Skeletal muscle-specific promoters

Abstract

The present disclosure relates to the fields of molecular biology and nucleic acid technology. The present disclosure also relates to therapy and prophylaxis of disease.

Description

[0001] SKELETAL MUSCLE-SPECIFIC PROMOTERS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the fields of molecular biology and nucleic acid technology. The present disclosure also relates to therapy and prophylaxis of disease.

[0004] BACKGROUND

[0005] The present disclosure related to novel skeletal muscle-specific promoters and their use in gene therapy.

[0006] Gene therapy as a modality aims at ameliorating disease-related phenotypes by using both viral and non-viral delivery systems. Significant efforts have been focusing on optimizing this therapy to be more efficient, non-immunogenic and less toxic while enabling a long-term expression of the gene of interest. One of the key challenges to achieve this goal is to regulate the expression of the transgene to meet the required level in the target cell / tissue.

[0007] For certain targets, ubiquitous expression in target and non-target tissues might lead to adverse events. Therefore, target cell / tissue specific expression might be beneficial which can be achieved using cell / tissue specific promoters.

[0008] In particular, novel, specific promoters with different degrees of tissue specificity and expression levels are required to modulate the expression of gene therapy targets for a variety of neuromuscular disease indications, while exhibiting limited activity in non-target tissues (Skopenkova et al, Acta Naturae 13 (2021) 47-58). There are different reasons why improved promoters are needed to regulate transgene expression: 1. specificity to skeletal and / or cardiac tissue only or in a specific ratio to improve the safety profile and ensure target specific efficacy, 2. ensure robust expression to ensure regulation of the gene in the desired range, 3. short regulatory elements would be beneficial to ensure maximum size for the transgene, 4. identification of promoters with cross-species activity to establish best exposure / efficacy / safety range for the desired target.

[0009] For some indications like Duchenne and Becker Muscular Dystrophy (DMD / BMD) a promoter expressing in both disease relevant tissues (skeletal and cardiac) are essential, while for targets for Facioscapulohumeral Dystrophy (FSHD) or some congenital myopathies a restricted expression to the skeletal muscle would most likely be more beneficial. An example is the modulation of expression of actin. Mutations in the skeletal actin gene (ACT Al) can lead to congenital myopathies (Nemaline). Actins in general are ubiquitously high expressed proteins and are highly conserved. ACT Al is expressed in skeletal muscle and heart, however cardiac actin (ACTC1) is mainly expressed in the heart (-80%). If ACTA1 is affected, it has been shown that most patients only show skeletal muscle specific symptoms. So a high expression of ACT Al in the heart could rather lead to potential safety liabilities in heart, while a high expression in the skeletal tissue is highly desired.

[0010] There are only few muscle-specific promoters known in the art with varying degree of specificity. These can be divide into natural promoters, creatine kinase promoter -MCK and desmin promoters and synthetic promoters, synthetic muscle-specific promoter C5-12 (SPC5- 12) and a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) (Toscano et al., Gene Ther. 18 (2011) 117-127).

[0011] While the natural promoters exhibit muscle specificity their expression levels are not sufficient to obtain a therapeutic effect.

[0012] While the synthetic promoters known in the art result in increased transgene expression achieving therapeutic effects, they are prone to off-target activity in other tissues and are active in skeletal as well as in cardiac muscle (Piekarowicz et al., Mol Ther Methods Clin Dev. 15 (2019) 157-169, 2019; Salva et al., Mol Ther. 15 (2007) 320-329).

[0013] Thus, muscular diseases which are limited to the skeletal muscle would benefit from new promoters which are specific to the skeletal muscle.

[0014] BRIEF SUMMARY

[0015] Herein disclosed are novel skeletal muscle specific promoters. Comparing the promoters according to the current invention with state-of-the-art promoter, such as MHCK7, showed a higher and more specific expression in primary skeletal muscle cells. In particular, the present inventors have shown that synthetic promoters comprising multiple MyoG elements more active and skeletal muscle-specific than all other constructs tested. In one embodiment, provided is a synthetic skeletal muscle-specific promoter comprising at least 6 MyoG elements operably linked to a promoter element.

[0016] In one embodiment, the synthetic skeletal muscle-specific comprises at least 8 MyoG elements operably linked to a promoter element.

[0017] In one embodiment, the synthetic skeletal muscle-specific promoter comprises 6 MyoG elements.

[0018] In one embodiment, the MyoG elements individually comprise, or consist of, the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

[0019] In one embodiment, the MyoG elements are connected through a linker.

[0020] In one embodiment, the at least one linker has a length of 4-14bp, preferably 4-6 bp, most preferably 4 bp.

[0021] In one embodiment, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO:25, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, and SEQ ID NO:99.

[0022] In one embodiment, the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:78, SEQ ID NO :80, SEQ ID NO: 82, and SEQ ID NO: 84.

[0023] In one embodiment, the promoter element is a minimal promoter.

[0024] In one embodiment, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0025] In one embodiment, provided is an expression system comprising a synthetic skeletal musclespecific promoter as described herein before operationally linked to a polynucleotide encoding a polypeptide of interest.

[0026] In one embodiment, provided is a vector comprising a synthetic skeletal muscle-specific promoter as described herein before operationally linked to a polynucleotide encoding a polypeptide of interest, optionally wherein the vector is an adeno -associated virus (AAV) vector.

[0027] In one embodiment, provided is the expression system as described herein above or the vector as described herein above, further comprising a 5'UTR sequence operationally linked to the synthetic skeletal muscle-specific promoter, wherein the 5'UTR sequence is located 3' to the synthetic skeletal muscle-specific promoter, optionally wherein the 5'UTR comprises an intron.

[0028] In one embodiment, the intron is not a complete naturally occurring intron.

[0029] In one embodiment, the intron is a truncated natural or synthetic intron.

[0030] In one embodiment, provided is an adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5’ to 3’ order:

[0031] (i) a 5' inverted terminal repeat (ITR) sequence;

[0032] (ii) a synthetic skeletal muscle-specific promoter as described herein before;

[0033] (iii) optionally, a 5' UTR sequence;

[0034] (iv) a polynucleotide encoding a therapeutic molecule;

[0035] (v) a 3' UTR sequence; and

[0036] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0037] In one embodiment, provided is a pharmaceutical composition comprising an expression system as described herein above, or a vector as described herein above, or an AAV vector as described herein above, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. In one embodiment, provided is an expression system as described herein above, or a vector as described herein above, or an AAV vector as described herein above, or a pharmaceutical composition as described herein above, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest in skeletal muscle cells.

[0038] In one embodiment, provided is use of an expression system as described herein above, or a vector as described herein above, or an AAV vector as described herein above, or a pharmaceutical composition as described herein above, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.

[0039] In one embodiment, provided is a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject an expression system as described herein above, or a vector as described herein above, or an AAV vector as described herein above, or a pharmaceutical composition as described herein above.

[0040] DETAILED DESCRIPTION

[0041] Herein disclosed are novel skeletal muscle specific promoters. Comparing the promoters according to the current invention with state-of-the-art promoter, such as MHCK7, showed a higher and more specific expression in primary skeletal muscle cells.

[0042] Without being bound by this theory, it is assumed that the muscle specific promoters according to the present invention allow for a reliable skeletal muscle-specific transgene expression which, inter alia, may result in decreased host immune response and increased therapeutic effect. In particular, the skeletal muscle-specific promoters according to the present invention in combination with muscle specific viral serotypes such as rAAV8 and rAAV9 would allow for a reliable skeletal muscle-specific transgene expression.

[0043] Aspects and embodiments of the present disclosure relate to synthetic skeletal muscle - specific promoters. Promoters according to the present disclosure comprise or consist of polynucleotides, in particular polynucleotides comprising or consisting of DNA or RNA. A 'polynucleotide' refers to a polymer chain of a plurality of nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g. in the case of polynucleotides formed by naturally-occurring nucleotide monomers). Polynucleotides include oligonucleotides, which generally comprise <50 nucleotides. A polynucleotide may be singlestranded, or may be double-stranded (i.e. may comprise a duplex formed by hydrogen-bonding between complementary nucleotides). Polynucleotides according to the present disclosure may comprise or consist of: single- stranded DNA, double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- stranded RNA, double- stranded RNA, RNA that is mixture of single- and double-stranded regions, single-stranded molecules comprising DNA and RNA, double-stranded molecules comprising DNA and RNA, and molecules comprising DNA and RNA having a mixture of single- and double-stranded regions.

[0044] In some aspects, a polynucleotide comprises or consists of DNA. In some embodiments, a polynucleotide is a polydeoxyribonucleotide. In some embodiments, a polynucleotide comprises or consists of RNA. In some embodiments, a polynucleotide is a polyribonucleotide.

[0045] In aspects wherein the polynucleotide of the present disclosure is defined by reference to a given nucleotide sequence, and wherein the given nucleotide sequence comprises or consists of RNA and / or is a polyribonucleotide, it will be appreciated that instances of ‘T’ for thymidine in such sequences are replaced with ‘U’, for uracil.

[0046] The present disclosure also contemplates polynucleotides comprising modified nucleotides, e.g. in which the phosphate and / or ribose and / or base of a deoxyribonucleotide or ribonucleotide is / are chemically modified. Nucleotide modifications contemplated in accordance with the present disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is hereby incorporated by reference in its entirety.

[0047] Phosphate modifications may be selected from phosphorothioate (e.g. Ap isomer, 5p isomer), phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 5' -(E)- vinylphosphonate, 5'-methylphosphonate, (5)-5'-C-methyl with phosphate, 5 ’-phosphorothioate, and peptide nucleic acid modifications. Ribose modifications may be selected from 2'-O- methyl, 2'-O-methoxyethyl, 2’-fluoro, 2’-deoxy-2’-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O- allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2’-O-benzyl, 2’-O-methyl-4-pyridine, locked nucleic acid, (5 -cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid and glycol nucleic acid modifications. Base modifications may be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5 ’-methylcytidine, 5’-fluoro-2’- deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'- triazole-modified adenine, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotoluyl ribonucleoside and 5'- nitroindole modifications.

[0048] In some aspects, a modified nucleotide may be selected from 2'-O-methyluridine-3'- phosphate, 2'-O-methyladenosine-3 '-phosphate, 2'-O-methylguanosine-3 '-phosphate, 2'-O- methylcytidine-3 '-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine- 3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'- phosphorothioate, 2'-fluorouridine-3 '-phosphate, 2'-fluoroadenosine-3 '-phosphate, 2'- fluoroguanosine-3 '-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'- phosphorothioate, 2'-fhioroguanosine-3'-phosphorothioate, 2'-fluoroadenosine-3'- phosphorothioate, and 2'-fhiorouridine-3'-phosphorothioate.

[0049] In some aspects, provided is a synthetic skeletal muscle-specific promoter. In a preferred embodiment, the synthetic skeletal muscle-specific promoter comprises Myogenin (MyoG) elements (alternatively referred to as MyoG binding sites). MyoG, also known as Myogenin, is a member of the myogenic regulatory factor family. It is a transcription factor that plays a crucial role in the development and differentiation of skeletal muscle. MyoG is involved in regulating the expression of genes that are essential for muscle cell formation and maturation. It is one of the key factors responsible for initiating the process of myogenesis, which is the formation of muscle tissue during embryonic development and regeneration. MyoG is expressed predominantly in skeletal muscle cells and is essential for their proper development and function. MyoG binding sites can be identified by methods know in the art and for example described in Cao et al., EMBO J. 2006 Feb 8; 25(3): 502-511).

