted as limiting or excluding the location within the nucleic acid expression cassette). However, this is not necessarily the case in vivo. For example, when located upstream of a promoter, a regulatory element sequence naturally present downstream of a gene can function in the same manner, affecting the transcription of the gene. Thus, according to a specific embodiment, the regulatory or enhancing effect of a regulatory element can be position-independent.

[0431] As used herein, a "spacer sequence" or "spacer" is a nucleic acid sequence that separates two functional nucleic acid sequences (e.g., TFBS, CRE, CRM, promoter element, etc.). It can essentially have any sequence, as long as it does not prevent the functional nucleic acid sequence (e.g., cis-regulatory element) from functioning as desired (e.g., if it includes a silencer sequence, prevents the binding of a desired transcription factor, etc., this may occur). Typically, it is non-functional because its presence is only to separate adjacent functional nucleic acid sequences from each other. In some embodiments, the spacer can have a length of 75, 50, 40, 30, 20, or 10 nucleotides or less.

[0432] As used herein, the term "pharmaceutically acceptable" is consistent with the art and means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0433] "Therapeutically effective amount" and similar phrases refer to a dosage or plasma concentration that provides a desired specific pharmacological effect (e.g., expression of a therapeutic gene in muscle) in a subject. A therapeutically effective amount may not always be effective in treating the conditions described herein, even if such a dosage is considered a therapeutically effective amount by one 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.

[0434] As used herein, the term "AAV vector" is well known in the art and generally refers to an AAV vector nucleic acid sequence comprising various nucleic acid sequences. As used herein, an AAV vector typically comprises a heterologous nucleic acid sequence of a non-AAV origin as part of the vector. The heterologous nucleic acid sequence typically comprises a promoter disclosed herein and other sequences of interest for genetic transformation of cells. Typically, the flank of the heterologous nucleic acid sequence is at least one, typically two, AAV inverted terminal repeats (ITRs).

[0435] "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid polypeptide (including variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an encapsidated polynucleotide AAV vector. If the particle contains a 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 "AAV vector." Therefore, the production of AAV virions or AAV particles necessarily includes the production of AAV vectors, because such vectors are contained in AAV virions or AAV particles.

[0436] "Small interfering" or "short interfering RNA" or siRNA is an RNA duplex of nucleotides that targets 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 the gene and the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, the length of 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 some embodiments, the length of the duplex is 19-25 nucleotides. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can include a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may further comprise a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0437] The term "treat" refers to the reduction, amelioration, or elimination of one or more signs, symptoms, or effects of a disease or condition. Thus, as used herein, "treat" includes any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the disease from occurring in a subject susceptible to or at risk of developing the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0438] "Administering" an agent to a subject includes any route of introducing or delivering the agent to the subject to perform its intended function. Administration can be by any suitable route, including oral, intranasal, intraocular, ocular, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another person. Intramuscular administration is of particular interest in the present invention.

[0439] The terms "individual," "subject," and "patient" are used interchangeably and refer to any individual subject having a disease or condition for which treatment is desired. For the 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, among others.

[0440] As used herein, the term "gene therapy vector" refers to a vector as described above that is suitable for or intended for use in gene therapy. A gene therapy vector typically comprises a promoter operably linked to a therapeutically useful expression product (e.g., a transgene that can be used to treat a disease or condition).

[0441] As used herein, the term "detargeting element" refers to a nucleic acid sequence that, when added to a CRM, synthetic promoter, expression cassette or vector, is intended to or is capable of reducing the expression of the CRM, synthetic promoter, expression cassette or vector in a specific tissue or cell. Examples of de-targeting elements can be found in the following literature: Kopp F, Schnoedt M, Haase R, Wagner E, Roidl A, Ogris M. De-targeting by miR-143 decreases unwanted transgene expression in non-tumorigenic cells. Gene Ther. 2013 Nov; 20(11): 1104-9. doi: 10.1038 / gt.2013.37. Epub 2013 Jun 27. PMID: 23804075, Dhungel B, Ramlogan-Steel CA, Layton CJ, Steel JC. MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma. Mol Ther Nucleic Acids. 2018 Dec 7;13:78-88.doi:10.1016 / j.omtn.2018.08.016.Epub 2018Aug 24.PMID:30245470;PMCID:PMC6148835 (especially Figure 2 and Materials and Methods) and Dhungel, B., Ramlogan-Steel, CA & Steel, JC Synergistic and independent action of endogenous microRNAs 122a and 199a for post-transcriptional liver detargeting of gene vectors. Sci Rep 8, 15539 (2018). https: / / doi.org / 10.1038 / s41598-018-33801-4 (especially Figure 2), Juliette Hordeaux, Elizabeth L. Buza, Brianne Jeffrey, Chunjuan Song, Tahsin Jahan and Yuan Yuan, Yanqing Zhu, Peter Bell, Mingyao Li, Jessica A. Chichester, Roberto Calcedo and James M. Wilson, MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates, Science Translational Medicine, Volume 12, 569, 2020, doi 10.1126 / scitranslmed.aba9188, which are incorporated herein by reference. The detargeting element can be a binding site for a protein or miRNA that is highly expressed in a specific cell or tissue. In some embodiments, the detargeting element can be a liver detargeting element. Adding a liver detargeting element to a CRM, synthetic promoter, expression cassette or vector may reduce the expression of the CRM, synthetic promoter, expression cassette or vector in liver tissue or cells.

