Skeletal muscle-specific promoters

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480080030.X (22) Application Date 2024.12.16 (30) Priority Data 23217483.9 2023.12.18 EP (85) PCT International Application Entering National Phase Date 2026.06.16 (86) PCT International Application Application Data PCT / EP2024 / 086429 2024.12.16 (87) PCT International Application Publication Data WO2025 / 132132 EN 2025.06.26 (71) Applicant: Hofmeister AG, Switzerland (72) Inventors: Simon Thomas Oslandes, Sara Gilles de Jong, Martin Eberlin, Cornelius Hermann Frank, Ulrich Martin Geppet, Nicol Helbach, Sebastian Königsberger, Oliver Protner, Ulrike Reif, Teresa Schmid (74) Patent Agency: China Patent & Trademark Agency Co., Ltd., 11021, Patent Attorney: Zhang Ying (51) Int.Cl. C12N 15 / 63 (2006.01) C12N 15 / 85 (2006.01) C12N 15 / 86 (2006.01) C12N 15 / 864 (2006.01) (54) Invention Title: Skeletal Muscle-Specific Promoter (57) Abstract: This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to therapies and prevention of diseases. Claims (2 pages), Description (86 pages), Sequence Listing (electronic publication), Drawings (12 pages), CN 122396772 A, 2026.07.14, CN 1 22 39 67 72 A 1. A synthetic skeletal muscle-specific promoter comprising at least six MyoG elements operatively linked to a promoter element. 2. The synthetic skeletal muscle-specific promoter of claim 1, comprising at least eight MyoG elements operatively linked to a promoter element. 3. The synthetic skeletal muscle-specific promoter of claim 1 or 2, wherein the synthetic skeletal muscle-specific promoter comprises six MyoG elements. 4. The synthetic skeletal muscle-specific promoter of any one of claims 1 to 3, wherein the MyoG element individually comprises or is composed of the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24). 5. The synthetic skeletal muscle-specific promoter of any one of claims 1 to 4, wherein the MyoG element is linked via a linker. 6. Claims 1 to 57. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 6, wherein the length of the linker is 4 to 14 bp, preferably 4 to 6 bp, and most preferably 4 bp. 8. 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 with 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 with 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 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 having at least 80%, 90%, 95%, 96%, 97%, 98%, and 99% of the sequence.Or a nucleotide sequence with 100% sequence identity. 11. An expression system comprising a synthetic skeletal muscle-specific promoter according to any one of claims 1 to 10, operably linked to a polynucleotide encoding a target polypeptide. 12. A vector comprising a synthetic skeletal muscle-specific promoter according to any one of claims 1 to 10, operably linked to a polynucleotide encoding a target polypeptide, optionally wherein the vector is an adeno-associated virus (AAV) vector. 13. The expression system of claim 11 or the vector of claim 12, further comprising a 5'UTR sequence operably linked to the synthetic skeletal muscle-specific promoter, wherein the 5'UTR sequence is located at the 3' of the synthetic skeletal muscle-specific promoter, optionally wherein the 5'UTR contains an intron. 14. The expression system or vector of claim 11 or 13, wherein the intron is not a complete, naturally occurring intron. Claims 1 / 2 Page 2 CN 122396772 A 15. An expression system or vector according to any one of claims 11, 13 or 14, wherein the introns are truncated natural or synthetic introns. 16. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises, in a 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 claims 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 load, diluent, excipient or adjuvant. 18. The expression system according to claims 11 to 15, or the vector according to any one of claims 12 to 15, or the AAV vector according to claim 16, or the pharmaceutical composition according to claim 17, for treating or preventing a disease or condition from which therapeutic or preventive benefits will be obtained from an increase in the expression level of the target polypeptide in skeletal muscle cells. 19. The expression system according to claims 11 to 15, or the vector according to any one of claims 12 to 15, or the AAV vector according to claim 16, or the pharmaceutical composition according to claim 17.The use of the pharmaceutical composition in the manufacture of a medicament for treating or preventing a disease or condition from which therapeutic or preventive benefits will be obtained from an increase in the expression level of a target polypeptide. 20. A method of treating or preventing a disease or condition from which therapeutic or preventive benefits will be obtained from an increase in the expression level of a target polypeptide, the method comprising administering to a subject the expression system according to claims 11 to 15, or the vector according to any one of claims 12 to 15, or the AAV vector according to claim 16, or the pharmaceutical composition according to claim 17. Claims 2 / 2 pages 3 CN 122396772 A Skeletal Muscle-Specific Promoter Technical Field

[0001] This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to therapies and prevention of diseases. Background Art

[0002] This disclosure relates to novel skeletal muscle-specific promoters and their use in gene therapy.

[0003] Gene therapy, as a mode, aims to improve disease-related phenotypes by using both viral and non-viral delivery systems. Much work has been devoted to optimizing such therapies to make them more effective, less immunogenic and less toxic, while achieving long-term expression of the target gene. One of the key challenges in achieving this goal is regulating the expression of transgenes to meet the required expression levels in target cells / tissues.

[0004] For some targets, pervasive expression in both target and non-target tissues can lead to adverse events. Therefore, target cell / tissue-specific expression may be beneficial, which can be achieved using cell / tissue-specific promoters.

[0005] Specifically, there is a need for novel specific promoters with varying degrees of tissue specificity and expression levels to regulate the expression of gene therapy targets for multiple neuromuscular disease indications, while exhibiting limited activity in non-target tissues (Skopenkova et al., Acta Naturae 13 (2021) 47-58). There are various reasons why improved promoters are needed to regulate transgene expression: 1. To be specific only to bone and / or heart tissue or to a specific ratio to improve safety distribution and ensure target-specific efficacy; 2. To ensure robust expression to ensure that gene regulation is within the required range; 3. Shorter regulatory elements will be beneficial to ensure the maximum size of the transgene; 4. To identify promoters with cross-species activity to determine the optimal exposure / efficacy / safety range for the desired target.

[0006] For some indications such as Duchenne and Becker muscular dystrophy (DMD / BMD), promoters expressing in both disease-related tissues (bone and heart) are necessary, while for targets of facioscapulohumeral muscular dystrophy (FSHD) or some congenital myopathy, restricting expression to skeletal muscle will most likely be more beneficial. One example is actin expression.Regulation of skeletal muscle actin gene (ACTA1). Mutations in the skeletal muscle actin gene (ACTA1) can lead to congenital myopathy (Nemaline). Generally, actin is a universally overexpressed protein and is highly conserved. ACTA1 is expressed in skeletal muscle and the heart, however, cardiac actin (ACTC1) is primarily expressed in the heart (approximately 80%). It has been shown that if ACTA1 is affected, most patients only exhibit skeletal muscle-specific symptoms. Therefore, high expression of ACTA1 in the heart may lead to potential cardiac safety risks, while high expression in bone tissue is highly desirable.