[0050] Exemplary MyoG elements are shown in the appended Examples and in the Exemplary Sequences further below. In a particular aspect, the MyoG element used according to the present invention comprises or consists of the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

[0051] In some aspects, the synthetic skeletal muscle-specific promoter comprises multiple MyoG elements. In some aspect, the synthetic skeletal muscle-specific promoter comprises at least 4, at least 5, at least 6, at least 7, or at least 8 MyoG elements. In a preferred aspect, the MyoG element used according to the present invention comprises or consists of 8 MyoG elements.

[0052] In some embodiments, the MyoG elements are separated from each other by a linker polynucleotide. The linker polynucleotide (sequence), also referred to as stuffer polynucleotide (sequence) are polynucleotide segments that are inserted between two other polynucleotide sequences. Preferably, linker polynucleotide sequences are non-coding. Linker polynucleotides are used to join two other DNA fragments together in a specific order or orientation, or to add space or flexibility between two functional elements. In the context of a promoter or gene, a stuffer or linker sequence might be used to ensure proper spacing and orientation of the elements for correct transcription or expression. Linker nucleotide sequences may comprise, or consist of, 1-11, e.g. one of 2-10, 3-9, 4-8, 5-7, or 6 nucleotides. Exemplary linker polynucleotide sequences are disclosed herein below in the Sequence Table (for example SEQ ID NOs:44-48, and in SEQ ID Nos: 100-109) and in the Examples, other suitable linker polynucleotide sequences can be readily identified and selected by the skilled person.

[0053] In a specific such aspect, the 8 MyoG elements comprise, or consist of, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25. This sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences individually selected from the group consisting of SEQ ID NOs:44-48.

[0054] In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25. This sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences, wherein the linker polynucleotide sequences each are 6 bp in length. In one aspect, the linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NOs:44-48. In another aspect, the linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NOs: 100-109.

[0055] In one aspect, the sequence comprises six copies of the MyoG element of SEQ ID NO:24 and six linker polynucleotide sequences. In one aspect, the six linker polynucleotide sequences each are 6 bp in length. In one aspect, the six linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 44-49. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:91.

[0056] In one aspect, the sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences. In one aspect, the eight linker polynucleotide sequences each are 4 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 100-104. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92.

[0057] In one aspect, the sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences. In one aspect, the eight linker polynucleotide sequences each are 8 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 105-109. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93.

[0058] In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0059] In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:95.

[0060] In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:96.

[0061] In one aspect, the sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences. In one aspect, the eight linker polynucleotide sequences each are 6 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 44-49. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:97.

[0062] In one aspect, the sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight linker polynucleotide sequences. In one aspect, eight six linker polynucleotide sequences each are 4 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 100-104. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:98.

[0063] In one aspect, the sequence comprises six copies of the MyoG element of SEQ ID NO:24 and six linker polynucleotide sequences. In one aspect, the six linker polynucleotide sequences each are 4 bp in length. In one aspect, the six linker polynucleotide sequences are individually selected from the group consisting of SEQ ID Nos 100-104. In a further such aspect, the synthetic skeletal muscle-specific promoter comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:99.

[0064] In further aspects, the MyoG elements are is operably linked to a promoter element. The current invention is based, at least in part, on the identification of muscle specific genes from databases and the identification of the promoter regions thereof. Additionally, specific transcription factor binding sites were identified within the promoter regions. In some aspects, the promoter element is a promoter element selected from the list consisting of the ACT Al promoter (NCBI gene ID 58, actin alpha 1), the CACNG1 promoter (NCBI gene ID 786, voltagedependent calcium channel gamma- 1 subunit), the MYBPC2 promoter (NCBI gene ID 4606, myosin binding protein C, fast type), the MYH2 promoter (NCBI gene ID 4620, myosin-2), the MYLPF promoter (NCBI gene ID 29895, and myosin regulatory light chain 2, skeletal muscle isoform), the MYBC1 promoter, the TTN promoter (NCBI gene ID 7273, titin), or fragments thereof that retain tissue specific expression, in particular skeletal muscle -specific expression.

[0065] In some embodiments, the promoter element is a minimal promoter. A ‘minimal promoter’ refers to the simplest and / or smallest polynucleotide sequence that is capable of binding RNA polymerase and other necessary transcription factors to initiate transcription, leading to the production of mRNA from a gene. A minimal promoter usually is the basic component of a larger promoter complex and is essential for gene expression. The minimal promoter typically includes a TATA box and other core promoter elements. Usually, a minimal promoter lacks enhancer or repressor binding sites.

[0066] In preferred embodiments, the promoter element comprises a TATA box. A ‘TATA box’ is a specific polynucleotide sequence found in the promoter region of many genes. It is typically composed of the nucleotide sequence TAT AAA (SEQ ID NO:51) and is located about 25-30 base pairs upstream from the transcription start site. The TATA box is recognized and bound by a transcription factor known as TATA-binding protein (TBP), which assists in the recruitment of RNA polymerase II and other factors necessary for the initiation of transcription. The TATA box is important for determining the direction of transcription and defining the exact point where transcription begins.

[0067] In some aspect, the promoter element comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 or SEQ ID NO:50.

[0068] In some aspects, the minimal promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 or SEQ ID NO:50.

[0069] In some aspects, the minimal promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:78, SEQ ID NO: 80, SEQ ID NO: 82, and SEQ ID NO: 84.

[0070] The minimal promoter is preferably provided immediately 3’ to (z.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the polynucleotide comprising the MyoG elements.

[0071] In some aspect, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column B of Table 1, or a fragment thereof that retains skeletal muscle-specific expression of a polypeptide of interest operably linked to the fragment.

[0072] In some aspects, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27 or SEQ ID NO:28.

[0073] In some aspects, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID N0:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0074] In some aspects, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0075] In some preferred aspects, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0076] In most preferred aspects, the synthetic skeletal muscle-specific promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. In a particular most preferred aspect, the synthetic skeletal muscle -specific promoter consists of a sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0077] In aspects as described herein above, the promoter and / or promoter element preferably exhibits functional properties as described herein below.

[0078] Functional properties of the synthetic promoters

[0079] In aspects and embodiments of the present disclosure, the polynucleotides of the present disclosure may be characterised by reference to one or more functional properties. One very important functional property of the new promoters according to the invention is tissuespecificity, in particular muscle-specificity.

[0080] In some embodiments, non-skeletal muscle cells comprising a synthetic skeletal muscle-specific promoter of the present disclosure operably linked to a polynucleotide encoding a protein of interest substantially do not express the polypeptide of interest. In some embodiments, skeletal muscle cells comprising a synthetic skeletal muscle-specific promoter of the present disclosure operably linked to a polynucleotide encoding a protein of interest express the polypeptide of interest.

[0081] While cardiac and skeletal muscle belong to the striated muscles and share some common properties (e.g. sarcomeres, rich in mitochondria and contain large extended myosin and action proteins), both have also distinct features. Skeletal muscles consist of parallel linear fibers while cardiac muscles are arranged in a cross-striation form. In addition to their similarities, the two cell types can be also differentiated by their function and their respective molecular constitution. Both can be differentiated by expression of specific genes and proteins which are in line with their physiological functions (Lindskog et al., BMC Genomics. 2015; 16(1): 475). Well-established markers to monitor the different steps of myogenic differentiation include PAX7, MYOD, MYOG, MRF4, MYH3 and MYH7, while examples of elevated proteins to identify skeletal muscles are MYH2, TNNT1, MYBPC1 and ACTA1 (Chai et al., Development (2017) 144 (12): 2104-2122; Lindskog et al., BMC Genomics. 2015; 16(1): 475). Hence monitoring of at least some of the proteins or their respective transcripts is recommended when using in vitro models to ensure correct cell specificity. Expression of a polypeptide of interest can be evaluated using any suitable technique for the detection and / or quantification of the relevant polypeptide. Such techniques include e.g. antibody-based methods, (for example flow cytometry, immunocytochemistry, Western blot, ELISA), fluorescence microscopy and flow cytometry. In some embodiments, expression of a polypeptide of interest can be evaluated as described in the experimental examples of the present disclosure.

[0082] For example, tissue specific expression, such as skeletal muscle-specific expression can be measured with assays known in the art and as described in the appended examples. For example, the expression level of a reporter gene under the control of the candidate promoter can be measured in a target cell (for example in a skeletal muscle cell or cell line) and in a non- target cell (for example a hepatocyte cell or cell line) and the expression in the target cell can be compared to the expression of the non-target cell. A skeletal muscle-specific promoter will exhibit high (or higher) expression in the skeletal muscle cell (such as in a HSMM cell) and low (or lower) expression in the non-target cell (such as in a Huh7 hepatocyte-derived cancer cell). Expression can be measured by measuring the level of the reporter gene, for example measuring fluorescence intensity of the expression of a fluorescent protein operably linked to the candidate promoter. A ratio between the expression of the reporter gene in the target cell and the expression of the reporter gene in the non-target cell can be calculated to illustrate the tissue specificity.

[0083] In some aspects, the ratio of i) the promoter activity in HSMM cells 5 days after transduction with an AAV of the serotype 2 at an MOI of 20,000 to ii) the promoter activity in Huh7 cells 3 days after transduction with the same AAV at an MOI of 20,000 is 10 or more, 100 or more, 1000 or more, 10'000 or more, or 20'000 or more. In some aspects, the promoter activity is determined as fluorescence intensity of the expression of a fluorescent protein operably linked to the synthetic promoter. In some aspect, the fluorescent protein is mGreenLantern. Such promoters may be referred to herein as ‘skeletal muscle-specific’ promoters.

[0084] In some aspect, the fluorescence intensity in Huh7 cells is 1000 RFU or less, 900 or less, 800 or less, 750 or less.

[0085] In some embodiments, cells that ‘substantially do not express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is less than 0.2 times, e.g. one of <0.1 times, <0.09 times, <0.08 times, <0.07 times, <0.06 times, <0.05 times, <0.04 times, <0.03 times, <0.02 times, or <0.01 times the level of expression by cells that express the polypeptide of interest. In some embodiments, cells that ‘express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is greater than 5 times, e.g. one of >10 times, >20 times, >50 times, >100 times, >1000 times, >5000 times or >10000 times level of expression by cells that ‘substantially do not express’ the polypeptide of interest.

[0086] Exemplary synthetic skeletal muscle-specific promoter according to the present disclosure include polynucleotides having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27 or SEQ ID NO:28.

[0087] It will be appreciated that the functional properties described herein are evaluated employing the similar (or the same) experimental conditions for the evaluation of cells comprising the different polynucleotides. However, some experimental conditions might need to be adjusted, for example depending on the type of cells. Adjustment of the experimental conditions to a specific type of cells can be done according to methods known in the field and as described in the experimental section.

[0088] Expression system

[0089] In some embodiments, provided is an expression system comprising a synthetic skeletal muscle-specific promoter as described hereinabove operationally linked to a polynucleotide encoding a polypeptide of interest. For example, the synthetic muscle-specific promoter as described hereinabove drives the expression of the polypeptide of interest in skeletal muscle cells.

[0090] The term ‘operably linked’ may include the situation where nucleic acid encoding a polypeptide of interest according to the present disclosure and regulatory nucleic acid sequence(s) e.g. a promoter and / or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into the desired polypeptide(s). A polypeptide of interest may be any polypeptide.