[0442] As used herein, the term "additional regulatory elements" refers to nucleic acid sequences that can be added to a CRM or synthetic promoter. Adding additional regulatory elements is expected to regulate the expression profile of a CRM or synthetic promoter (i.e., increase, decrease, or maintain the activity level in any particular cell type or tissue) without rendering the CRM or synthetic promoter essentially nonfunctional. Additional regulatory elements may need to be added to increase or decrease the activity level in the desired one or more tissues. For example, adding the tMCK SA / SD intron (SEQ ID NO: 29) to SP0524 (synthetic muscle-specific promoter SP0527) would increase activity in the heart. The additional regulatory elements may be selected from: CRE, CRM, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, detargeting elements, liver detargeting elements, introns, UTRs, and splicing elements. The term "abnormal gene expression" refers to gene expression associated with a disease or pathology. Abnormal gene expression may be caused by gene or mRNA mutations.

[0443] The invention will now be described with reference to the following non-limiting examples:

[0444] Example 1 - In vivo data

[0445] The strength of synthetic muscle-specific promoters according to embodiments of the present invention was tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase.

[0446] Materials and methods

[0447] Synthetic muscle-specific promoters were selected for in vivo testing.

[0448] In vivo experiments

[0449] AAV containing a synthetic promoter (e.g., SP0527) operably linked to luciferase was diluted in 0.9% saline and injected via the tail vein at 1 e per mouse. 11 A dose of 500 vg / 200 μl (6 mice per group) was delivered to 8-week-old male Balb / c mice. Mice were sacrificed 6 weeks after injection using the protocol 1 method. AAV9 was used.

[0450] Six weeks after injection, diaphragm (skeletal muscle), heart (myocardial muscle), gastrocnemius (skeletal muscle), soleus (skeletal muscle), tibialis anterior (TA) (skeletal muscle) and liver were collected from each mouse. The following tissues were collected for IHC: heart, TA, soleus, liver, gastrocnemius. For luciferase (Luc) expression and vector copy number (VCN) analysis, all samples were snap-frozen in liquid nitrogen immediately after dissection and then moved to dry ice before storage at -80°C. Tissues used for IHC were fixed on cork, slowly frozen in isopentane on liquid nitrogen, and then moved to dry ice. The gastrocnemius muscle of mice includes fast muscle fibers (about 80%) and slow muscle fibers (about 20%).

[0451] Real-time imaging

[0452] At 4 weeks post-injection, all 6 mice in each group were imaged to examine luciferase expression. For imaging, the Spectra Lumina III Series In Vivo Imaging System. IVIS machines detect bioluminescence by imaging the light emitted by enzyme-catalyzed reactions, thereby reporting activity at the molecular level. In cell biology and small animal research, bioluminescent reporters require a small chemical substrate for non-invasive imaging. This protocol specifically requires IP injection of D-luciferin and induction of anesthesia with isoflurane. The imaging procedure is as follows:

[0453] A stock solution of D-luciferin potassium salt (as substrate) was prepared by using luciferin from (cat. no. MB000102, Syd Labs, USA) lot no. Ro405-017 and (cat. no. LUCK-1G, GoldBio, USA) lot no. 016067LUCK. For reconstitution, PBS from Gibco cat. no. 14190-094, lot no. 2241142 was used.

[0454] - Prepare 15 mg / ml (W / V) fluorescein stock solution (1500 mg / 100 ml PBS), aliquot, label, wrap in foil to protect from light, and store in a -20 / -80°C freezer.

[0455] - D-luciferin substrate was administered intraperitoneally to mice at 300 μl / mouse of luciferin stock solution.

[0456] -Anesthetic agent mixer filled with isoflurane / isoflurane.

[0457] - Move the mouse into the anesthesia induction chamber.

[0458] - Oxygen supply was set at 2.5 L / min, and mice were observed as they were about to fall asleep.

[0459] - Once the mice are under anesthesia, during a 5-minute countdown, move the mice from the induction chamber to the bioimaging IVIS chamber in the order corresponding to the animal experimental number (group).