[0007] Only a few muscle-specific promoters are known in the art, and these promoters have varying degrees of specificity. These can be categorized into natural promoters, creatine kinase promoter-MCK and desmin promoters and synthetic promoters, synthetic muscle-specific promoter C5-12 (SPC5-12) and α-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) (Toscano et al., Gene Ther. 18 (2011) 117-127).

[0008] Although natural promoters exhibit muscle specificity, their expression levels are insufficient to achieve therapeutic effects.

[0009] Although synthetic promoters known in the art lead to increased transgene expression for therapeutic effects, they are prone to off-target activity in other tissues and are active in skeletal muscle and cardiac muscle (Piekarowicz et al., Mol Ther Methods Clin Dev. 15 (2019) 157-169, 2019; Salva et al., Mol Ther. 15 (2007) 320-329).

[0010] Therefore, muscle diseases limited to skeletal muscle will benefit from novel promoters that are specific to skeletal muscle. Specification 1 / 86 pages 4 CN 122396772 A Summary of the Invention

[0011] This document discloses novel skeletal muscle-specific promoters. The promoters according to the invention, compared with prior art promoters such as MHCK7, show higher and more specific expression in primary skeletal muscle cells. Specifically, the inventors have shown that synthetic promoters comprising multiple MyoG elements have higher activity and skeletal muscle specificity than all other constructs tested.

[0012] In one embodiment, a synthetic skeletal muscle-specific promoter is provided comprising at least 6 MyoG elements operatively linked to promoter elements.

[0013] In one embodiment, the synthetic skeletal muscle-specific promoter comprises at least 8 MyoG elements operatively linked to promoter elements.

[0014] In one embodiment, the synthetic skeletal muscle-specific promoter comprises 6 MyoG elements operatively linked to promoter elements.MyoG elements.

[0015] In one embodiment, these MyoG elements individually comprise or consist of the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

[0016] In one embodiment, these MyoG elements are linked by a linker.

[0017] In one embodiment, at least one linker has a length of 4 bp to 14 bp, preferably 4 bp to 6 bp, most preferably 4 bp.

[0018] 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 with 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.

[0019] In one embodiment, the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0020] In one embodiment, the promoter element is a minimal promoter.

[0021] 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 with 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: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:7 ...78, SEQ ID NO:79, SEQ ID NO:79, 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 NONO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0022] In one embodiment, an expression system is provided comprising a synthetic skeletal muscle-specific promoter as described above, operably linked to a polynucleotide encoding a polypeptide of interest.

[0023] In one embodiment, a vector is provided comprising a synthetic skeletal muscle-specific promoter as described above, operably linked to a polynucleotide encoding a polypeptide of interest, optionally wherein the vector is an adeno-associated virus (AAV) vector.

[0024] In one embodiment, an expression system or a vector as described above is provided, the expression system or vector further comprising a 5'UTR sequence operably linked to a synthetic skeletal muscle-specific promoter, wherein the 5'UTR sequence is located at the 3' of the synthetic skeletal muscle-specific promoter, optionally wherein the 5'UTR contains an intron.

[0025] In one embodiment, the intron is not a complete, naturally occurring intron. Specification 2 / 86 pages 5 CN 122396772 A

[0026] In one embodiment, the intron is a truncated natural or synthetic intron.

[0027] In one embodiment, an adeno-associated virus (AAV) vector comprising a vector genome is provided, wherein the vector genome comprises, in 5' to 3' order:

[0028] (i) a 5' inverted terminal repeat (ITR) sequence;

[0029] (ii) a synthetic skeletal muscle-specific promoter as described above;

[0030] (iii) optionally, a 5' UTR sequence;

[0031] (iv) a polynucleotide encoding a therapeutic molecule;

[0032] (v) a 3' UTR sequence; and

[0033] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0034] In one embodiment, a pharmaceutical composition is provided comprising an expression system as described above, or a carrier as described above, or an AAV carrier as described above, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0035] In one embodiment, an expression system as described above, or a carrier as described above, or an AAV carrier as described above, or a pharmaceutical composition as described above is provided for treating or preventing a disease or condition from which therapeutic or preventive benefits will be derived from an increase in the expression level of the polypeptide of interest in skeletal muscle cells.

[0036] In one embodiment, use of an expression system as described above, or a carrier as described above, or an AAV carrier as described above, or a pharmaceutical composition as described above is provided in the manufacture of a medicament for treating or preventingDiseases or conditions from which therapeutic or preventive benefits will be obtained by increasing the expression level of the polypeptide of interest.

[0037] In one embodiment, a method is provided for treating or preventing diseases or conditions from which therapeutic or preventive benefits will be obtained by increasing the expression level of the polypeptide of interest, the method comprising administering to a subject an expression system as described above, or a vector as described above, or an AAV vector as described above, or a pharmaceutical composition as described above. Detailed Description

[0038] Novel skeletal muscle-specific promoters are disclosed herein. The promoters according to the invention are compared with prior art promoters such as MHCK7, showing higher and more specific expression in primary skeletal muscle cells.

[0039] Without being bound by this theory, it is believed that the muscle-specific promoters according to the invention allow reliable skeletal muscle-specific transgene expression, which in particular can lead to reduced host immune response and increased therapeutic effect. Specifically, the skeletal muscle-specific promoters according to the invention, when combined with muscle-specific viral serotypes such as rAAV8 and rAAV9, will allow reliable skeletal muscle-specific transgene expression.

[0040] Aspects and embodiments of this disclosure relate to the synthesis of skeletal muscle-specific promoters. The promoters according to this disclosure comprise or consist of polynucleotides, particularly polynucleotides comprising or consisting of DNA or RNA.

[0041] A “polynucleotide” is a polymer chain of multiple nucleotide monomers linked by bonds between monomers, typically phosphodiester bonds (e.g., in the case of polynucleotides formed from naturally occurring nucleotide monomers). Polynucleotides include oligonucleotides, which typically contain ≤50 nucleotides. Polynucleotides may be single-stranded or double-stranded (i.e., may comprise a double strand formed by hydrogen bonds between complementary nucleotides). Polynucleotides according to this disclosure may comprise or consist of: single-stranded DNA, double-stranded DNA, DNA being a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA being a mixture of single-stranded 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-stranded and double-stranded regions.