[0091] In some embodiments, a polypeptide of interest according to the present disclosure may be an antigen-binding polypeptide, an aptamer, an antigen-binding polypeptide complex, an antibody or an antigen-binding fragment or derivative thereof, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, a decoy receptor for a ligand, a decoy ligand for a receptor, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, a transcription factor, an epigenetic modifier, a constituent protein of a site-specific nuclease nucleic acid editing system (e.g. a CRISPR / Cas9 system, a CRISPR / Cpfl system, a CRISPR / C2cl system, a CRISPR / C2c2 system, a CRISPR / C2c3 system, a ZFN system or a TALEN system), a constituent protein of a ribonucleoprotein, or a viral protein (e.g. a capsid protein or a viral enzyme).

[0092] In some embodiments, a polypeptide of interest is a polypeptide suitable for use in therapy or prophylaxis of a disease / condition. In some embodiments, a polypeptide of interest is a detectable polypeptide or a polypeptide having detectable activity.

[0093] A polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be any polypeptide whose administration is useful for the treatment or prevention of a disease / condition. In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be a polypeptide for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition. By way of illustration, in some embodiments, the polypeptide of interest may be ACTA1 deficiency of ACTA1 is associated with myopathy.

[0094] In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be a polypeptide which inhibits the expression and / or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition.

[0095] A detectable polypeptide may be or comprise a fluorescent polypeptide. Fluorescent polypeptides include green fluorescent protein and variants thereof (e.g. enhanced green fluorescent protein), yellow fluorescent protein (e.g. citrine), red fluorescent protein and variants thereof (e.g. mOrange, mCherry), blue fluorescent protein and variants thereof (e.g. TagBFP), cyan fluorescent protein and variants thereof (e.g. mTurquoise, cerulean), allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin. In some aspect, the fluorescent protein is mGreenLantern.

[0096] A detectable polypeptide may be or comprise an epitope tag. Epitope tags include e.g. His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione- s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S- peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol).

[0097] In some aspect, the detectable polypeptide comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column D of Table 1, or a fragment thereof that retains its detectable activity.

[0098] A polypeptide having detectable activity may be or comprise an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. betagalactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

[0099] A polypeptide of interest expressed from a polynucleotide of the present disclosure may additionally comprises one or more extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. Such extraneous amino acids may be characterised as forming an N-terminal tag on the polypeptide of interest. It may be desirable to minimise the size of, or completely remove, such extraneous amino acids / N-terminal tags on the polypeptide of interest.

[0100] In some embodiments, the N-terminal tag consists of fewer than 50 amino acids, e.g. one of <40 amino acids, <30 amino acids, <25 amino acids, <20 amino acids, <15 amino acids, <10 amino acids, <9 amino acids, <8 amino acids, <7 amino acids, <6 amino acids, <5 amino acids, <4 amino acids, <3 amino acids, <2 amino acids or 1 amino acid. In some embodiments, the polypeptide of interest lacks an N-terminal tag.

[0101] The expression system according to the present invention may comprise additional nucleotide sequences and / or sequence features in addition to the synthetic muscle-specific promoter and the polynucleotide encoding a polypeptide of interest. The expression system of the present disclosure comprise a start codon 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest. The start codon is preferably the trinucleotide ‘ATG’.

[0102] In some embodiments, the expression system of the present invention further comprises a Kozak sequence. In preferred embodiments, the Kozak sequence is provided immediately upstream of the start codon for initiating translation of the polypeptide of interest.

[0103] In some embodiments, the expression system of the present invention further comprises one or more enhancer sequences. The one or more enhancer sequences are preferably 5’ to the synthetic muscle-specific promoter sequence.

[0104] In some embodiments, the expression system of the present invention comprises a 5'UTR. The term 5'UTR stands for 5' Untranslated Region. This region is transcribed but is not translated into protein. The 5'UTR can contain regulatory elements and can modulate the stability of the mRNA, its localization, and the rate of protein synthesis. In some embodiments, the 5'UTR is operationally linked to the synthetic skeletal muscle-specific promoter of the present disclosure.

[0105] In some embodiments, the 5'UTR comprises an intron. In preferred embodiments, the intron consists of fewer nucleotides than the number of nucleotides of a known (e.g. naturally- occuring) intron. In some aspect, the 5'UTR does not comprise a complete naturally occurring promoter intron. In some aspects, the intron is a truncated natural or synthetic intron. In some aspect, 5'UTR does not comprise an intron.

[0106] In some embodiments, the 5'UTR comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column C of Table 1.

[0107] In some embodiments, the expression system of the present invention further comprises a stop codon. The stop codon is preferably provided immediately 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the trinucleotide encoding the terminal amino acid of a polypeptide of interest. In some embodiments, the expression system of the present invention further comprises a polyadenylation signal sequence. In preferred embodiments, the polyadenylation signal sequence is provided 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest. In some embodiments, the expression system of the present invention further comprises a terminator sequence. The terminator sequence is preferably 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the polyadenylation signal sequence, when present).

[0108] In some embodiments, the expression system of the present invention comprises a 3'UTR. The term 3'UTR stands for 3' Untranslated Region. This region is transcribed but is not translated into protein. The 3'UTR refers to the polynucleotide sequence that follows the stop codon and typically includes the polyadenylation signal.

[0109] In some embodiments, the 3'UTR comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column E of Table 1.

[0110] In preferred embodiments, the constituent nucleotide sequences of the expression system of the present invention are provided immediately adjacent to one another. However, in some embodiments, the polynucleotide further comprises one or more linker nucleotide sequences between one or more of the constituent nucleotide sequences of the expression system of the present invention.

[0111] Linker nucleotide sequences may comprise, or consist of, 1 -10, e.g. one of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides.

[0112] Where an expression system of the present invention according to the present disclosure comprises one more linker nucleotide sequences, the linker sequences are preferably selected such that they do not alter the amino acid sequence of a polypeptide encoded by the polynucleotide. In preferred embodiments, where an expression system of the present invention according to the present disclosure comprises one more linker sequences, the expression system of the present invention encodes the same polypeptide as the equivalent expression system of the present invention lacking the linker nucleotide sequence(s). In some embodiments, the expression system of the present invention further comprises inverted terminal repeat (ITR) sequences.

[0113] In some embodiments, the polynucleotide comprises an ITR 5’ to the promoter and / or enhancer sequences, when present. In some embodiments, the polynucleotide comprises an ITR 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the stop codon, polyadenylation signal sequence and / or terminator sequence, when present).

[0114] In some embodiments, the 5'ITR comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column A of Table 1.

[0115] In some embodiments, the 3 'ITR comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column G of Table 1.

[0116] In some embodiments, the expression system of the present disclosure has a size permitting its delivery as a gene therapy, i.e. in a suitable vector. In some embodiments, the vector consists of a nucleotide sequence having a size within the packaging limit of a vector for delivering the polynucleotide.

[0117] In some embodiments, the expression system has a size within the packaging limit of an AAV vector. In some embodiments, the expression system has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.eB, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74.

[0118] In some embodiments, the expression system of the present disclosure consists of fewer than 6,000 nucleotides, e.g. one of <5,000, <4,750, <4,500, <4,250, <4,000, <3,750, <3,500, <3,250, <3,000, <2,750, <2,500, <2,250, <2,000, <1,750, <1,500, <1,250 or <1,000 nucleotides.

[0119] In some embodiment, the expression system comprises an AAV stuffer sequence. Such stuffer sequence may be required for example to accommodate for different sizes of promoter element or of the polynucleotide encoding the protein of interest. Exemplary AAV stuffer sequences comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column F of Table 1. In some embodiments, the expression system of the present disclosure comprises one or more nucleotide sequences encoding a selectable marker, to facilitate identification and / or selection of cells comprising / expressing a polypeptide of interest. Selectable markers include proteins that confer resistance to antibiotics or other toxins, e.g., blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, and proteins that complement auxotrophic deficiencies.

[0120] In some embodiments, the expression system of the present disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the expression system of the present disclosure comprises a nucleotide sequence permitting two or more polypeptides to be translated separately from a single polyribonucleotide.

[0121] The expression system of the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0122] In some embodiments, the expression system of the present disclosure is a vector.

[0123] A ‘vector’ as used herein refers to a polynucleotide used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell (i.e. the vector may be an expression vector). Such vectors may include a promoter sequence operably linked to the nucleotide sequence to be expressed. Vectors may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used in a vector according to the present disclosure.

[0124] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, baculoviral vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32: 189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, the vector may be a eukaryotic vector, i. e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. In some embodiments, the vector comprises a CMV (e.g. mCMV), SV40, RSV or PGK promoter.

[0125] In some embodiments, a vector is selected based on tropism for a cell type / tissue / organ to which it is desired to deliver the expression system according to the present disclosure. In some embodiments, a vector is selected based on tropism for a cell type / tissue / organ in which it is desired to express the polypeptide of interest. For example, it may be desired to deliver the expression system to, and / or express the polypeptide of interest in, a cell type / tissue / organ affected by a disease / condition to be treated / prevented in accordance with the present disclosure (e.g. a cell type / tissue / organ in which the symptoms of the disease / condition manifest).

[0126] For example, it might be desirable to deliver an expression system of the present disclosure encoding a polypeptide of interest to skeletal muscle tissue, and vectors having a tropism for such cells / tissue may be employed in such instances.

[0127] In preferred embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus vectors and their use to vector gene therapy is reviewed e.g. in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, both of which are hereby incorporated by reference in their entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, a vector may be an adeno- associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0128] In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described e.g. in McCarty, Mol Ther. (2008) 16(10): 1648-56, which is hereby incorporated by reference in its entirety. Conventional AAV have a single-stranded DNA genome, and depend on the DNA replication machinery of a transduced cell to synthesize the complementary strand, delaying transgene expression. By contrast, scAAV contain complementary sequences that spontaneously anneal upon infection, eliminating the requirement for DNA synthesis in the transduced host cell. Compared to classical, single-stranded AAV vectors, scAAV vectors have been shown to provide for accelerated onset of transgene expression, and an increased level of transgene expression.

[0129] In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV -PHP. eB and AAV9.45), AAV1, AAV2 (including AAV2 variant AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the vector is an AAV9 vector.

[0130] In some embodiments a vector comprises modification to increase binding to and / or transduction of a cell-type of interest (i.e. as compared to the level of binding / transduction by the unmodified vector). In some embodiments modification is to a capsid protein.

[0131] In some embodiments a vector comprises a capsid protein comprising a cell -targeting peptide. In some embodiments the cell-targeting peptide is a cell-targeting peptide described in Biining and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248- 265, which is hereby incorporated by reference in its entirety, e.g. a cell-targeting peptide shown in Table 1, 2, 3 or 4 thereof.

[0132] In some embodiments a vector comprises a capsid protein comprising substitution to one or more tyrosine residues, e.g. one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments a vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in lida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby incorporated by reference in its entirety.

[0133] In some embodiments, a vector may be an adeno-associated virus vector described in Biining and Srivastava, supra. In some embodiments, a vector may be an adeno-associated virus vector described in lida et al., supra.

[0134] In some embodiments the vector comprises a control element for inducible expression of the expression system of the disclosure.