[0460] -Capture an image.

[0461] - Comparison of luciferase expression between the upper half of the animal (including the heart, diaphragm, and liver) and the lower half of the animal (including the soleus, tibialis anterior, and gastrocnemius muscles).

[0462] Tissue homogenization and lysis

[0463] Tissues stored at -80°C were thawed at room temperature. Reporter gene lysis buffer (Promega, catalog number #E4030) was pipetted into each tube containing sample tissue. 1-2 Qiagen carbide beads (Qiagen, catalog number #69997) were added to each tube. The tube was placed in a Qiagen tissue lyser II (QIAGEN, catalog number #85220) and homogenized at a frequency of 25.0 Hz for 75 seconds. After 75 seconds, the adapter was rotated and the sample was further homogenized for 75 seconds. The sample was then quickly frozen at -80°C for 10-15 minutes and subsequently thawed at 37°C for 10 minutes using a dry heat bath system. The tube was centrifuged at 10,000xg for 3 minutes. The supernatant was transferred to a new 1.5 ml Eppendorf tube without disturbing the precipitate. The above steps were repeated for a second time by adding additional reporter gene lysis buffer to the precipitated tissue. The supernatant from the repeated process was added to the same 1.5 ml Eppendorf tube. The tube was vortexed thoroughly and stored at -80°C.

[0464] Measurement of luciferase activity

[0465] - Luciferase activity was measured using Promega's Luciferase Assay System (Promega, catalog # E4550).

[0466] - Working luciferase (LAR) reagent solution was thawed at room temperature 30 minutes before use, while sample tissue lysates were thawed on ice before use.

[0467] - Dilute the sample of interest 1:10 in a new 1.5 ml Eppendorf tube.

[0468] - 10 μl of diluted samples of interest and negative controls (reporter lysis buffer) were manually pipetted into 96-well flat bottom solid white plates in triplicate and luminescence (RLU) was measured by injecting 50 μl of LAR into each well on a BMG Fluostar plate reader.

[0469] -Luciferase expression (RLU per μg / μL) was calculated by normalizing relative light units to protein concentration.Data are presented as mean ± standard deviation using GraphPad Prism 9.

[0470] Protein quantification

[0471] Protein was extracted from the collected tissues and quantified using the BCA Pierce Protein Assay Kit (ThermoFisher 23225). The manufacturer's instructions are as follows. Bovine serum albumin (BSA) standards were prepared by diluting albumin to a working range of 20-20,000 μg / ml. An appropriate volume of BCA working reagent (WR) was prepared by mixing 50 parts BCA reagent A and 1 part BCA reagent B (50:1, reagent A:B). The microplate procedure was followed (the sample to WR ratio was 1:8):

[0472] - Samples were diluted 1:10 in reporter lysis buffer (1x)

[0473] - Pipette 10 μL of each standard or unknown sample into the wells of a 96-well plate in duplicate

[0474] - Add 200 μL of WR to each well and place the plate on a plate shaker for 30 seconds to promote mixing

[0475] - Cover the plate and incubate at 37°C for 30 minutes

[0476] - Cool the plate to room temperature and measure the absorbance at 562 nm on a BMG Fluostar plate reader

[0477] Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm absorbance measurements of all other BSA standard replicates and sample replicates. Prepare a standard curve by plotting the average blank-corrected 562 nm measurement of each BSA standard against its concentration (μg / ml). Use the standard curve to determine the protein concentration of each sample.

[0478] Vector copy number

[0479] The vector copy number was determined by duplex Taqman qPCR. DNA was extracted using the Blood and Tissue Kit (250) (QIAGEN, catalog #69506). Taqman qPCR was performed on each sample using luciferase and GAPDH specific primer and probe sets:

[0480]

[0481] Standard curves were used for Luc and GAPDH analysis. In the multiplex qPCR protocol, the following final concentrations of reagents and DNA were used: 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 cycling protocol was as follows: 95°C for 20 seconds, PCR: 40 cycles, 95°C for 1 second, 60°C for 20 seconds. ΔΔCt VCN (amount) was calculated for each genome by subtracting the average VCN (amount) for each genome of the saline sample (start).

[0482] result

[0483] SP0525 drives high expression in cardiac muscle (heart) and high expression in skeletal muscle (e.g., gastrocnemius, soleus, tibialis anterior). SP0525 drives lower expression in the liver. Figure 3 Displayed in.

[0484] SP0526 drives high expression in cardiac muscle (heart) and high expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0526 drives lower expression in the liver. Figure 4 Displayed in.

[0485] SP0527 drives high expression in cardiac muscle (heart) and high expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0527 drives lower expression in the liver. Figure 5 Displayed in.