[0042] In some aspects, the polynucleotide comprises or is composed of DNA. In some embodiments, the polynucleotide is a polydeoxyribonucleotide. In some embodiments, the polynucleotide comprises RNA or is composed of DNA. In some embodiments, the polynucleotide is a polynucleotide.

[0043] In which the polynucleotides of this disclosure are defined by reference to a given nucleotide sequence and wherein the given nucleotide sequence comprises RNAIn aspects that are composed of and / or are polynucleotides, it should be understood that instances of “T” representing thymidine in such sequences are replaced by “U” representing uracil.

[0044] This disclosure also contemplates polynucleotides comprising modified nucleotides (e.g., in which the phosphate esters of deoxyribonucleotides or ribonucleotides and / or the ribose and / or bases are chemically modified). Nucleotide modifications contemplated according to this disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which are hereby incorporated in their entirety by reference.

[0045] Phosphate modifications may be selected from thiophosphates (e.g., Rp isomers, Sp isomers), dithiophosphates, methylphosphonates, methoxypropylphosphonates, 5'-(E)-vinylphosphonates, 5'-methylphosphonates, (S)-5'-C-methyl phosphates, 5'-thiophosphates, and peptide nucleic acid modifications. Riboside modification can be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluorine, 2'-deoxy-2'-fluorine, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, 2'-arabinose-fluorine, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acids, (S)-cEt-BNA, tricyclic DNA, PMO, non-locked nucleic acids, hexitol nucleic acids, and ethylene glycol nucleic acid modifications. The base modification can be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, adenine modified with N-ethylpiperidine 7'-EAA triazole, adenine modified with N-ethylpiperidine 6'-triazole, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotolyl ribonucleoside, and 5'-nitroindole.

[0046] In some aspects, the 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'-thiophosphate, 2'-O-methyladenosine-3'-thiophosphate, 2'-O-methylguanosine-3'-thiophosphate, 2'-O-methylcytidine-3'-thiophosphate, 2'-fluorouridine-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-thiophosphate, 2'-fluorocytidine-3'-thiophosphate, 2'-fluoroguanosine-3'-thiophosphate, 2'-fluoroguanosine-3'-thiophosphate, 2'- Fluoroadenosine-3'-thiophosphate and 2'-fluorouridine-3'-thiophosphate.

[0047] In some aspects, a synthetic skeletal muscle-specific promoter is provided. In a preferred embodiment, the synthetic skeletal muscle-specific promoter comprises myocyte cytopoietin.(MyoG) element (or MyoG binding site). MyoG, also known as myopoietin, is a member of the myogenic regulatory factor family. It is a transcription factor that plays a key role in the development and differentiation of skeletal muscle. MyoG is involved in regulating the expression of genes necessary for muscle cell formation and maturation. It is one of the key factors that trigger the process of myogenesis (the formation of muscle tissue during embryonic development and regeneration). MyoG is mainly expressed in skeletal muscle cells and is essential for the normal development and function of skeletal muscle cells. MyoG binding sites can be identified by methods known in the art and described, for example, in the following literature: Cao et al., EMBO J. 8 Feb 2006; 25(3): 502-511).

[0048] Exemplary MyoG elements are shown in the appended examples and exemplary sequences further described below. In one particular aspect, the MyoG element used according to the invention comprises or is composed of the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

[0049] In some aspects, synthetic skeletal muscle-specific promoters comprise multiple MyoG elements. In some aspects, synthetic skeletal muscle-specific promoters comprise 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 invention comprises 8 MyoG elements or is composed of such MyoG elements.

[0050] In some embodiments, MyoG elements are separated from each other by a linker polynucleotide. A linker polynucleotide (sequence), also called a filler polynucleotide (sequence), is a polynucleotide fragment inserted between two other polynucleotide sequences. Preferably, the linker polynucleotide sequence is non-coding. Linker polynucleotides are used to link 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 promoters or genes, filler sequences or linker sequences can be used to ensure proper spacing and orientation of elements to achieve correct transcription or expression. The linker nucleotide sequence may comprise or consist of 1 to 11 nucleotides (e.g., one of 2 to 10, 3 to 9, 4 to 8, 5 to 7, or 6). Exemplary linker polynucleotide sequences are disclosed in the sequence listing below (e.g., SEQ ID NO:44 to SEQ ID NO:48, and in SEQ ID NO:100 to SEQ ID NO:109) and examples; other suitable linker polynucleotide sequences can be readily identified and selected by those skilled in the art.

[0051] In one specific aspect of this kind, the eight 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 with SEQ ID NO:25. This sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight adapter polynucleotide sequences individually selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:48.

[0052] In another aspect of this kind, 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 with SEQ ID NO:25. This sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight adapter polynucleotide sequences, each adapter polynucleotide sequence being 6 bp in length. In one aspect, the linker polynucleotide sequence is individually selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:48. In another aspect, the linker polynucleotide sequence is individually selected from the group consisting of SEQ ID NO:100 to SEQ ID NO:109.

[0053] 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, each of the six linker polynucleotide sequences is 6 bp in length. In one aspect, the six linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:49. In another 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 with SEQ ID NO:91.

[0054] 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, each of the eight linker polynucleotide sequences is 4 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:100 to SEQ ID NO:104. In another such aspect, the synthetic skeletal muscle-specific promoter comprises having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the element in SEQ ID NO:92.Nucleotide sequences with sequence identity.

[0055] 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, each of the eight linker polynucleotide sequences is 8 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:105 to SEQ ID NO:109. In another 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 with SEQ ID NO:93.

[0056] In another 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 with SEQ ID NO:94. Specification 5 / 86 pages 8 CN 122396772 A

[0057] In another aspect of this, 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 with SEQ ID NO:95.

[0058] In another aspect of this, 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 with SEQ ID NO:96.

[0059] In one aspect, the sequence comprises eight copies of the MyoG element of SEQ ID NO:24 and eight adapter polynucleotide sequences. In one aspect, each of the eight adapter polynucleotide sequences is 6 bp in length. In one aspect, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:44 to SEQ ID NO:49. In another 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 with SEQ ID NO:97.