[0135] In some embodiment, provided is an adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5' to 3' order: (i) a 5' inverted terminal repeat (ITR) sequence;

[0136] (ii) a synthetic skeletal muscle-specific promoter;

[0137] (iii) optionally, a 5' UTR sequence;

[0138] (iv) a polynucleotide encoding a protein of interest;

[0139] (v) a 3' UTR sequence; and

[0140] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0141] In some embodiment, provided is an adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5' to 3' order:

[0142] (i) a 5' inverted terminal repeat (ITR) sequence;

[0143] (ii) a synthetic skeletal muscle-specific promoter;

[0144] (iii) optionally, a 5' UTR sequence;

[0145] (iv) a polynucleotide encoding a therapeutic molecule;

[0146] (v) a 3' UTR sequence; and

[0147] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0148] In some embodiments, the AAV comprises (i) a 5' ITR sequence comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column A of Table 1.

[0149] In some embodiments, the AAV comprises (ii) a promoter comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column B of Table 1. Preferably, the promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28. Most preferably, the promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0150] In some embodiments, the AAV comprises (iii) a 5'UTR comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column C of Table 1.

[0151] In some embodiments, the AAV comprises (iv) a polynucleotide encoding a protein of interest comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column D of Table 1.

[0152] In some embodiments, the AAV comprises (v) a 3'UTR comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column E of Table 1.

[0153] In some embodiments, the AAV comprises (vi) a 3'ITR comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column G of Table 1.

[0154] In some embodiments, the AAV additionally comprises a polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from Column F of Table 1.

[0155] In some embodiments, provided is an adeno-associated virus (AAV) vector, comprising a vector genome, wherein the vector genome comprises from 5' to 3' the sequences A-G from one row selected from Table 1, optionally connected by a linker polynucleotide sequence.

[0156] Cells

[0157] The present disclosure also provides a cell comprising a synthetic skeletal muscle - specific promoter according to the present disclosure. Also provided is a cell comprising an expression systems (such as one or more vectors) according to the present disclosure.

[0158] The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal e.g. rabbit, guinea pig, rat, mouse, hamster or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In preferred embodiments, the cell is a human cell.

[0159] The present disclosure also provides a method for producing a cell comprising an expressing system / vector according to the present disclosure, the method comprising introducing an expression system / vector of the present disclosure into a cell. In some embodiments, introducing an expression system / vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. adeno- associated viral transduction). In some embodiments, the expression system / vector is introduced to the cell in vivo, e.g. by administration of a vector according to the present disclosure (e.g. a viral vector, e.g. an adeno-associated viral vector) to a subject. In some embodiments, the expression system / vector is introduced into cells in culture ex vivo or in vitro.

[0160] Any suitable method may be employed to produce a cell according to the present disclosure. Such methods may comprise nucleic acid transfer for permanent (i.e. stable) or transient expression of the polynucleotide of the present disclosure. In some embodiments, following introduction into a cell, the polynucleotide may be integrated into or form part of the genomic DNA of the cell. In some embodiments, following introduction into a cell, the expression system / vector may be maintained extrachromosomally.

[0161] Any suitable genetic engineering platform may be used, and include gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol. (2014) 32: 189-225, hereby incorporated by reference in its entirety. Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3: 16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s) / vector(s) into cells include transduction, transfection and electroporation.

[0162] In some embodiments, the methods additionally comprise maintaining the cell under conditions suitable for expression of the vector / polypeptide of interest by the cell. The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.

[0163] Compositions

[0164] The present disclosure also provides compositions comprising the synthetic skeletal muscle-specific promoters, expression systems, vectors and cells described herein. In particular, the present disclosure provides pharmaceutical compositions and medicaments comprising the synthetic skeletal muscle-specific promoters, expression systems, vectors and cells of the present disclosure.

[0165] Such compositions may comprise the relevant article (z.e. the expression system / vector / cell) in a formulation suitable for clinical use. The present disclosure is concerned in particular with pharmaceutical compositions / medicaments comprising vectors according to the present disclosure.

[0166] The compositions of the present disclosure may comprise one or more pharmaceutically - acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g.titanium oxide).

[0167] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed.(A)Adejare), 23rd Edition (2020), Academic Press.

[0168] The pharmaceutical compositions / medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, a pharmaceutical composition / medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0169] Medicaments and pharmaceutical compositions may be formulated for administration to a blood vessel, or to a tissue / organ of interest (e.g. a tissue / organ affected by a disease / condition, e.g. a tissue / organ in which symptoms of the disease / condition manifest).

[0170] The pharmaceutical compositions / medicaments may comprise the expression system / vector / cell in a sterile or isotonic medium. The pharmaceutical compositions / medicaments may be provided in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via cannula) to a blood vessel, or a selected region of the human or animal body. The pharmaceutical compositions / medicaments may be provided in solid form, e.g. in lyophilised form.

[0171] The present disclosure also provides methods for producing pharmaceutical compositions / medicaments according to the present disclosure. Such methods may comprise mixing an expression system / vector / cell described herein with a pharmaceutically-acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent. Such methods generally include the step of bringing into association the expression system / vector / cell with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

[0172] Expression systems, vectors, cells and compositions according to the present disclosure may be modified and / or formulated to facilitate delivery to, and / or uptake by, a cell type / tissue / organ of interest (e.g. a cell type / tissue / organ in which symptoms of a disease / condition manifest).

[0173] Strategies for targeted delivery of polynucleotides are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.

[0174] In some embodiments, articles of the present disclosure may be encapsulated in a nanoparticle or a liposome. In some embodiments, articles of the present disclosure may be (covalently or non-covalently) associated with a cell -penetrating peptide (e.g a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.

[0175] Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3: 16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a PLGA, polypeptide, poly(P -amino ester), DOPE, P- cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle.

[0176] In some embodiments, expression systems and vectors according to the present disclosure comprise modification to incorporate one or more moieties facilitating delivery to, and / or uptake by, a cell type, organ or tissue of interest (e.g. a cell type / tissue / organ in which symptoms of a disease / condition manifest). In some embodiments, polynucleotides or vectors according to the present disclosure are linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and / or uptake by, a cell type, tissue or organ of interest. Therapeutic / prophylactic applications

[0177] The synthetic skeletal muscle-specific promoters, expression systems, vectors and cells and compositions of the present disclosure find use in therapy and prophylaxis.

[0178] Accordingly, the present disclosure provides an expression system, vector, cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an expression system, vector, cell or composition described herein for use in a method of treating or preventing a disease / condition described herein. Also provided is the use of an expression system, vector, cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of an expression system, vector, cell or composition described herein.

[0179] The intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition or reduce the pathology of a disease / condition. The intervention may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the intervention may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the intervention may prevent progression / development of the disease / condition a later stage e.g. a chronic stage).

[0180] It will be appreciated that the expression systems, vectors, cells and compositions described herein may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from an increase in the level of the polypeptide of interest (i.e. the polypeptide of interest encoded by the polynucleotide).

[0181] For example, the disease / condition may be a disease / condition associated with and / or characterised by deficiency / insufficiency of the polypeptide of interest. Deficiency / insufficiency of the polypeptide of interest may be positively associated with the onset, development or progression of the disease / condition, and / or positively associated with the severity of one or more symptoms of the disease / condition. Deficiency / insufficiency of the polypeptide of interest may be a risk factor for the onset, development or progression of the disease / condition. The disease / condition may be characterised by a decreased level of expression or activity of the polypeptide of interest, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition may be characterised by a decrease in the number / proportion / activity of cells expressing the polypeptide of interest, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue).

[0182] By way of illustration, in some embodiments, the polypeptide of interest may be ACTA1, and the disease / condition to be treated / prevented in accordance with the present disclosure may be a disease / condition caused by deficiency / insufficiency of ACTA1, e.g. a congenital myopathy, such as for example nemaline myopathy, intranuclear rod myopathy, actin filament aggregate myopathy, congenital fibre type disproportion and myopathy with core-like areas (Laing et al, Hum Mutat. 2009 Sep; 30(9): 1267-1277).

[0183] By way of further example, in embodiments wherein the polypeptide of interest is a polypeptide capable of inhibiting the expression and / or activity of a target antigen of interest, the disease / condition may be a disease / condition in which the target antigen, or cells comprising / expressing the target antigen are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing the target antigen is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing the target antigen may be a risk factor for the onset, development or progression of the disease / condition. The disease / condition may be characterised by an increase in the level of expression or activity of the target antigen, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition may be characterised by an increase in the number / proportion / activity of cells expressing the target antigen, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Therapeutic / prophylactic intervention in accordance with the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells comprising / expressing the target antigen.

[0184] By way of further example, the disease / condition may be a disease / condition to be treated by nucleic acid editing, and the polypeptide of interest may be a constituent protein of an appropriate site-specific nuclease nucleic acid editing system.

[0185] The present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering polynucleotides, vectors, cells and compositions according to the present disclosure to a subject (e.g. a subject in need of treatment).

[0186] Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed.(A)Adejare), 23rd Edition (2020), Academic Press.

[0187] Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intra-arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion.

[0188] Multiple doses of an article of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. Further methods

[0189] The present disclosure also provides a method for modifying a cell to express a therapeutic molecule, comprising introducing into a cell an expression system or vector according to the present disclosure. The present disclosure also provides a method for modifying a cell to express a protein of interest according to the present disclosure, comprising introducing into a cell an expression system or vector according to the present disclosure.

[0190] In some embodiments, introducing an expression systems or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).

[0191] Transfection relates to the process of introducing nucleic acid into cells using means other than viral infection and is hence a non-viral method. Transfection may be performed by physical / mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection and laser irradiation) or chemical methods (liposomal-based or non-liposomal based). Liposomal-based transfection reagents are chemicals which enable the formation of positively charged lipid aggregates, which can then merge with the phospholipid bilayer of the cell to facilitate the entry of foreign genetic material. Examples of liposomal-based transfection reagents include, but are not limited to Oligofectamine®, Lipofectamine® and DharmaFECT®. Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers and non-liposomal lipids. One example of a non-liposomal transfection reagent is polyethylenimine (PEI).

[0192] Electroporation may be performed e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, el 14, which is hereby incorporated by reference in its entirety.

[0193] Transduction is a process by which nucleic acids may be introduced into a cell by a virus or a viral vector. Accordingly, in some embodiments the polynucleotide is, or is comprised in, a viral vector, or the vector is a viral vector. Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety. Agents may be employed in the methods of the present disclosure to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0194] In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce an expression system or vector according to the present disclosure in the presence of cell culture medium comprising viral vector(s) comprising the expression system (referred to in the art as ‘spinfection’).

[0195] In some embodiments, the methods comprise culturing the cell under conditions suitable for expression of the polypeptide of interest the cell. In some embodiments, the methods comprise culturing the cell under conditions suitable for transcription of a polydeoxyribonucleotide. In some embodiments, the methods comprise culturing the cell under conditions suitable for post -transcriptional processing e.g. splicing) of a polyribonucleotide. In some embodiments, the methods comprise culturing the cell under conditions suitable for translation of a polypeptide from a polyribonucleotide.

[0196] Methods for culturing (including generating and / or expanding) populations of cells in vitro! ex vivo - including suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods etc. - are well known to the skilled person. Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0197] The present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell an expression system or vector according to the present disclosure. Where the polynucleotide is, or wherein the vector comprises / encodes, an OFF-switch, the cell may express the polypeptide of interest following introduction of the expression system / vector into the cell.

[0198] Subjects

[0199] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use). The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0200] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease / condition.