[0486] SP0528 drives high expression in cardiac muscle (heart) and high expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0528 drives lower expression in the liver. Figure 6 Displayed in.

[0487] like Figure 7 As shown, expression cassette 529 drives high expression in cardiac muscle (heart). Expression cassette 529 also drives expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm, and soleus), but this is lower than expression in cardiac muscle (heart). Expression cassette 529 drives low expression in liver. This is Figure 7 Displayed in.

[0488] like Figure 8As shown, the synthetic muscle-specific promoter SP0530 drives high expression in cardiac muscle (heart). The synthetic muscle-specific promoter SP0530 also drives expression in skeletal muscle (such as gastrocnemius, tibialis anterior, diaphragm and soleus), but this is lower than the expression in cardiac muscle (heart). The synthetic muscle-specific promoter SP0530 drives low expression in the liver. It is noteworthy that, compared with the original design SP0524 (SP0530 is SP0524+tMCK SA / SD intron), adding tMCK SA / SD intron (SEQ ID NO:29) seems to cause increased expression in the heart.

[0489] like Figure 9 As shown, the synthetic muscle-specific promoter SP0531 drives high expression in cardiac muscle (heart) and skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm, and soleus). The synthetic muscle-specific promoter SP0531 drives low expression in the liver.

[0490] Background (saline) expression in Figure 10 Displayed in. Figure 11 and 12 It was shown that the vector copy number was similar between the different tested promoters and expression cassette 529 in the heart and liver, respectively.

[0491] In vivo imaging of mice injected with AAV containing a synthetic promoter (e.g., SP0525) operably linked to luciferase Figure 13 Displayed in.

[0492] Example 2 - Further in vivo studies

[0493] The strength of synthetic muscle-specific promoters according to embodiments of the present invention was tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase.

[0494] Materials and methods

[0495] Synthetic muscle-specific promoters were selected for in vivo testing at various doses.

[0496] In vivo experiments

[0497] AAV containing a synthetic promoter (e.g., SP0525) operably linked to luciferase was diluted in 0.9% saline and delivered to 8-week-old male Balb / c mice via tail vein injection at three doses: 1 e per mouse. 11 vg / 200μl (6 mice per group), 6e per mouse 11 vg / 200ul (5 or 6 mice per group), 1.2e per mouse 12vg / 200ul (6 mice per group). Mice were sacrificed 6 weeks after injection using the protocol 1 method. AAV9 was used.

[0498] At 6 weeks after injection, diaphragm (skeletal muscle), heart (myocardial muscle), gastrocnemius (skeletal muscle), soleus (skeletal muscle), tibialis anterior (TA) (skeletal muscle), quadriceps (skeletal muscle), psoas (skeletal muscle) and liver were collected from each mouse at all doses and saline. 12 vg / 200ul) and saline only, the following tissues were additionally collected: brain, kidney, spleen, lymph node, gall bladder, colon, adrenal gland, lung and testis. The following tissues were collected for IHC: TA, soleus, heart, quadriceps, psoas and gastrocnemius. For luciferase (Luc) expression and vector copy number (VCN) analysis, all samples were snap-frozen in liquid nitrogen immediately after dissection and then moved to dry ice before storage at -80°C. Tissues used for IHC were fixed on cork, slowly frozen in isopentane on liquid nitrogen, and then moved to dry ice. The gastrocnemius muscle of mice consists of fast muscle fibers (approximately 80%) and slow muscle fibers (approximately 20%).

[0499] Tissue homogenization and lysis

[0500] Tissues stored at -80°C were thawed at room temperature. 1x reporter gene lysis buffer (RLB) (Promega, catalog number #E4030) was pipetted into each tube containing sample tissue. 1-2 Qiagen carbide beads (Qiagen, catalog number #69997) were added to each tube. The tube was placed in a Qiagen tissue lyser II (QIAGEN, catalog number #85220) and homogenized at a frequency of 25.0 Hz for 75 seconds. After 75 seconds, the joint was rotated and the sample was further homogenized for 75 seconds. The sample was then quickly frozen at -80°C for 10-15 minutes and subsequently thawed at 37°C for 10 minutes using a dry heat bath system. The tube was centrifuged at 10,000xg for 3 minutes. The supernatant was transferred to a new 1.5 ml Eppendorf tube without disturbing the precipitate. The above steps were repeated for a second time by adding additional reporter gene lysis buffer to the precipitated tissue. The supernatant produced by the repeated process was added to the same 1.5 ml Eppendorf tube. The tubes were vortexed thoroughly and stored at -80 °C.

[0501] Measurement of luciferase activity

[0502] - Luciferase activity was measured using Promega's Luciferase Assay System (Promega, catalog # E4550).