[0060] 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, each of the eight six-linker polynucleotide sequences is 4 bp in length. In one aspect...In this embodiment, the eight linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:100 to SEQ ID NO:104. In another aspect of this 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 with SEQ ID NO:98.

[0061] 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, each of the six linker polynucleotide sequences is 4 bp in length. In one aspect, the six linker polynucleotide sequences are individually selected from the group consisting of SEQ ID NO:100 to SEQ ID NO:104. In another aspect of this invention, 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 with SEQ ID NO:99.

[0062] In another aspect, the MyoG element is operatively linked to a promoter element. The invention is based, at least in part, on the identification of muscle-specific genes and their promoter regions from databases. Furthermore, specific transcription factor binding sites have been identified within the promoter regions. In some aspects, the promoter element is selected from a list consisting of the following: ACTA1 promoter (NCBI gene ID 58, actin α1), CACNG1 promoter (NCBI gene ID 786, voltage-dependent calcium channel γ-1 subunit), MYBPC2 promoter (NCBI gene ID 4606, myosin-binding protein C, fast type), MYH2 promoter (NCBI gene ID 4620, myosin-2), MYLPF promoter (NCBI gene ID 29895, and myosin regulatory light chain 2, skeletal muscle isotype), MYBC1 promoter, TTN promoter (NCBI gene ID 7273, titin), or fragments thereof that retain tissue-specific expression (specifically, skeletal muscle-specific expression).

[0063] In some embodiments, the promoter element is a minimal promoter. A "minimal promoter" is the simplest and / or smallest polynucleotide sequence capable of binding RNA polymerase and other necessary transcription factors to initiate transcription, leading to the production of mRNA from a gene. Minimal promoters are typically fundamental components of larger promoter complexes and are crucial for gene expression. Minimal promoters usually include a TATA box and other core promoter elements. Typically, minimal promoters lack enhancement...

[0064] In a preferred embodiment, the promoter element comprises a TATA box. A “TATA box” is a specific polynucleotide sequence found in the promoter regions of many genes. It typically consists of the nucleotide sequence TATAAA (SEQ ID NO:51) and is located approximately 25 to 30 base pairs upstream of the transcription start site. The TATA box is recognized and bound by transcription factors known as TATA-binding proteins (TBPs), which help recruit RNA polymerase II and other factors required for transcription initiation. The TATA box is important for determining the direction of transcription and defining the exact site of transcription initiation.

[0065] In some aspects, the promoter element comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:49 or SEQ ID NO:50. Specification 6 / 86 pages 9 CN 122396772 A

[0066] 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 with SEQ ID NO:49 or SEQ ID NO:50.

[0067] 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 with 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.

[0068] The minimal promoter is preferably provided immediately adjacent to the 3' of the polynucleotide containing the MyoG element (i.e., downstream of it in the context of the nucleotide sequence of the polynucleotide).

[0069] 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 with a sequence selected from Column B of Table 1, wherein the fragment preserves skeletal muscle-specific expression of the polypeptide of interest operably linked to the fragment.

[0070] 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 with SEQ ID NO: 27 or SEQ ID NO: 28.

[0071] In some respects, 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 with 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, SEQ ID NO:8 ... NO:88 and SEQ ID NO:89.

[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 with 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.

[0073] 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 with 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:59, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:69 ...NO:77, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89.

[0074] In a most preferred 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 with 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.

[0075] In a particularly 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.

[0076] In the aspects described above, the promoter and / or promoter elements preferably exhibit the functional characteristics described below.

[0077] Functional Properties of Synthetic Promoters 7 / 86 pages 10 CN 122396772 A

[0078] In aspects and embodiments of this disclosure, the polynucleotides of this disclosure can be characterized by reference to one or more functional properties. A very important functional property of the novel promoters according to the invention is tissue specificity, specifically muscle specificity.

[0079] In some embodiments, non-skeletal muscle cells containing a synthetic skeletal muscle-specific promoter of this disclosure operably linked to a polynucleotide encoding a protein of interest do not substantially express the polypeptide of interest. In some embodiments, skeletal muscle cells containing a synthetic skeletal muscle-specific promoter of this disclosure operably linked to a polynucleotide encoding a protein of interest express the polypeptide of interest.

[0080] Although cardiac muscle and skeletal muscle belong to striated muscle and share some common properties (e.g., sarcomeres, rich in mitochondria and containing large molecularly extended myosin and action proteins), they also have different characteristics. Skeletal muscle is composed of parallel linear fibers, while cardiac muscle is arranged in a cross-striped pattern. In addition to their similarities, these two cell types can also be distinguished by their functions and their respective molecular compositions. The two can be distinguished by the expression of specific genes and proteins corresponding to their physiological functions (Lindskog et al., BMC Genomics. 2015; 16(1): 475). Maturation markers used to monitor different stages of myogenic differentiation include PAX7, MYOD, MYOG, MRF4, MYH3, and MYH7, while examples of proteins used to identify elevated levels in skeletal muscle include MYH2, TNNT1, MYBPC1, and ACTA1 (Chal et al., Development (2017) 144 (12):2104-2122; Lindskog et al., BMC Genomics. 2015; 16(1): 475). Therefore, when using in vitro models, it is recommended to monitor at least a portion of the protein or its corresponding transcript to ensure proper cell specificity.

[0081] The expression of the target peptide can be assessed using any suitable technique for detecting and / or quantifying the relevant peptide. Such techniques include, for example, antibody-based methods (e.g., flow cytometry, immunocytochemistry, Western blotting, ELISA), fluorescence microscopy, and flow cytometry. In some embodiments, the expression of the peptide of interest can be assessed as described in the experimental examples of this disclosure.

[0082] For example, tissue-specific expression, such as skeletal muscle-specific expression, can be measured using 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 a candidate promoter can be measured in target cells (e.g., in skeletal muscle cells or cell lines) and non-target cells (e.g., in hepatocytes or cell lines), and the expression in target cells can be compared with the expression in non-target cells. Skeletal muscle-specific promoters will exhibit high (or higher) expression in skeletal muscle cells (such as in HSMM cells) and low (or lower) expression in non-target cells (such as in Huh7 hepatocellular carcinoma cells). Expression can be measured by measuring the level of the reporter gene (e.g., measuring the fluorescence intensity of the expression of a fluorescent protein operatively linked to a candidate promoter). The ratio between reporter gene expression in target cells and reporter gene expression in non-target cells can be calculated to illustrate tissue specificity.