[0201] Sequence identity

[0202] The ‘sequence identity’ between a given nucleotide sequence (e.g. of a polynucleotide) and a reference nucleotide sequence is calculated by determining the percentage of the nucleotides in the given nucleotide sequence that are identical to those of the reference nucleotide sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences. Similarly, ‘sequence identity’ between a given amino acid sequence (e.g. of a polypeptide) and a reference amino acid sequence is calculated by determining the percentage of the amino acids in the given amino acid sequence that are identical to those of the reference amino acid sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences.

[0203] Pairwise and multiple sequence alignment for the purposes of evaluating sequence identity between two or more nucleotide or amino acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21, 951 -960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0204] Exemplary sequences

[0205]

[0206] Table 1

[0207] NUMBERED PARAGRAPHS

[0208] The following numbered paragraphs (paras) describe particular aspects and embodiments of the present disclosure:

[0209] 1. A synthetic skeletal muscle-specific promoter comprising at least 6 MyoG elements operably linked to a promoter element.

[0210] 2. The synthetic skeletal muscle-specific promoter according to para 1, comprising at least 8 MyoG elements operably linked to a promoter element.

[0211] 3. The synthetic skeletal muscle-specific promoter according to para 1 or 2, comprising 6 MyoG elements or 8 MyoG elements, operably linked to a promoter element.

[0212] 4. The synthetic skeletal muscle-specific promoter according to any one of para 1 to 3, wherein the synthetic skeletal muscle-specific promoter has 8 MyoG elements.

[0213] 5. The synthetic skeletal muscle-specific promoter according to any one of para 1 to 4, wherein the synthetic skeletal muscle-specific promoter comprises 8 MyoG elements.

[0214] 6. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 5, wherein the MyoG elements individually comprise, or consist of, the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24). 7. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 6, wherein the 8 MyoG elements comprise, or consist of, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25.

[0215] 8. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 7, wherein the MyoG elements are connected through at least one linker.

[0216] 9. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 8, wherein the MyoG elements are connected through a linker.

[0217] 10. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 9, wherein the MyoG elements are connected through a linker of the same length.

[0218] 11. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0219] 10, wherein the at least one linker has a length of 4-14bp, preferably 4-6 bp, most preferably 4 bp.

[0220] 12. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0221] 11, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO:25, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99.

[0222] 13. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0223] 12, wherein the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 or SEQ ID NO:50.

[0224] 14. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0225] 13, wherein the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, and SEQ ID NO: 84. 15. A synthetic skeletal muscle-specific promoter comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27 or SEQ ID NO:28.

[0226] 16. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to 15, wherein the synthetic skeletal muscle-specific promoter comprises or consists of the sequence of SEQ ID NO:27 or SEQ ID NO:28.

[0227] 17. A synthetic skeletal muscle-specific promoter comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0228] 18. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0229] 17, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO: 89.

[0230] 19. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0231] 18, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0232] 20. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0233] 19, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. 21. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0234] 20, wherein the synthetic skeletal muscle-specific promoter consists of a sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0235] 22. The synthetic skeletal muscle-specific promoter according to any one of paras 1 to

[0236] 21, wherein the ratio of i) the promoter activity in HSMM cells 5 days after transduction with an AAV of the serotype 2 at an MOI of 20,000 to ii) the promoter activity in Huh7 cells 3 days after transduction with the same AAV at an MOI of 20,000 is 10 or more.

[0237] 23. The synthetic skeletal muscle-specific promoter according to para 22, wherein the ratio is 100 or more, 1000 or more, 10'000 or more, or 20'000 or more.

[0238] 24. The synthetic skeletal muscle-specific promoter according to any one of para 22 or 23, wherein the promoter activity is determined as fluorescence intensity of the expression of a fluorescent protein operably linked to the synthetic promoter, wherein the fluorescent protein and the synthetic promoter are included in the transgene of the AAV.

[0239] 25. The synthetic skeletal muscle-specific promoter according to para 24, wherein the fluorescent protein is mGreenLantern.

[0240] 26. An expression system comprising a synthetic skeletal muscle-specific promoter according to any one of paras 1 to 25 operationally linked to a polynucleotide encoding a polypeptide of interest.

[0241] 27. A vector comprising a synthetic skeletal muscle-specific promoter according to any one of paras 1 to 25 operationally linked to a polynucleotide encoding a polypeptide of interest, optionally wherein the vector is an adeno-associated virus (AAV) vector.

[0242] 28. The expression system according to para 26 or the vector according to para 27, wherein the polynucleotide encoding the polypeptide of interest comprises a start codon located 3' to the synthetic skeletal muscle-specific promoter.

[0243] 29. The expression system or the vector according to any one of paras 26 to 28, further comprising a polyadenylation sequence 3’ of the polynucleotide encoding the polypeptide of interest. 30. The expression system or the vector according to any one of paras 26 to 29, further comprising a 5'UTR sequence operationally linked to the synthetic skeletal muscle-specific promoter, wherein the 5'UTR sequence is located 3' to the synthetic skeletal muscle-specific promoter, optionally wherein the 5'UTR comprises an intron.

[0244] 31. The expression system or the vector according to para 30, wherein the intron is not a complete naturally occurring promoter intron, or wherein the intron is not a complete naturally occurring intron.

[0245] 32. The expression system or the vector according to para 30 or 31, wherein the intron is a truncated natural or synthetic intron.

[0246] 33. The vector according to any one of paras 27 to 32, further comprising an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end.

[0247] 34. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5’ to 3’ order:

[0248] (i) a 5' inverted terminal repeat (ITR) sequence;

[0249] (ii) a synthetic skeletal muscle-specific promoter according to any one of paras 1 to 25;

[0250] (iii) optionally, a 5' UTR sequence;

[0251] (iv) a polynucleotide encoding a therapeutic molecule;

[0252] (v) a 3' UTR sequence; and

[0253] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0254] 35. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5’ to 3’ order:

[0255] (i) a 5' inverted terminal repeat (ITR) sequence;

[0256] (ii) a synthetic skeletal muscle-specific promoter according to any one of paras 1 to 25;

[0257] (iii) optionally, a 5' UTR sequence;

[0258] (iv) a polynucleotide encoding a protein of interest;

[0259] (v) a 3' UTR sequence; and

[0260] (vi) a 3' inverted terminal repeat (ITR) sequence. 36. An AAV vector according to para 34 or 35, wherein the 5' ITR sequence comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence according to SEQ ID NO: 1.

[0261] 37. An AAV vector according to any one of paras 34 to 36, wherein the 5' UTR sequence comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO 35.

[0262] 38. An AAV vector according to any one of paras 34 to 37, wherein the polynucleotide encoding the protein of interest comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence according to SEQ ID NO:36 or SEQ ID NO:37.

[0263] 39. An AAV vector according to any one of paras 34 to 38, wherein the 3' UTR sequence comprises a polyadenylation sequence.

[0264] 40. An AAV vector according to any one of paras 34 to 39, wherein the 3' UTR sequence comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence according to SEQ ID NO:38 or SEQ ID NO: 39.

[0265] 41. An AAV vector according to any one of paras 34 to 40, wherein the 3' ITR sequence comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence according to SEQ ID NO:2.

[0266] 42. A cell comprising an expression system according to any one of paras 26 to 32, or the vector according to any one of paras 27 to 33, or the AAV vector according to any one of paras 34 to 41.

[0267] 43. A pharmaceutical composition comprising the expression system according to any one of paras 26 to 32, or the vector according to any one of paras 27 to 33, or the AAV vector according to any one of paras 34 to 41, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 44. An expression system according to any one of paras 26 to 32, or the vector according to any one of paras 27 to 33, or the AAV vector according to any one of paras 34 to 41, or a pharmaceutical composition according to para 43, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest in skeletal muscle cells.

[0268] 45. Use of an expression system according to any one of paras 26 to 32, or the vector according to any one of paras 27 to 33, or the AAV vector according to any one of paras 34 to 41, or a pharmaceutical composition according to para 43, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.

[0269] 46. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject an expression system according to any one of paras 26 to 32, or the vector according to any one of paras 27 to 33, or the AAV vector according to any one of paras 34 to 41, or a pharmaceutical composition according to para 43.

[0270] 47. The invention as hereinbefore described with reference to the Examples and Figures.

[0271] ***

[0272] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0273] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0274] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0275] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0276] As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. Reference herein to peptides, polypeptides and proteins also includes glycopeptides / glycopolypeptides / glycoproteins, lipopeptides / lipopolypeptides / lipoproteins, nucleopeptides / nucleopolypeptides / nucleoproteins, etc.

[0277] As used herein, an amino acid sequence, or a region of a polypeptide, which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97 %, >98%, >99% or 100% sequence identity to the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21, 951 -960).

[0278] Similarly, a nucleotide sequence, or a region of a polynucleotide, which ‘corresponds’ to a specified reference nucleotide sequence or region of a polynucleotide has at least 60%, e.g. one of at least >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97 %, >98%, >99% or 100% sequence identity to the amino acid sequence of the nucleotide sequence / polynucleotide / region. A polynucleotide / region / position of a polynucleotide / nucleotide sequence which ‘corresponds’ to a specified reference nucleotide sequence / region / position of a polynucleotide / nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21, 951 -960). As used herein, an amino acid sequence (e.g. the amino acid sequence of a peptide / polypeptide / domain / region) which is ‘derived from’ a reference amino acid sequence e.g. the amino acid sequence of a reference peptide / polypeptide / domain / region) comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference amino acid sequence. Similarly, a nucleotide sequence (e.g. a nucleotide sequence of a polynucleotide) which is ‘derived from’ a reference nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference nucleotide sequence.

[0279] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0280] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0281] Methods described herein may preferably be performed in vitro. The term ‘in vitro" is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo" is intended to encompass procedures with / on intact multi-cellular organisms.

[0282] BRIEF DESCRIPTION OF THE FIGURES

[0283] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0284] Figure 1 : Reporter gene expression of first -generation AAV vectors in human skeletal muscle myocytes (HSMM). Reporter AAVs were transduced into HSMM at MOIs ranging from 100 to 400 000 and eGFP levels were measured using cytometry. Cells transduced with a promoterless reporter construct (with mGL instead of eGFP) were used as controls. Mean fluorescence indices (MFI) are plotted.

[0285] Figure 2: Reporter gene expression of first -generation AAV vectors in hepatocellular carcinoma cell lines. Reporter AAVs were transduced into HepG2 (2A) and Huh7 (2B) at MOIs ranging from 100 to 400 000 and eGFP levels were measured using cytometry. Cells transduced with a promoterless construct (with mGL instead of eGFP) were used as controls. Mean fluorescence indices (MFI) are plotted.

[0286] Figure 3: Maps of synthetic promoters comprising motifs of muscle-specific transcription factors and minimal promoter elements. The localization of the transcription factor binding motifs (black arrows) and minimal promoters (gray arrows) are indicated below the nucleotide sequences.

[0287] Figure 4: Reporter gene expression of second and third-generation AAV vectors in human skeletal muscle myocytes (HSMM). Reporter AAVs were transduced into HSMM at MOIs 400 000 and mGL levels were measured using cytometry. Cells transduced with a promoterless reporter construct were used as controls. Mean fluorescence indices (MFI) are plotted.

[0288] Figure 5: Reporter gene expression of second and third -generation AAV vectors in hepatocellular carcinoma cell lines. Reporter AAVs were transduced into HepG2 (A) and Huh7 (B) at MOI 250 000 and mGL levels were measured using cytometry. Cells transduced with a promoterless construct were used as controls. Mean fluorescence indices (MFI) are plotted.