[0503] - Working luciferase (LAR) reagent solution was thawed at room temperature 30 minutes before use, while sample tissue lysates were thawed on ice before use.

[0504] - Dilute the sample of interest 1:10 in a new 1.5 ml Eppendorf tube.

[0505] - 10 μl of diluted samples of interest and negative controls (reporter lysis buffer) were manually pipetted into 96-well flat bottom solid white plates in triplicate and luminescence (RLU) was measured by injecting 50 μl of LAR into each well on a BMG Fluostar plate reader.

[0506] -Luciferase expression (RLU per μg / μL) was calculated by normalizing relative light units to protein concentration.Data are presented as mean ± standard deviation using GraphPad Prism 9.

[0507] Protein quantification

[0508] Protein was extracted from the collected tissues and quantified using the BCA Pierce Protein Assay Kit (ThermoFisher #23225). The manufacturer's instructions are as follows. Bovine serum albumin (BSA) standards were prepared by diluting albumin to a working range of 20-20,000 μg / ml. An appropriate volume of BCA working reagent (WR) was prepared by mixing 50 parts BCA reagent A and 1 part BCA reagent B (50:1, reagent A:B). The microplate procedure was followed (sample to WR ratio was 1:8):

[0509] - Samples were diluted 1:10 in reporter lysis buffer (1x)

[0510] - Pipette 10 μL of each standard or unknown sample into the wells of a 96-well plate in duplicate

[0511] - Add 200 μL of WR to each well and place the plate on a plate shaker for 30 seconds to promote mixing

[0512] - Cover the plate and incubate at 37°C for 30 minutes

[0513] - Cool the plate to room temperature and measure the absorbance at 562 nm on a BMG Fluostar plate reader

[0514] Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm absorbance measurements of all other BSA standard replicates and sample replicates. Prepare a standard curve by plotting the average blank-corrected 562 nm measurement of each BSA standard against its concentration (μg / ml). Use the standard curve to determine the protein concentration of each sample.

[0515] Vector copy number

[0516] The vector copy number was determined by duplex Taqman qPCR. DNA was extracted using the automated Maxwell RSC tissue DNA kit (Promega #AS1610). Multiplex Taqman qPCR was performed on each sample using the following primers: Fast Advanced Master Mix (#4444557, ThermoFisher) with a luciferase-specific primer and probe set for the target and a GAPDH or HPRT1-specific primer and probe set for the housekeeping gene:

[0517]

[0518] Standard curves were used for Luc and GAPDH analysis. In the multiplex qPCR protocol, the following final concentrations of reagents and DNA were used: 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 cycling protocol was as follows: 95°C for 20 seconds, PCR: 40 cycles, 95°C for 1 second, 60°C for 20 seconds. ΔΔCt VCN (amount) was calculated for each genome by subtracting the average VCN (amount) for each genome of the saline sample (start).

[0519] mRNA

[0520] RNA was isolated using the Maxwell simplyRNA tissue kit (Promega #AS1340) according to the manufacturer's instructions. The isolated RNA was treated with DNase I and reverse transcribed into its complementary cDNA using Superscript III reverse transcriptase (Life technologies #18418020). Amplification was performed using Fast Advanced Master Mix (#4444557, ThermoFisher) on a Life Technology QuantStudio 7. The primer and probe sets used are detailed in the previous table.

[0521] result

[0522] Expression of CK7 (low and medium doses) in different muscle tissues and non-muscle tissues (liver) Figure 14 and 15 The expression of SP0527 (low, medium and high doses) in different muscle tissues and non-muscle tissues (liver) is shown in Figure 16-18 SP0527 drives high expression in cardiac muscle (heart) and high expression in skeletal muscle (e.g., gastrocnemius, tibialis anterior, diaphragm). SP0527 drives lower expression in the liver.

[0523] Background (saline) expression in Figure 19 Displayed in. Figure 20 The vector copy numbers of CK7 and SP0527 in the liver at different doses are shown. A dose-dependent pattern can be seen in this figure, with lower doses having lower vector copy numbers and higher doses having higher vector copy numbers.

[0524] Figure 21 The expression of SP0527 (high dose) in muscle tissue and various non-muscle tissues such as brain, liver, lung, colon, spleen, testis and kidney is shown. SP0527 drives high expression in muscle tissue and lower expression in non-muscle tissues such as brain, liver, lung, colon, spleen, testis and kidney. SP0527 showed low expression in all non-muscle tissues tested. Background (saline) expression was Figure 22 Displayed in.

[0525] Example 3 - Liver detargeting

[0526] Synthetic promoters SP0525-SP0528 were tested by operably linking each synthetic muscle-specific promoter to the reporter gene luciferase. Expression cassette 529 was also tested by replacing the expression product (eg, gene of interest) with the reporter gene luciferase.