[0083] In some aspects, i) the ratio of promoter activity in HSMM cells 5 days after transduction with serotype 2 AAV at an MOI of 20,000 to ii) 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, promoter activity is determined by the fluorescence intensity of the expression of a fluorescent protein operatively linked to a synthetic promoter. In some aspects, the fluorescent protein is mGreenLantern. Such promoters may be referred to herein as “skeletal muscle-specific” promoters.

[0084] In some aspects, 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” the polypeptide of interest may exhibit an expression level of the polypeptide of interest that is less than 0.2 times, for example ≤0.1 times, ≤0.09 times, of the expression level of cells expressing the polypeptide of interest.Page 8 / 86, 11 CN 122396772 A ≤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. In some embodiments, cells "expressing" the polypeptide of interest may show an expression level of the polypeptide of interest that is greater than 5 times (e.g., ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times) than cells that "substantially do not express" the polypeptide of interest.

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

[0087] It should be understood that the functional properties described herein are evaluated using similar (or identical) experimental conditions to evaluate cells containing different polynucleotides. However, some experimental conditions may need to be adjusted, for example, depending on the cell type. Adjustments to experimental conditions for a specific cell type can be made according to methods known in the art and as described in the Experimental section.

[0088] Expression System

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

[0090] The term "operably linked" can include situations where a nucleic acid encoding a polypeptide of interest according to this disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner that the expression of the nucleic acid encoding the polypeptide is placed under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, if the regulatory sequence is capable of influencing the transcription of a selected nucleic acid sequence, the regulatory sequence is operably linked to that nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0091] The target polypeptide can be any polypeptide.

[0092] In some embodiments, the polypeptide of interest according to this disclosure may be an antigen-binding polypeptide, an aptamer, an antigen-binding polypeptide complex, an antibody or its antigen-binding fragment or derivative, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, a ligand decoy receptor, a receptor decoy ligand, an enzyme, a growth factor, a hormone, interferon, interleukin, a thrombolytic agent, a transcription factor, an epigenetic modifier, a site-specific nuclease, or a nucleic acid editing system (e.g., CRISPR / Cas9 system, CRISPR / Cpf1 system, CRISPR / C2c1).The target peptide is a constituent protein of a system (such as 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).

[0093] In some embodiments, the target peptide is a peptide suitable for the treatment or prevention of a disease / condition. In some embodiments, the target peptide is a detectable peptide or a peptide with detectable activity.

[0094] A peptide suitable for the treatment or prevention of a disease / condition can be any peptide whose administration can be used for the treatment or prevention of a disease / condition. In some embodiments, a peptide suitable for the treatment or prevention of a disease / condition can be a peptide whose deficiency is positively correlated with or involves the pathology of a disease or condition. For example, in some embodiments, the peptide of interest can be ACTA1, the deficiency of which is associated with myopathy.

[0095] In some embodiments, a peptide suitable for the treatment or prevention of a disease / symptom may be a peptide that inhibits the expression or activity of factors whose expression or activity is positively correlated with or involved in the pathology of the disease or symptom.

[0096] The detectable peptide may be or contains a fluorescent peptide. Fluorescent peptides include green fluorescent protein and its variants (e.g., enhanced green fluorescent protein), yellow fluorescent protein (e.g., citrine), red fluorescent protein and its variants (e.g., mOrange, mCherry), blue fluorescent protein and its variants (e.g., TagBFP), cyan fluorescent protein and its variants (e.g., mTurquoise, cerulean), allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin. In some aspects, the fluorescent protein is mGreenLantern.

[0097] The detectable peptide may be or contains an epitope tag. Epitope tags include, for example, His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, 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).

[0098] In some aspects, the detectable polypeptide comprises, or consists of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column D of Table 1, or a fragment thereof retaining its detectable activity.

[0099] A polypeptide having detectable activity may be or comprises an enzymatic moiety. Enzymatic moieties include, for example, luciferase, glucose oxidase, galactosidase (e.g.,β-galactosidase), glucosidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0100] The target polypeptide expressed by the polynucleotide of this disclosure may additionally include one or more foreign amino acids added at the N-terminus of the polypeptide (i.e., immediately upstream of the amino acid sequence of the target polypeptide). Such foreign amino acids may be characterized as forming an N-terminal tag on the target polypeptide. It may be necessary to minimize the size of such foreign amino acid / N-terminal tag on the target polypeptide or to completely remove such foreign amino acid / N-terminal tag.

[0101] In some embodiments, the N-terminal tag consists of less than 50 amino acids, such as ≤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 target polypeptide lacks an N-terminal tag.

[0102] In addition to the synthesized muscle-specific promoter and the polynucleotide encoding the polypeptide of interest, the expression system according to the present invention may also contain additional nucleotide sequences and / or sequence features.

[0103] The expression system of this disclosure includes a start codon at the 5' position of the nucleotide sequence encoding the polypeptide of interest (i.e., upstream of it in the context of the nucleotide sequence of the polynucleotide). The start codon is preferably a trinucleotide 'ATG'.

[0104] In some embodiments, the expression system of the present invention further includes a Kozak sequence. In a preferred embodiment, the Kozak sequence is provided upstream of the start codon used to initiate the translation of the polypeptide of interest.

[0105] In some embodiments, the expression system of the present invention further includes one or more enhancer sequences. One or more enhancer sequences are preferably located at the 5' position of the synthesized muscle-specific promoter sequence.

[0106] In some embodiments, the expression system of the present invention includes a 5'UTR. The term 5'UTR stands for 5' untranslated region. This region is transcribed but not translated into protein. The 5'UTR may contain regulatory elements and may regulate the stability of mRNA, its localization, and the rate of protein synthesis. In some embodiments, the 5'UTR is operatively linked to a synthetic skeletal muscle-specific promoter of this disclosure.

[0107] In some embodiments, the 5'UTR contains introns. In a preferred embodiment, the introns consist of fewer nucleotides than the number of nucleotides in known (e.g., naturally occurring) introns. In some aspects, the 5'UTR does not contain complete...The naturally occurring promoter introns. In some aspects, the introns are truncated natural or synthetic introns. In some aspects, the 5'UTR does not contain introns.

[0108] In some embodiments, the 5'UTR comprises, or is composed of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column C of Table 1.

[0109] In some embodiments, the expression system of the present invention further comprises a stop codon. The stop codon is preferably provided at the 3' of the trinucleotide encoding the terminal amino acid of the polypeptide of interest (i.e., downstream of it in the context of a polynucleotide nucleotide sequence).