[0289] Figure 6: Reporter gene expression of a subset of second- and third-generation AAV vectors in HSMM, HepG2 and Huh7 cells. Reporter AAVs were transduced into HSMM, HepG2 and Huh7 at MOI 20 000 and mGL levels were measured using cytometry. Cells transduced with a promoterless reporter construct were used as controls. (6A) Mean fluorescence indices (MFI); (6B) Ratio of reporter gene expression in HSMM and Huh7 cells. For each construct, the MFI in HSMM was divided by the MFI in Huh7. Before calculating the ratio, the MFI of the promoterless control was subtracted from the MFI of the test constructs.

[0290] Figure 7: Relative gene expression of fourth-generation designs optimizing 8x MyoG motif CMV2 reporter. Adherent HEK293 cells were co -transfected with a 4thgeneration promoter construct and MyoG transcription factor or control plasmid, respectively. The impact of nonameric MyoG binding motif quantity (Ox, 2x, 4x, 6x, 8x, 12x, 14x, 16x) on gene expression was investigated (7 A). The highest expression was observed with 6 motifs, showing a 1.73-fold increase compared to the original 8x-motif construct. Bidirectional clusters or altered nucleotides surrounding the core binding motif (7B) did not result in a relevant increase of gene expression. The effect of the distances between motifs were measured for 4bp, 6bp (8xMoG CMV2), 8bp, lObp, 12bp, and 14bp (7C). The highest geometric mean fluorescence indices (MFI) was achieved with a 4 bp distance between motifs (1.39-fold increase), with decreasing expression observed at longer distances. Reducing the spacer distance between the last motif and the minimal promoter from 44bp to Obp led to a 2.10-fold increase in expression (7D). Lastly, the additional minimal promoters tested did not outperform the original minimal CMV promoter 2 (7E). Data represent the mean ± SD of three biological replicates, each with three technical replicates.

[0291] Figure 8: Evaluation of improved fifth-generation reporter design relative to 8x MyoG motif CMV2 reporter. Reducing the distance between the last MyoG binding motif and the minimal promoter had the most significant impact on gene expression (8 bp: 1.52 -fold increase, 4 bp: 1.75-fold increase, 0 bp: 2.09-fold increase), followed by the ideal number of motifs (8x MyoG motifs: 1.72-fold increase, 6x MyoG motifs: 2.09-fold increase). The optimal configuration combined 6 MyoG binding motifs with no spacer, resulting in a 2.09-fold increase in expression. Data represent the mean ± SD of two biological replicates.

[0292] EXAMPLES

[0293] EXAMPLE 1 : GENERAL METHODS

[0294] 1.1 Recombinant DNA techniques Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer’s instructions. Desired gene segments were synthesized at Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning / sequencing plasmids. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the cloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow transfer into the respective reporter vectors.

[0295] 1.2 Production of rAAV by triple transfection

[0296] Recombinant AAVs were produced by triple transfection in Expi293F (Thermo -Fisher Scientific) cells using PEI MAX (Polysciences) as a transfection reagent according to the manufacturers’ recommendation. In brief, Expi293F cells were grown in 500 mL shake flasks and a culture volume of 125 mL to a density of 3xl06cells / mL. For transfection, equimolar amounts of a first plasmid carrying the rep-cap gene of AAV2 (pAAV2 rep / cap), a second plasmid carrying the adenoviral helper genes E4, E2a and VA (pHelper) and a third plasmid carrying the reporter gene of interest flanked by AAV2 ITRs (pTransgene) were combined and diluted in 2.5 mL Expi293 expression medium. In total 0.6 pg DNA per 106cells were used. PEI MAX was separately mixed with 2.5 mL Expi293 expression medium and combined with the previously prepared DNA mix at a ratio of 1 :2.5 (DNA:PEI). After incubation the DNA-PEI complexes were added dropwise to the cells. Finally, transfected cell cultures were incubated at 37°C, 125 rpm and 8% CO2 for 72 h.

[0297] To harvest recombinant AAVs, cells were centrifuged at 300 g for 5 min and chemically lysed using a Triton CG-110-based lysis buffer containing 50 U / mL DENARASE (c-LEcta). For complete lysis, the cells were incubated and agitated for 1.5 h at 37°C and 180 rpm. To remove cell debris after the lysis, the suspension was centrifuged at 4,000 x g for 60 min. At last, the supernatant was passed through a 0.8 / 0.2 pm dual filter unit and collected. The sterile and clarified cell lysates were stored at -80°C until the iodixanol gradient purification was performed.

[0298] 1.3 Purification of rAAV by iodixanol gradient ultracentrifugation

[0299] For purification and separation of desired full rAAV capsids, iodixanol density-gradient ultracentrifugation was applied, using layers of 60%, 40%, 25% and 15% iodixanol. Filtrated virus lysates were thawed and transferred to an ultracentrifugation tube. Subsequently, the differently concentrated iodixanol gradients were added on top. Tubes were mass balanced, sealed and centrifuged at 230,000 x g for 2 h at 4°C in a Beckman 50.2 Ti Rotor. Upon centrifugation, tubes were fixed well and punctured on the top with a 21G needle to allow air inflow. A second 21G needle attached to a syringe was carefully inserted below the 60-40% interface and 1.0 to 2.5 mL were collected from the 40% phase. The resulting vector stocks were either stored at -80°C or further processed to exchange the iodixanol against PBS and increase the virus concentration. This was achieved by ultrafiltration using Amicon Ultra- 15 centrifugal filter units. Concentrated samples were stored at -80°C.

[0300] 1.4 rAAV transduction and analysis of reporter gene expression

[0301] Primary human skeletal muscle myoblasts (HSMM) were used as a cellular model to measure promoter activity in human skeletal muscle. Two hepatocellular carcinoma cell lines, HepG2 and Huh7 served as control cell lines to evaluate non-muscle specific promoter activity.

[0302] For the transduction of HSMM, cells were seeded at 3 to 4.5xl04cells / cm2(typically in 96 well format) in growth medium (Lonza SkBM-2 or PromoCell skeletal muscle growth medium plus respective supplements as provided by the vendor) and checked for proper morphology. On the next day, the cells were transduced by constructs diluted in MEM (L- Glutamine, Pen / Strep, 10 pM Etoposide, w / o phenol red). 4-5 hours post-transduction, cells were supplemented with fetal calf serum (cf=2.5% v / v). On day 3, etoposide was withdrawn by growth medium exchange. On day 5 the medium was changed against differentiation medium (Lonza DMEM / BioWhittaker F-12 [1 : 1], L-Glutamine, heat inactivated horse serum plus Pen / Strep). Between d7 and d9 cells were typically analyzed on an Incucyte (Sartorius) or via flow cytometry for fluorophore expression (e.g. eGFP, mGreenLantern). For flow cytometry, cells were washed twice with PBS and detached with 0.05% Trypsin / EDTA. Detachment was stopped by addition of growth medium or PBS / 10% FBS. Cells were then centrifuged, washed and co-stained for viability (7AAD) for 15 min.

[0303] For the transduction of HepG2 or Huh7 cells, 4.5xl04(HepG2) or 6x104(Huh7) cells were seeded per cm2(96 well format). Cells were transduced with different MOIs (see figure legends) by medium aspiration, followed by addition of 100 pL fresh cultivation medium plus 100 pL of the respective AAV dilutions in cultivation medium. Cells were analyzed on day 3 for transgene expression.

[0304] EXAMPLE 2

[0305] 2.1 Database search for skeletal muscle-specific promoters and construction of first generation reporter constructs

[0306] Potential skeletal muscle-specific promoters were identified in a three-step process combining the information of different genomics databases. First, the GTEx tissue expression database (Nat Genet. 2013 Jun; 45(6): 580-585.) was screened for skeletal muscle specific genes. Second, annotated promoter regions were identified using the FANTOM5 sequence collection (Nature. 2014 Mar 27;507(7493):462-70). Third, in order to narrow the potential promoter boundaries, binding sites of known skeletal muscle-specific transcription factors (MYOD, MYOG, MYF5, MYF6) were mapped. In this way, 8 candidate promoters with either 1.9, 1.2 or 0.5 kb length were defined. These promoter elements were combined in 5’ to 3’ direction with the 5’UTR of the ACT Al gene, either with or without the first intron, an open reading frame coding for eGFP, and a 3’UTR consisting of the bovine growth hormone poly A signal (bGH poly A; J Biol Chem. 1992 Aug 15;267(23): 16330-4) and the human gastrin terminator element (HGT; Mol Cell Biol 1986 Apr; 6(4): 1032-1043). These reporter genes were inserted between the AAV2 inverted terminal repeats of an rAAV transgene plasmid. Two corresponding plasmids that either comprise the well -characterized muscle-specific promoter MHCK7 (Mol Ther. 2007 Feb;15(2):320-9) or the strong ubiquitous promoter CAG (Gene. 1991 Dec 15; 108(2): 193-9) were generated for comparison. 2.2 Evaluation of first generation reporter constructs rAAV particles were generated and purified as described and transduced into HSMM cells. Constructs carrying eGFP under control of potential skeletal muscle -specific promoters were transduced at MOI between 100 and 400 000 to assess the dynamic range of the assay. The rAAV carrying the strong ubiquitous CAG promoter was transduced at a single intermediate MOI of 20 000 whereas as for the no -promoter construct the maximal MOI of 400 000 was applied. Reporter gene expression was measured by flow cytometry.

[0307] As expected, increasing fluorescence was measured as the MOI increased (Figure 1). However, at MOIs of 100 000 and higher the signal tended to reach a plateau. Apart from the MHCK7 and C AG-based control constructs, only the constructs driven by the ACTA1 or the MYH2 promoter showed considerably higher eGFP expression than the no-promoter control. The expression of the CAG-driven reporter rAAV was considerably higher than with any other construct.

[0308] When the same constructs were transduced into HepG2 or Huh7 strong reporter expression was only observed with the CAG promoter (Figure 2). The expression of the ACTA1 promoter construct was slightly above the no promoter control, whereas all other constructs expressed at background level.

[0309] Based on these results the ACTA1 promoter and the MYH2 promoter were selected for further engineering to increase strength and specificity of skeletal muscle -specific expression.

[0310] EXAMPLE 3

[0311] 3.1 Construction of second generation reporter constructs

[0312] Three additional versions of the MYH2 promoter were created. In one version (MYH2 0.7 kb), the 5’-terminal 500 bp were deleted from the 1.2 kb-fragment resulting in a fragment of 0.7 kb. In a second version (MYH2 0.7 kb doubled), this 0.7 kb fragment was duplicated. In a third version (MYH2 1.2 kb min CMV5'), a DNA stretch of 31 bp upstream of the TATA box was replaced by the corresponding sequence of the human CMV major immediate early promoter. These fragments were combined in 5’ to 3’ direction with a short synthetic 5’UTR, a short human beta globin / human Ig heavy chain chimeric intron, an open reading frame coding for mGreenLantern (mGL; PNAS. 2020 Dec 1;117(48) : 30710 -30721 ), the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Hum Gene Ther. 1999 Sep 20;10(14):2295-305) and the human growth hormone poly A signal (hGH poly A, GenBank Accession NG_011676). These reporter genes were inserted between the AAV2 ITRs of an rAAV transgene plasmid. For comparison, five corresponding plasmids were generated either comprising no-promoter, the 1.9 kb ACTA1 promoter, the 1.2 kb MYH2 promoter, the MHCK7 promoter or the ubiquitous and strong CB A promoter which is a variant of the CAG promoter lacking the first 78 bp and differing in four other nucleotide positions. One additional MHCK7- based construct was created comprising the SV40 late 16s minimal intron as described in European patent application EP3442600 instead of the human beta globin / human Ig heavy chain chimeric intron.