[0527] In vivo experiments

[0528] AAV containing a synthetic promoter (e.g., SP0525) or expression cassette 529 operably linked to luciferase was diluted in 0.9% saline and delivered to 8-week-old male Balb / c mice via tail vein injection at different doses. The doses included 1 e per mouse. 11 vg / 200μl, 6e 11 vg / 200μl, 1e 12 vg / 200μl, 6e 12 vg / 200μl, 1e 13 vg / 200μl and 6e 13 vg / 200 μl (6 mice per group). Mice were sacrificed 6 weeks after injection using the protocol 1 method. AAV9 was used. Tissues were collected and processed as detailed in Example 1.

[0529] SP0525, SP0526, SP0527, SP0528, and expression cassette 529 showed muscle-specific activity at all doses tested. The expression profile of SP0525 in different muscle tissues was similar to that of Figure 3 The expression profiles of SP0526 in different muscle tissues were similar at all tested doses. Figure 4 The expression profiles of SP0527 in different muscle tissues were similar at all tested doses. Figure 5 The expression profiles of SP0528 in different muscle tissues were similar at all tested doses. Figure 6 At all tested doses, the expression profiles of expression cassette 529 in different muscle tissues were similar to those of Figure 7 Expression profiles shown.

[0530] In 6e 12 At a dose of 100 vg / 200 μl, SP0525, SP0526, SP0527, SP0528, and expression cassette 529 showed lower activity in the liver than CK7 and / or CK8. 13 At a dose of 6e vg / 200 μl, SP0525, SP0526, SP0527, SP0528, and expression cassette 529 showed lower activity in the liver than CK7 and / or CK8. 13 At a dose of 100 vg / 200 μl, SP0525, SP0526, SP0527, SP0528 and expression cassette 529 showed lower activity in the liver than CK7 and / or CK8.

[0531] Sequence information

[0532] Table 1 - Muscle-specific synthetic promoters

[0533]

[0534]

[0535] Table 2 - CRMs from synthetic promoters in Table 1

[0536]

[0537]

[0538] Table 3 - CREs from synthetic promoters in Table 1

[0539]

[0540] Table 4 - Promoter Elements

[0541]

[0542] Table 5 - Liver detargeting elements from synthetic promoters in Table 1

[0543] Table 6 - Schematic representation of a muscle-specific promoter according to an embodiment of the present invention showing cis-regulatory elements, promoter elements, and other elements

[0544]

[0545]

[0546] Table 7 - Schematic representation of muscle-specific expression cassettes with liver de-targeting elements

[0547]

[0548] Expression cassette 529 is represented as follows:

[0549]

[0550] Table 8 - Introns from the synthetic promoters in Table 1

[0551]

[0552] Terms

[0553] 1. A synthetic muscle-specific cis-regulatory module (CRM) comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof, DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof and at least one additional regulatory element, optionally wherein the additional regulatory element is a detargeting element or a functional variant thereof.

[0554] 2. The synthetic muscle-specific CRM according to clause 1, wherein the at least one additional regulatory element is a detargeting element or a functional variant thereof, optionally a liver detargeting element or a functional variant thereof.

[0555] 3. The synthetic muscle-specific CRM according to clause 2, wherein the liver detargeting element or a functional variant thereof is selected from the group consisting of:

[0556] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0557] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0558] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0559] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0560] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0561] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof.

[0562] 4. The synthetic muscle-specific CRM according to any of the preceding clauses, wherein the CRM comprises a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0563] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 1 (SEQ ID NO: 13);

[0564] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 2 (SEQ ID NO: 14);

[0565] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 3 (SEQ ID NO: 17);

[0566] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and ZBTB20 binding site (SEQ ID NO: 15);

[0567] - CRE0145 (SEQ ID NO: 10), liver detargeting sequence 3 (SEQ ID NO: 17), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); and

[0568] - CRE0145 (SEQ ID NO: 10), ZBTB20 binding site (SEQ ID NO: 15), DES_MT_enhancer_48bp (SEQ ID NO: 11) and ZBTB20 binding site (SEQ ID NO: 15), optionally, wherein the regulatory elements are present in the CRM in the order listed and are adjacent to each other.

[0569] 5. A synthetic muscle-specific CRM according to any of the preceding clauses, comprising or consisting of SEQ ID NOs: 20, 21, 22 and 9 or functional variants thereof, optionally comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 20, 21, 22 and 9.

[0570] 6. A synthetic muscle-specific promoter comprising a CRM according to any preceding clause, said CRM being operably linked to a promoter element, optionally wherein said promoter element is SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0571] 7. A synthetic muscle-specific promoter according to clause 6, comprising a synthetic muscle-specific CRM comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof, DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof and at least one additional regulatory element, optionally wherein the additional regulatory element is a liver detargeting element selected from the group consisting of:

[0572] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0573] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0574] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof; and

[0575] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0576] It is operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof, or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof.