[0110] In some embodiments, the expression system of the present invention further comprises a polyadenylation signal sequence. In a preferred embodiment, the polyadenylation signal sequence is provided at the 3' of the nucleotide sequence encoding the polypeptide of interest (i.e., downstream of the polynucleotide sequence in the context of the polynucleotide sequence). In some embodiments, the expression system of the present invention further comprises a terminator sequence. The terminator sequence is preferably at the 3' of the nucleotide sequence encoding the polypeptide of interest (and at the 3' of the polyadenylation signal sequence (if present)).

[0111] 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 not translated into protein. The 3'UTR refers to the polynucleotide sequence following the stop codon and typically includes a polyadenylation signal.

[0112] In some embodiments, the 3'UTR comprises, or is composed of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column E of Table 1.

[0113] In a preferred embodiment, the constituent nucleotide sequences of the expression system of the present invention are provided adjacent to each other. However, in some embodiments, the polynucleotide is further included among one or more adapter nucleotide sequences in one or more of the constituent nucleotide sequences of the expression system of the present invention.

[0114] The adapter nucleotide sequence may comprise or consist of 1 to 10 nucleotides (e.g., one of 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides).

[0115] When the expression system of the present invention according to this disclosure includes one or more adapter nucleotide sequences, the adapter sequences are preferably selected such that they do not alter the amino acid sequence of the polypeptide encoded by the polynucleotide. In a preferred embodiment,In the case where the expression system of the present invention according to this disclosure contains one or more adapter sequences, the expression system of the present invention encodes the same polypeptide as the equivalent expression system of the present invention lacking the adapter nucleotide sequence.

[0116] In some embodiments, the expression system of the present invention further comprises an inverted terminal repeat (ITR) sequence.

[0117] In some embodiments, the polynucleotide is contained in the ITR at the 5' of the promoter and / or enhancer sequence (when present). In some embodiments, the polynucleotide is contained in the ITR at the 3' of the nucleotide sequence encoding the polypeptide of interest (and at the 3' of the stop codon, polyadenylation signal sequence, and / or terminator sequence (when present).

[0118] In some embodiments, the 5' ITR comprises, or is composed of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column A of Table 1.

[0119] In some embodiments, the 3'ITR comprises, or is composed of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column G of Table 1.

[0120] In some embodiments, the expression system of this disclosure has a size that allows it to be delivered 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 limits of a vector for delivering polynucleotides.

[0121] In some embodiments, the expression system has a size within the packaging limits of an AAV vector. In some embodiments, the expression system has a size within the packaging limits of an AAV vector for one of the following serotypes: AAV9, AAV9.45, AAV-PHP.eB, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10, or AAVrh74.

[0122] In some embodiments, the expression system of this disclosure comprises fewer than 6,000 nucleotides (e.g., ≤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).

[0123] In some embodiments, the expression system includes an AAV filler sequence. For example, such filler sequences may be needed to adapt to promoter elements of different sizes or polynucleotides encoding proteins of interest. Exemplary AAV filler sequences comprise sequences selected from the table.The sequence of column F of 1 has, or is composed of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0124] In some embodiments, the expression system of this disclosure comprises one or more nucleotide sequences encoding selection markers to facilitate the identification and / or selection of cells containing / expressing the polypeptide of interest. Selection markers include proteins conferring resistance to antibiotics or other toxins (e.g., blastcin, ampicillin, neomycin, methotrexate, or tetracycline) and proteins supplementing nutritional deficiencies.

[0125] In some embodiments, the expression system of this disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the expression system of this disclosure comprises a nucleotide sequence that allows two or more polypeptides to be translated separately from a single polynucleotide.

[0126] The expression system of this disclosure may be provided in purified or isolated form, i.e., isolated from other nucleic acids or naturally occurring biological materials.

[0127] In some embodiments, the expression system of this disclosure is a vector.

[0128] As used herein, “vector” refers to a polynucleotide used as a medium for transferring exogenous nucleic acids into cells. A vector can be a vector for expressing nucleic acids in cells (i.e., a vector can be an expression vector). Such vectors can include a promoter sequence operatively linked to a nucleotide sequence to be expressed. Vectors can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon can be used for vectors according to this disclosure.

[0129] Vectors contemplated in connection with this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugating plasmids (e.g., F plasmids), non-conjugating plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, such as γ-retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV), such as SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, baculovirus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are hereby incorporated herein by reference in their entirety.

[0130] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector containing elements necessary for protein expression from a vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, which may contain, for example, cytomegalovirus (CMV) or SV40.A promoter drives protein expression. In some embodiments, the vector contains a CMV (e.g., mCMV), SV40, RSV, or PGK promoter.

[0131] In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ to which the expression system according to the present disclosure is to be delivered. In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ in which the polypeptide of interest is to be expressed. For example, it may be desirable to deliver the expression system to a cell type / tissue / organ affected by a disease / condition to be treated / prevented according to the present disclosure (e.g., a cell type / tissue / organ exhibiting symptoms of the disease / condition), and / or to express the polypeptide of interest in that cell type / tissue / organ.

[0132] For example, it may be desirable to deliver the expression system of the present disclosure encoding the polypeptide of interest to skeletal muscle tissue, and in such cases a vector tropistic to such cells / tissues may be used.

[0133] In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus vectors and their applications in vector gene therapy are reviewed, for example, 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 referenced in their entirety on page 12 / 86 of CN 122396772 A. In some embodiments, the vector may be the adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, the vector may be the adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0134] In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described, for example, in McCarty, Mol Ther. (2008) 16 (10): 1648–56, which are hereby incorporated in their entirety by reference. Conventional AAVs have a single-stranded DNA genome and rely on the DNA replication mechanism of the transduced cell to synthesize the complementary strand, thereby delaying transgene expression. In contrast, scAAVs contain a complementary sequence that anneals spontaneously upon infection, thereby eliminating the need for DNA synthesis in the transduced host cell. Compared to classic single-stranded AAV vectors, scAAV vectors have been shown to provide accelerated transgene expression and increased transgene expression levels.

[0135] In some embodiments, the vector may be an adeno-associated virus 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.

[0136] In some embodiments, the vector contains modifications to increase binding and / or transduction with the cell type of interest (i.e., binding / transduction levels compared to unmodified vectors). In some embodiments, the modification is a modification of the capsid protein.