[0313] In order to compensate for the different lengths of the promoters and to keep the size of the recombinant AAV genomes approximately constant, 500, 1000 or 1481 bp non-coding stuffer DNA were inserted upstream of the 3’ ITR sequence.

[0314] In addition, a comparative promoter occupancy analysis (Nat Rev Genet. 2009 Jul;10(7):443-456) was performed based on transcriptomics data of HepG2 and HSMM cells for the first generation of promoters. The goal was to strengthen activating and to weaken repressive transcription factor activities in skeletal muscle cells, whereas in other cells, explicitly liver cells, the opposite effect was intended. GTEx tissue expression data of various transcription factors were also considered.

[0315] Based on the result, four additional ACTA1 promoter variants were designed. First, the 1.9 kb ACTA1 promoter was shortened to the 3 ’-terminal 800 bp (ACTA1 A400bp). Second, within this 800 bp ACT Al promoter the three binding motifs for MYOG were strengthened (ACTA1 3xMY0G). Third, the binding site of transcription repressor ZNF619 (G3 (Bethesda). 2018 Jan 8;8(l):219-229) in ACTA1 A400bp was replaced by a binding site of ZNF121 (Genome Res. 2016 Dec;26( 12): 1742-1752), which is not expressed in HSMM and skeletal muscle. This modification resulted in promoter ACTA1 ZNF619 / ZNF121. Fourth, a proximal SRF site (Cell.2013 Jan 17;152(l-2):327-39) was removed in ACTA1 ZNF619 / ZNF121 yielding ACT Al ASRF site.

[0316] Furthermore, four additional MYH2 promoter variants were designed. First, the MYH2 promoter was shortened to the 3’-terminal 500 bp, yielding MYH2 0.5 kb. Second, the MYOG binding site within the 500 bp fragment was strengthened, yielding MYH2 strengthened MYOG. Third, the binding site of the transcriptional repressor ZNF136 (G3 (Bethesda). 2018 Jan 8;8(l):219-229) in the latter version was replaced with a binding site for the transcriptional repressor ZNF439 (G3 (Bethesda). 2018 Jan 8;8(l):219-229), which is highly expressed in most human tissues but not skeletal muscle or HSMM. This resulted in version MYH2 ZNF136 / ZNF439. Finally, a weak EGR1 site (Cell. 2013 Jan 17;152(1 -2):327-39) around position 240 of MYH2 ZNF136 / ZNF439 was replaced with a moderately strong MEF2D site (Cell.2013 Jan 17;152(l-2):327-39), yielding MYH2 EGR1 / MEF2D.

[0317] 3.2 Construction of third generation reporter constructs

[0318] Four synthetic promoters were designed by combining binding motifs of skeletal muscle-specific transcription factors with minimal promoters (Figure 3).

[0319] In one version, four copies of the nonameric binding motif of MYOG (AGCAGCTGC; Nucleic Acids Res. 2022 Jan 7;50(Dl):D165-D173) separated from each other by a hexameric random sequence (AGCCTT) were combined with short spacer sequence and a minimal promoter sequence comprising a TATA box and an Inr initiator element, yielding Pmin 4xMyoG. In the same way, an additional version with eight MYOG binding sites was created and named Pmin 8xMyoG. In a third synthetic promoter, called CMV2 8xMyoG, the same fragment comprising the eight MYOG binding motifs and the spacer was combined with a minimal CMV MIE promoter sequence, extending from position - 54 to position +66. A fourth promoter called CMV2 MyoG&MyoD&Mef2D was obtained by replacing four MYOG binding motifs in CMV2 8xMyoG by two octameric binding motifs for the muscle specific transcription factor MYOD (GCACCTGT; Nucleic Acids Res. 2022 Jan 7;50(Dl):D165-D173) and two decameric binding motifs of the muscle specific transcriptional activator MEF2D (CTATAAATAG, Nucleic Acids Res. 2022 Jan 7;50(Dl):D165-D173), respectively. In the same way as for the second generation reporter constructs, these promoters were combined in 5’ to 3’ direction with a short synthetic 5’UTR, a short human beta globin / human Ig heavy chain chimeric intron, an open reading frame coding for mGreenLantern, the woodchuck hepatitis virus posttranscriptional regulatory element and the human growth hormone poly A signal. These reporter genes were inserted between the AAV2 ITRs of an rAAV transgene plasmid. In these constructs the 1481 bp stuffer sequence was placed upstream of the 3’ ITR.

[0320] 3.3 Evaluation of second and third generation reporter constructs rAAV particles carrying an mGL reporter gene under control of a second and third generation promoter were generated and purified as described and transduced into HSMM cells at an MOI of 400 000. Reporter gene expression was measured by flow cytometry. A corresponding rAAV with a promoterless mGL reporter was transduced as well to assess the background signal of the assay. In a first experiment the corresponding construct comprising the CB A promoter was not transduced because the reporter signal was expected to be out of range (Figure 4).

[0321] As for the new MYH2 promoter variants, none of them showed higher reporter gene expression than the first generation 1.2 kb version. This was against the expectation, because the modifications were aiming at increasing the expression in HSMM. The modifications that were applied to the 1.9 kB ACTA1 promoter did not drastically change the expression of the reporter gene either, apart from ACT Al ASRF site. This one unintendedly showed much lower expression than all the other versions. Both MHCK7 promoter constructs and the 1.9 kb ACTA1 promoter showed similar expression levels, which were higher than those of the MYH2 constructs.

[0322] All synthetic promoters were active in HSMM cells and drove higher reporter gene expression than the no-promoter control. Especially, the two promoters comprising eight MyoG binding motifs (herein referred to as MyoG elements), Pmin 8xMyoG and CMV2 8xMyoG were more active than all other constructs tested. Surprisingly, the replacement of four MYOG motifs in CMV2 8xMyoG with two MYOD and two MEFD2 motifs yielding CMV2 MyoG&MyoD&Mef2D strongly decreased reporter gene expression in HSMM cells. The same constructs were transduced into HepG2 and Huh7 cells at an MOI of 250000 (Figure 5). In HepG2 cells, reporter gene expression of the promoterless control was remarkably high and the expression of most constructs was similar or only slightly higher. Relatively strong reporter expression was only observed with the ubiquitous CBA promoter. In Huh7, the expression of the CBA promoter construct was also strong. Apart from that, elevated expression compared to the no-promoter controls was observed for ACTA1 1.9kb, ACTA1 A400bp and ACTA1 3MY0G. Interestingly, the expression driven by ACTA1 ZNF619 / ZNF121 was decreased compared to ACTA1 A400bp suggesting that the replacement of the ZNF619 binding motif by a motif for ZNF121 decreased undesirable non-muscular expression.

[0323] In summary, we observed remarkable muscle-cell specific expression with the synthetic promoters Pmin 8xMyoG and CMV2 8xMyoG and a reduction of non-muscular expression with ACTA1 ZNF619 / ZNF121. In contrast, any modifications made to MYH2 1.2kb did not show the intended effects. Therefore we decided to further investigate Pmin 8xMyoG, CMV2 8xMyoG, ACTA1 ZNF619 / 121 and MYH2 1.2 kb together with respective control constructs for their capability to drive muscle specific gene expression. To this end, the respective AAV vectors carrying the mGL reporter gene were transduced into HSMM, HepG2 and Huh7 cells at an MOI of 20000 and the expression of mGL was determined cytometrically (Figure 6A).

[0324] The results largely confirmed previous observations: the ubiquitous CBA promoter showed the strongest expression of all promoters in all three cell lines; the synthetic promoters Pmin 8xMyoG and CMV2 8xMyoG showed strong expression in HSMM cells at levels that were about 2-fold (Pmin 8xMyoG) or 6 fold (CMV2 8xMyoG) higher compared to the muscle specific benchmark promoter MHCK7 SV40 intron. Importantly Pmin 8xMyoG and CMV2 8xMyoG showed almost no reporter gene expression in both liver carcinoma cell lines.

[0325] Similar to previous experiments, the expression in HepG2 was generally low compared to the no-promoter control, even with the CBA promoter. Therefore we focused our analysis on HSMM and Huh7 cells. As a quantitative measure of muscle-specificity we calculated the ratio of expression levels in HSMM cells and Huh7 cells (Figure 6B). The expression ratio HSMM / Huh7 of Pmin 8xMyoG was about fourfold higher than with MHCK7 SV40 intron and even tenfold higher than with the CBA promoter. With CMV2 8xMyoG the expression ratio was even more shifted toward HSMM being sixfold higher than with MHCK7 SV40 intron and 15-fold higher than with CBA.

[0326] EXAMPLE 4

[0327] 4.1 Construction of fourth generation reporter constructs

[0328] For further optimization, additional constructs were designed based on the most successful 3rd generation reporter construct, the highly specific CMV2 8xMyoG reporter. The design was carefully selected not only for its success in Example 3, but also for its exclusive specificity to skeletal muscle. This specificity is particularly important because certain AAV capsids can transduce not only skeletal muscle cells but also cardiac and smooth muscle cells, as well as off-target tissues such as the liver, lymphocytes, and testis. By utilizing a skeletal muscle - specific promoter, gene expression is confined to muscle cells, thereby minimizing off-target effects and significantly enhancing the precision and efficacy of the therapeutic approach. Multiple aspects, such as i) quantity of the nonameric MyoG binding motifs (AGCAGCTGC; Nucleic Acids Res. 2022 Jan 7;50(Dl):D165-D173; SEQ ID NO:28, 50, 52-57), ii) directionality and positioning of such motifs (SEQ ID NO:58-61), iii) length of sequence separating MyoG binding motifs (SEQ ID NO:28, 62-66), iv) length of spacer sequence between last binding motif and minimal promoter (SEQ ID NO:28, 67-77), and v) design of minimal promoters were investigated (SEQ ID NO:28, 50, 78-85). The synthetic 5’UTR (pxlOOO, SEQ ID NOVO), a short human beta globin / human Ig heavy chain chimeric intron, the open reading frame coding for mGreenLantern (mGL; PNAS. 2020 Dec 1 ; 117(48):30710 - 30721), the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Hum Gene Ther. 1999 Sep 20;10(14):2295-305) and the human growth hormone poly A signal (hGH poly A, GenBank Accession NG_011676) were not altered.

[0329] 4.2 Transfection of adherent HEK293

[0330] In order to improve the CMV2 8xMyoG reporter construct, adherent HEK293 cells were co-transfected with reporter variants as well as the transcription factor MyoG or a negative control respectively, using Lipofectamine™ 3000 Transfection Reagent (Invitrogen™) according to the manufacturer’s protocol. In brief, adherent HEK293 cells were seeded in a 96- well plate to achieve 70-90% confluency at the time of transfection. Each sample included either the transcription factor MyoG or a negative control, one of the previously mentioned reporter variants (3.5 fmol), and a transfection control. The total DNA amount per well was 93 ng and 70,6 respectively. The DNA was diluted in Opti-MEM™ Reduced Serum Medium, and 0.2 pL of P3000™ Reagent was added to the DNA solution. Separately, Lipofectamine™ 3000 Reagent was diluted in Opti-MEM™ Reduced Serum Medium at 0.2 pL per well. The diluted DNA / P3000™ mixture was combined with the diluted Lipofectamine™ 3000 Reagent and incubated for 15 minutes at room temperature to form complexes. The DNA- Lipofectamine™ 3000 complexes were then added to the HEK293 cells in the 96 -well plate, and the plate was gently rocked to ensure even distribution. The cells were incubated at 37°C in a CO2 incubator for 72 hours. Post-incubation, transfection efficiency and gene expression were analyzed using appropriate assays such as fluorescence microscopy and flow cytometry.