[0577] 8. A synthetic muscle-specific promoter according to any one of clauses 6-7, comprising a synthetic muscle-specific CRM comprising a combination of regulatory elements or functional variants thereof selected from the group consisting of:

[0578] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 1 (SEQ ID NO: 13);

[0579] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 2 (SEQ ID NO: 14);

[0580] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and liver detargeting sequence 3 (SEQ ID NO: 17);

[0581] - CRE0145 (SEQ ID NO: 10), DES_MT_enhancer_48bp (SEQ ID NO: 11) and ZBTB20 binding site (SEQ ID NO: 15);

[0582] - CRE0145 (SEQ ID NO: 10), liver detargeting sequence 3 (SEQ ID NO: 17), DES_MT_enhancer_48bp (SEQ ID NO: 11), and liver detargeting sequence 3 (SEQ ID NO: 17); and

[0583] - CRE0145 (SEQ ID NO: 10), ZBTB20 binding site (SEQ ID NO: 15), DES_MT_enhancer_48bp (SEQ ID NO: 11) and ZBTB20 binding site (SEQ ID NO: 15);

[0584] is operably linked to promoter element SCP1 (SEQ ID NO: 12) or a functional variant thereof, or promoter element CRE0053 (SEQ ID NO: 26) or a functional variant thereof,

[0585] Optionally, wherein the regulatory elements are present in the CRM in the order listed and are adjacent to each other.

[0586] 9. A synthetic muscle-specific promoter according to any one of clauses 6 to 8, wherein the synthetic muscle-specific promoter comprises or consists of SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3) or SP0528 (SEQ ID NO: 4) or a functional variant of any one thereof.

[0587] 10. A synthetic muscle-specific promoter according to any one of clauses 6 to 9, comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1 to 4.

[0588] 11. A synthetic muscle-specific promoter according to any one of clauses 6 to 10, wherein the synthetic muscle-specific promoter comprises or consists of SP0527 (SEQ ID NO: 3) or a functional variant of any one thereof, optionally wherein the synthetic muscle-specific promoter comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.

[0589] 12. A synthetic muscle-specific promoter comprising a liver detargeting element.

[0590] 13. The synthetic muscle-specific promoter according to clause 12, wherein the liver detargeting element is selected from the group consisting of:

[0591] -SEQ ID NO: 15 or a functional variant thereof;

[0592] -SEQ ID NO: 13 or a functional variant thereof;

[0593] - SEQ ID NO: 17 or a functional variant thereof; or

[0594] - SEQ ID NO: 14 or a functional variant thereof.

[0595] 14. A synthetic muscle-specific promoter according to clause 12 or 13, wherein the liver detargeting element comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 14.

[0596] 15. A synthetic muscle-specific promoter comprising or consisting of:

[0597] - CRE0145 (SEQ ID NO: 10) or a functional variant thereof,

[0598] -DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof,

[0599] - SCP1 (SEQ ID NO: 12) or a functional variant thereof, or CRE0053 (SEQ ID NO: 26) or a functional variant thereof; and

[0600] - at least one additional regulatory element.

[0601] 16. The synthetic muscle-specific promoter according to clause 15, wherein the at least one additional regulatory element is a detargeting element, optionally a liver detargeting element, wherein the liver detargeting element is selected from the group consisting of:

[0602] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0603] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0604] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0605] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0606] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0607] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof.

[0608] 17. A synthetic muscle-specific promoter according to clause 15, wherein the at least one additional regulatory element is an intron, optionally wherein the intron is selected from the tMCK SA / SD intron (SEQ ID NO: 29) or the MVM truncation intron (SEQ ID NO: 30).

[0609] 18. The synthetic muscle-specific promoter according to clause 15, wherein the additional regulatory element is

[0610] A liver detargeting element selected from the group consisting of:

[0611] - liver detargeting sequence 1 (SEQ ID NO: 13) or a functional variant thereof;

[0612] - liver detargeting sequence 2 (SEQ ID NO: 14) or a functional variant thereof;

[0613] - liver detargeting sequence 3 (SEQ ID NO: 17) or a functional variant thereof;

[0614] -ZBTB20 binding site (SEQ ID NO: 15) or a functional variant thereof;

[0615] - Mir122 miRNA target sequence 1 (SEQ ID NO: 16) or a functional variant thereof; and

[0616] - Mir122 miRNA target sequence 2 (SEQ ID NO: 19) or a functional variant thereof; or

[0617] An intron selected from the group consisting of: tMCK SA / SD intron (SEQ ID NO: 29) and MVM truncation intron (SEQ ID NO: 30).