[0137] In some embodiments, the vector contains a capsid protein with a cell-targeting peptide. In some embodiments, the cell-targeting peptide is the cell-targeting peptide described herein by reference in Büning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248–265, such as the cell-targeting peptides shown in Tables 1, 2, 3, or 4 thereto.

[0138] In some embodiments, the vector comprises a capsid protein comprising substitutions for one or more tyrosine residues, such as 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, the vector comprises a capsid protein wherein one or more tyrosine residues are substituted with another amino acid, as described in Iida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby referenced herein by reference in its entirety.

[0139] In some embodiments, the vector may be an adeno-associated virus vector as described above in Büning and Srivastava. In some embodiments, the vector may be an adeno-associated virus vector as described above by Iida et al.

[0140] In some embodiments, the vector contains control elements for inducible expression of the expression system of this disclosure.

[0141] In some embodiments, an adeno-associated virus (AAV) vector comprising a vector genome is provided, 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 the protein of interest;

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

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

[0148] In some embodiments, an adeno-associated virus (AAV) vector comprising a vector genome is provided, wherein the vector genome comprises, in 5' to 3' order:

[0149] (i) a 5' inverted terminal repeat (ITR) sequence;

[0150] (ii) a synthetic skeletal muscle-specific promoter;

[0151] (iii) optionally, a 5' UTR sequence;

[0152] (iv) a polynucleotide encoding a therapeutic molecule;

[0153] (v) a 3' UTR sequence; and

[0154] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0155] In some embodiments, the AAV comprises (i) a 5' ITR sequence, which comprises or is composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column A of Table 1.

[0156] In some embodiments, the AAV comprises (ii) a promoter, which comprises or is composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column B of Table 1. Preferably, the promoter comprises or is composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 with 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.

[0157] In some embodiments, the AAV comprises (iii) a 5'UTR, the 5'UTR comprising or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column C of Table 1.

[0158] In some embodiments, the AAV comprises (iv) a polynucleotide encoding the protein of interest, the polynucleotide comprising or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column D of Table 1.

[0159] In some embodiments, AAV includes (v) 3'UTR, the 3'UTR including and selected from Table 1The sequence of column E has at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or is composed of therefrom.

[0160] In some embodiments, AAV comprises (vi) a 3'ITR, the 3'ITR comprising or being composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence selected from column G of Table 1.

[0161] In some embodiments, AAV further comprises a polynucleotide, the polynucleotide comprising or being composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence selected from column F of Table 1.

[0162] In some embodiments, an adeno-associated virus (AAV) vector is provided, comprising a vector genome, wherein the vector genome comprises, from 5' to 3', sequences A to G selected from one row of Table 1, optionally linked by a linker polynucleotide sequence.

[0163] Cells

[0164] This disclosure also provides cells comprising a synthetic skeletal muscle-specific promoter according to this disclosure. Cells comprising an expression system (such as one or more vectors) according to this disclosure are also provided.

[0165] The cells may be eukaryotic cells, such as mammalian cells. Mammals may be primates (rhesus monkeys, cynomolgus monkeys, non-human primates, or humans) or non-human mammals (e.g., rabbits, guinea pigs, rats, mice, hamsters, or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cattle, such as dairy cows, or any animal in the genus Bos), horses (including any animal in the family Equidae), donkeys, and non-human primates). In a preferred embodiment, the cells are human cells.

[0166] This disclosure also provides a method for producing cells comprising an expression system / vector according to the present disclosure, as described on pages 14 / 86 of CN 122396772 A, which includes introducing the expression system / vector of the present disclosure into cells. In some embodiments, introducing the expression system / vector according to the present disclosure into cells includes transformation, transfection, electroporation, or transduction (e.g., adeno-associated virus transduction). In some embodiments, the expression system / vector is introduced into cells in vivo, for example by administering a vector (e.g., a viral vector, such as an adeno-associated virus vector) according to the present disclosure to a subject. In some embodiments, the expression system / vector is introduced into cells in vitro or in vitro cultured.

[0167] Any suitable method can be used to produce cells according to the present disclosure. Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of polynucleotides according to the present disclosure. In some embodiments, upon introduction into cells...Once inside the cell, the polynucleotide can be integrated into or form part of the cell's genomic DNA. In some embodiments, the expression system / vector can be maintained extrachromosomally after introduction into the cell.

[0168] Any suitable genetic engineering platform can be used, including gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, such as those described in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is hereby incorporated in its entirety by reference. Methods also include those described, for example, in Wang and Rivière Mol Ther Oncolytics (2016) 3:16015, the entire contents of which are incorporated herein by reference. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0169] In some embodiments, the method further includes maintaining the cells under conditions suitable for the expression of the vector / peptide of interest by the cells.

[0170] This disclosure also provides cells obtained or available by the methods according to this disclosure.

[0171] Compositions

[0172] This disclosure also provides compositions comprising the synthetic skeletal muscle-specific promoters, expression systems, vectors, and cells described herein. Specifically, this disclosure provides pharmaceutical compositions and pharmaceuticals comprising the synthetic skeletal muscle-specific promoters, expression systems, vectors, and cells of this disclosure.

[0173] Such compositions may comprise related articles (i.e., expression systems / vectors / cells) suitable for clinical use in formulations. This disclosure specifically relates to pharmaceutical compositions / pharmaceuticals comprising vectors according to this disclosure.

[0174] The pharmaceutical compositions / medications disclosed herein may comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (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), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0175] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, to the extent of reasonable medical judgment, for contact with the tissues of a relevant subject (e.g., a human subject) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener in the compositions according to this disclosure must also be "acceptable" in the sense of compatibility with the other components of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in standard pharmacy textbooks (e.g., Remington's "The Science and Practice of Instructions 15 / 86 pages 18 CN 122396772 A Pharmacy" (edited by (A) Adejare), 23rd edition (2020), Academic Press).

[0176] The pharmaceutical compositions / medications according to this disclosure can be formulated for administration to a subject, for example, via a route of administration appropriate to the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, the pharmaceutical compositions / medications can be formulated for parenteral, systemic, local, intracavitary, intravascular, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. In some embodiments, the pharmaceutical composition / medication may be formulated for administration by injection or infusion, or by ingestion.

[0177] Medications and pharmaceutical compositions may be formulated for administration to a blood vessel or to a target tissue / organ (e.g., a tissue / organ affected by a disease / condition, such as a tissue / organ where symptoms of the disease / condition are manifest).