[0331] Each biological replicate included three technical replicates, and a total of three biological replicates were performed to ensure robustness and reproducibility.

[0332] 4.3 Assessing Reporter Gene Expression: Critical Parameter Determination

[0333] In this example of the invention, the impact of varying numbers of the nonameric MyoG binding motif (Ox, 2x, 4x, 6x, 8x, 12x, 14x, 16x) on gene expression was investigated (Figure 7A). The starting construct contained 8 motifs. It was observed that the highest expression level was achieved with the construct containing 6 motif repeats (SEQ ID NO: 54) , which exhibited a 1.73-fold increase in expression compared to the original CMV2 8xMyoG reporter construct (8-motif construct, SEQ ID NO:28). Surprisingly, this finding shows that reducing the number of motifs to six can enhance gene expression, highlighting the importance of an optimized quantity of binding motifs. In contrast, no critical changes in expression were observe by the design of bidirectional clusters or altering nucleotides surrounding the core binding sequence (JASPAR: agCAGCTGc, HOCOMOCO: cgCAGCTGcc; Figure 7B).

[0334] Next, the distances between motifs were examined (Figure 7C). It was observed that a distance of 4 bp (SEQ ID NO: 62) resulted in the highest expression, with nearly equal expression levels observed for distances of 6 and 8 bp between motifs. In contrast, decreasing expression levels were noted for increasing distances such as 10, 12, and 14 bp between motifs, suggesting that shorter distances between motifs are more favorable for optimal gene expression.

[0335] The distance between motifs and the promoter sequence was thoroughly investigated, revealing some noteworthy results (Figure 7D). The original reporter construct featured a 44 bp spacer. From this starting point, the distance was systematically reduced. While distances of 42 bp, 36 bp, and 20 bp maintained expression levels similar to the original construct, distances of 40 bp, 28 bp, 24 bp, and 16 bp led to a decrease in expression. Notably, shorter distances yielded increased expression: a 12 bp spacer resulted in a 1.23 -fold increase, an 8 bp spacer led to a 1.75-fold increase, and a 0 bp spacer achieved a 2.10-fold increase in expression. These findings highlight the significant impact of reducing the distance between motifs and the promoter sequence on enhancing gene expression.

[0336] Furthermore, the efficacy of multiple additional promoters was evaluated to identify candidates that could outperform the originally chosen minimal promoter (Minimal promoter 2, SEQ ID NO:50). Five minimal promoter pairs, including shorter versions of synthetic (SEQ ID NO: 28, 50, 78-81) and endogenous promoters (SEQ ID NO:82-85), were tested. Despite rigorous testing, none of the five promoter pairs demonstrated superior performance compared to the original minimal promoter 2.

[0337] The data suggest that careful consideration of both the positioning and the number of binding motifs is essential for maximizing transcriptional efficiency. These findings provide a robust framework for the development of highly efficient synthetic cell -specific regulatory elements, which can be tailored for various applications in gene therapy, synthetic biology, and biotechnology. The insights gained from the 4th generation construct design will inform future iterations and refinements of the previously identified CMV2 8 MyoG reporter construct.

[0338] EXAMPLE 5

[0339] 5.1 Construction of fifth generation reporter constructs

[0340] Following the learnings of Example 4, further reporter constructs were designed. This included one construct (SEQ ID NO: 86) with 8 nonameric MyoG binding motifs (AGCAGCTGC; Nucleic Acids Res. 2022 Jan 7;50(Dl):D165-D173) with 4 bp between each motif and no base pairs between the last motif and the CMV2 promoter (SEQ ID NO:50). The remaining three designs contained 6 MyoG binding motifs with 4 bp between each motif and 8 bp, 4 bp, or 0 bp between the last motif and the CMV2 promoter (SEQ ID NO:50). The synthetic 5’UTR, a short human beta globin / human Ig heavy chain chimeric intron, the open reading frame coding for mGreenLantern (mGL; PNAS. 2020 Dec 1;117(48):30710-30721), the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Hum Gene Ther. 1999 Sep 20;10(14):2295-305) and the human growth hormone poly A signal (hGH poly A, GenBank Accession NG 011676) were used as described above.

[0341] 5.2 Evaluating novel designs to selected fourth generation reporter constructs

[0342] The co-transfection of all reporter variants as well as measurements were performed as described 4.2. Two biological replicates were preformed to ensure a robust data evaluation. The 5th generation reporter construct design supported the findings of the 4th generation design (Figure 8).

[0343] From left to right, the following constructs are included in Figure 8:

[0344] OxMotif, 44bp Distance: SEQ ID NO:50

[0345] 8xMotif, 8bp, 44bp Distance: SEQ ID NO:63

[0346] 8xMotif, 6bp, 44bp Distance: SEQ ID NO:28 8xMotif, 6bp, 42bp Distance: SEQ ID NO:67 8xMotif, 6bp, 8bp Distance: SEQ ID NO:76 8xMotif, 6bp, Obp Distance :SEQ ID NO:77 8xMotif, 4bp, 44bp Distance :SEQ ID NO: 62 6xMotif, 6bp, 44bp Distance : SEQ ID NO: 54 8xMotif, 4bp, Obp Distance : SEQ ID NO: 86 6xMotif, 4bp, 8bp Distance : SEQ ID NO: 87 6xMotif, 4bp, 4bp Distance : SEQ ID NO:88 6xMotif, 4bp, Obp Distance : SEQ ID NO :89.

[0347] A reduced distance between the last MyoG binding motif and the minimal promoter resulted in the largest impact of a single parameter (SEQ ID NO: 87 (8 bp, 1.52-fold increase) vs SEQ ID NO:88 (6bp, 1.75-fold increase) vs SEQ ID NO:89 (Obp, 2.09-fold increase)), followed by the ideal number of motifs (SEQ ID NO: 86 (8x MyoG motifs; 1.72 -fold increase) vs SEQ ID NO:89 (6x MyoG motifs, 2.09-fold increase)). However, the highest impact was observed when combining all prime conditions. Specifically, the optimal configuration was achieved by minimizing the distance between the MyoG binding motifs and the minimal promoter (0 bp), while simultaneously optimizing the number of MyoG binding motifs (6x MyoG binding motifs). This combinatorial effect (SEQ ID NO:89 (2.09-fold increase)) underscores the importance of both spatial arrangement and motif quantity in enhancing promoter activity. Notably, this optimization not only significantly increased gene expression but also resulted in a more compact reporter construct, with a final length of just 195 base pairs.

[0348] EXAMPLE 6

[0349] In vivo validation of fifth generation reporter constructs

[0350] In the present study, a robust in vivo model is utilized to evaluate the efficacy and specificity of three distinct promoters for gene expression using the AAVrh74 capsid. C57BL / 6 mice are divided into three groups (n=4 per group) and are administered intravenously with AAV vectors at a dose of lxlOA12 vector genomes (vg) per mouse (5xl0A13 vg / mL). The promoters tested include a constitutive CAG promoter (SEQ ID NO: 12), a state-of-the-art MHCK7 promoter (Toscano et al., Gene Ther. 18 (2011) 117-127), and a novel 5th generation MyoG promoter (SEQ ID NO:89), each driving the expression of a reporter gene (e.g., mGreenLantern or luciferase). After a 4-week period, three mice from each group are sacrificed to perform comprehensive immunohistochemistry (IHC) and in situ hybridization (ISH) analyses across a variety of tissues, including heart, skeletal muscle, and intestinal smooth muscle, as well as off- target tissues such as liver, spleen, and lung. The remaining mouse in each group is subjected to a whole-animal clearance study using bioluminescence or fluorescence imaging to assess the biodistribution and persistence of the AAV vectors. Quantitative measures of transgene expression levels and tissue distribution are obtained to compare promoter performance. This validation study aims to confirm the effectiveness of the designed 5th generation promoters in driving skeletal muscle-specific gene expression, providing critical insights for the development of targeted gene therapies and supporting the advancement of these promoter designs towards clinical applications.

Claims

CLAIMS1. A synthetic skeletal muscle-specific promoter comprising at least 6 MyoG elements operably linked to a promoter element.

2. The synthetic skeletal muscle-specific promoter according to claim 1, comprising at least 8 MyoG elements operably linked to a promoter element.

3. The synthetic skeletal muscle-specific promoter according to claim 1 or 2, wherein the synthetic skeletal muscle-specific promoter comprises 6 MyoG elements.

4. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 3, wherein the MyoG elements individually comprise, or consist of, the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

5. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 4, wherein the MyoG elements are connected through a linker.

6. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 5, wherein the linker has a length of 4-14bp, preferably 4-6 bp, most preferably 4 bp.

7. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 6, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO:25, SEQ ID NO:91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99.

8. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 7, wherein the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, and SEQ ID NO: 84.

9. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 8, wherein the promoter element is a minimal promoter.

10. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 9, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

11. An expression system comprising a synthetic skeletal muscle-specific promoter according to any one of claims 1 to 10 operationally linked to a polynucleotide encoding a polypeptide of interest.

12. A vector comprising a synthetic skeletal muscle-specific promoter according to any one of claims 1 to 10 operationally linked to a polynucleotide encoding a polypeptide of interest, optionally wherein the vector is an adeno-associated virus (AAV) vector.

13. The expression system according to claim 11 or the vector according to claims 12, further comprising a 5'UTR sequence operationally linked to the synthetic skeletal muscle -specific promoter, wherein the 5'UTR sequence is located 3' to the synthetic skeletal muscle-specific promoter, optionally wherein the 5'UTR comprises an intron.

14. The expression system or the vector according to claim 1 1 or 13, wherein the intron is not a complete naturally occurring intron.

15. The expression system or the vector according to any one of claims 11, 13 or 14, wherein the intron is a truncated natural or synthetic intron.

16. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises in 5’ to 3’ order:(i) a 5' inverted terminal repeat (ITR) sequence;(ii) a synthetic skeletal muscle-specific promoter according to any one of claims 1 to 10;(iii) optionally, a 5' UTR sequence;(iv) a polynucleotide encoding a therapeutic molecule;(v) a 3' UTR sequence; and(vi) a 3' inverted terminal repeat (ITR) sequence.

17. A pharmaceutical composition comprising an expression system according to claim 11 to 15, or a vector according to any one of claims 12 to 15, or an AAV vector according to any one of claims 13, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.

18. An expression system according to claim 11 to 15, or a vector according to any one of claims 12 to 15, or an AAV vector according to claim 16, or a pharmaceutical composition according to claim 17, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest in skeletal muscle cells.

19. Use of an expression system according to claim 11 to 15, or a vector according to any one of claims 12 to 15, or an AAV vector according to claim 16, or a pharmaceutical composition according to claim 17, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.

20. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject an expression system according to claim 11 to 15, or a vector according to any one of claims 12 to 15, or an AAV vector according to claim 16, or a pharmaceutical composition according to claim 17.

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