[0618] 19. A synthetic muscle-specific promoter according to any one of clauses 15 to 18, wherein the synthetic muscle-specific promoter comprises SP0525 (SEQ ID NO: 1), SP0526 (SEQ ID NO: 2), SP0527 (SEQ ID NO: 3), SP0528 (SEQ ID NO: 4), SP0530 (SEQ ID NO: 27) or SP0531 (SEQ ID NO: 28) or a combination thereof. or consisting of a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of ID NOs: 1-4, 27-28.

[0619] 20. An expression cassette comprising a synthetic muscle-specific promoter according to any one of clauses 6 to 19, said synthetic muscle-specific promoter being operably linked to a sequence encoding an expression product, or an expression cassette comprising a synthetic muscle-specific promoter comprising CRE0145 (SEQ ID NO: 10) or a functional variant thereof and DES_MT_enhancer_48bp (SEQ ID NO: 11) or a functional variant thereof, wherein said synthetic muscle-specific promoter is operably linked to an expression product and a target sequence of miR122.

[0620] 21. A vector comprising the synthetic muscle-specific promoter according to any one of clauses 6 to 19 or the expression cassette according to clause 20.

[0621] 22. The vector according to clause 21, which is an AAV vector, an adenoviral vector, a retroviral vector or a lentiviral vector.

[0622] 23. A gene therapy vector comprising a synthetic promoter comprising a liver detargeting element selected from the group consisting of:

[0623] -SEQ ID NO: 15 or a functional variant thereof;

[0624] -SEQ ID NO: 13 or a functional variant thereof;

[0625] - SEQ ID NO: 17 or a functional variant thereof; or

[0626] - SEQ ID NO: 14 or a functional variant thereof.

[0627] 24. The gene therapy vector of clause 23, comprising or consisting of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 13 or SEQ ID NO: 14.

[0628] 25. The gene therapy vector of clause 23 or 24, wherein the gene therapy vector is an AAV vector.

[0629] 26. A gene therapy AAV vector comprising an expression cassette comprising a synthetic promoter operably linked to a sequence encoding an expression product and a target sequence for miR122.

[0630] 27. The gene therapy AAV vector according to clause 26, wherein the target sequence of miR122 comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 19 or a functional variant thereof.

[0631] 28. The gene therapy AAV vector of clause 26 or 27, wherein the target sequence of miR122 comprises or consists of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 19.

[0632] 29. A virion comprising the vector according to any one of clauses 21-28.

[0633] 30. A pharmaceutical composition comprising the synthetic muscle-specific promoter according to any one of clauses 6-19, the expression cassette according to clause 20, the vector according to any one of clauses 21-28, or the virosome according to clause 29.

[0634] 31. The synthetic muscle-specific promoter according to any one of clauses 6 to 19, the expression cassette according to clause 20, the vector according to any one of clauses 21 to 28, the virosome according to clause 29 or the pharmaceutical composition according to clause 30, for use in therapy.

[0635] 32. A cell comprising the synthetic muscle-specific promoter according to any one of clauses 6 to 19, the expression cassette according to clause 20, the vector according to any one of clauses 21 to 28, or the virion according to clause 29.

[0636] 33. The synthetic muscle-specific promoter according to any one of clauses 6 to 19, the expression cassette according to clause 20, the vector according to any one of clauses 21 to 28, the virosome according to clause 29 or the pharmaceutical composition according to clause 30, for use in the preparation of a pharmaceutical composition for treating a medical condition or disease.

[0637] 34. A method for producing an expression product, the method comprising providing an expression cassette according to clause 20 in a muscle cell, and expressing the expression product present in the expression cassette.

[0638] 35. A method for expressing a therapeutic transgene in a muscle cell, the method comprising introducing into the muscle cell an expression cassette according to clause 20, a vector according to any one of clauses 21 to 28, or a virosome according to clause 29.

[0639] 36. A method of treating a subject, preferably a human, in need thereof, comprising:

[0640] administering to a subject an expression cassette according to clause 20, a vector according to any one of clauses 21 to 28, a virosome according to clause 29, or a pharmaceutical composition according to clause 30, comprising a sequence encoding a therapeutic product operably linked to a promoter according to any one of clauses 6 to 19; and

[0641] A therapeutic amount of a therapeutic product is expressed in the muscle of the subject.

[0642] 37. A method of treating a subject according to clause 36, wherein the therapeutic amount of the therapeutic product is expressed in skeletal muscle and / or cardiac muscle.

Claims

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

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

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

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

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

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

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

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

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

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

19.

11. A virosome comprising the vector according to any one of claims 9-10.

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

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

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

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

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

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