[0178] The pharmaceutical composition / medication may comprise an expression system / vector / cell in a sterile or isotonic medium. The pharmaceutical composition / medication may be provided in a fluid form, including gels. Fluid formulations may be formulated for administration to a blood vessel or selected area of ​​a human or animal body by injection or infusion (e.g., via a catheter). The pharmaceutical composition / medication may be provided in a solid form, such as lyophilized form.

[0179] This disclosure also provides methods for producing pharmaceutical compositions / medications according to this disclosure. Such methods may include combining the expression system / vector / cell described herein with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, etc.Mixing agents, flavoring agents, or sweeteners. Such methods typically involve the step of associating the expression system / vector / cell with a carrier constituting one or more auxiliary components. Typically, compositions are prepared by uniformly and tightly associating the active compound with a carrier (e.g., a liquid carrier, a finely dispersed solid carrier, etc.) and then shaping the product if necessary.

[0180] The expression systems, carriers, cells, and compositions according to this disclosure can be modified and / or formulated to facilitate delivery to and / or uptake by cell types / tissues / organs of interest (e.g., cell types / tissues / organs exhibiting symptoms of disease / symptoms).

[0181] Strategies for targeted delivery of polynucleotides are reviewed, for example, 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 herein by reference in their entirety.

[0182] In some embodiments, the articles of this disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of this disclosure may be associated (covalently or non-covalently) with cell-penetrating peptides (e.g., protein transduction domains, trojan peptides, arginine-rich peptides, vectocell peptides), cationic polymers, cationic lipids, or viral vectors.

[0183] Nanoparticles may be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendritic molecules, and polymeric therapeutic agents. Nanoparticles may be inorganic, such as nanotubes or metal particles, optionally with added organic molecules. In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, the entire contents of which are incorporated herein by reference. In some embodiments, the nanoparticles are PLGA, peptides, poly(β-amino esters), DOPE, β-cyclodextrin-containing polycationic, linear PEI, PAMAM dendrimers, branched PEI, chitosan, or polyphosphate nanoparticles.

[0184] In some embodiments, the expression system and vector according to this disclosure include modifications to incorporate one or more portions to facilitate delivery to and / or uptake by a cell type, organ, or tissue of interest (e.g., a cell type / tissue / organ exhibiting symptoms of a disease / symptom). In some embodiments, a polynucleotide or vector according to this disclosure is linked (e.g., chemically conjugated) to one or more portions to facilitate delivery to and / or uptake by a cell type, tissue, or organ of interest.

[0185] Therapeutic / Prophylactic Application Instructions 16 / 86 pages 19 CN 122396772 A

[0186] The synthetic skeletal muscle-specific promoters, expression systems, vectors, cells, and compositions disclosed herein can be used in treatment and prevention.

[0187] Therefore, this disclosure provides the expression systems, vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. It also provides the expression systems, vectors, cells, or compositions described herein for use in methods of treating or preventing the diseases / conditions described herein. It also provides the use of the expression systems, vectors, cells, or compositions described herein in the manufacture of medicaments for treating or preventing the diseases or conditions described herein. It also provides a method of treating or preventing the diseases or conditions described herein, comprising administering to a subject a therapeutically or preventively effective amount of the expression system, vector, cell, or composition described herein.

[0188] The interventions described in the preceding paragraph can effectively reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition, or reduce the pathology of a disease / condition. Interventions can effectively prevent the progression of a disease / condition, such as preventing the worsening of a disease / condition or slowing the rate of disease / condition development. In some embodiments, the intervention can result in an improvement in the disease / condition, such as a reduction in the symptoms of the disease / condition or a reduction in some other associated severity / activity of the disease / condition. In some implementations, the intervention can prevent the progression / development of the disease / symptom in a later stage (e.g., the chronic phase).

[0189] It should be understood that the expression systems, vectors, cells, and compositions described herein can be used to treat / prevent any disease / symptom that will derive therapeutic or preventive benefit from an increase in the expression level of the polypeptide of interest (i.e., the polypeptide of interest encoded by a polynucleotide).

[0190] For example, a disease / symptom can be associated with and / or characterized by a deficiency / insufficiency of the target polypeptide. A deficiency / insufficiency of the target polypeptide may be positively correlated with the onset, development, or progression of the disease / symptom, and / or positively correlated with the severity of one or more symptoms of the disease / symptom. A deficiency / insufficiency of the target polypeptide may be a risk factor for the onset, development, or progression of the disease / symptom. A disease / symptom is characterized by a reduced expression or activity level of the target polypeptide, for example, compared to the expression / activity level in the absence of the disease / symptom. In some embodiments, the disease / symptom may be characterized by a reduced number / proportion / activity of cells expressing the target peptide, for example, compared to the level / number / proportion / activity in the absence of the disease / symptom (e.g., in healthy subjects or equivalent non-disease tissues).

[0191] For example, in some embodiments, the peptide of interest may be ACTA1, and the disease / symptom to be treated / prevented according to this disclosure may be a disease / symptom caused by a deficiency / insufficiency of ACTA1, such as congenital myopathy, such as, for example, linear myopathy, intranuclear rod myopathy, actin filament aggregate myopathy, congenital fiber type imbalance, and myopathy with a core-like region (Laing

[0192] As a further example, in embodiments in which the target polypeptide is a polypeptide capable of inhibiting the expression and / or activity of a target antigen of interest, the disease / symptom can be a disease / symptom in which the target antigen is pathologically involved or cells containing / expressing the target antigen are involved, for example, a disease / symptom in which an increase in the level / activity of the target antigen or an increase in the number / proportion / activity of cells containing / expressing the target antigen is positively correlated with the onset, development or progression of the disease / symptom and / or the severity of one or more symptoms of the disease / symptom. In some embodiments, an increase in the level / activity of the target antigen or an increase in the number / proportion / activity of cells containing / expressing the target antigen can be a risk factor for the onset, development or progression of the disease / symptom. The disease / symptom can be characterized by an increase in the level of expression or activity of the target antigen, for example, compared to the level of expression / activity in the absence of the disease / symptom. In some embodiments, the disease / symptom may be characterized by an increase in the number / proportion / activity of cells expressing the target antigen, for example, compared to the level / number / proportion / activity in the absence of the disease / symptom (e.g., in healthy subjects or equivalent non-disease tissues). The therapeutic / preventive intervention according to this disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject or a subject treated with an appropriate control): a decrease in the level of the target antigen; a decrease in the activity of the target antigen; and / or a decrease in the number / proportion / activity of cells containing / expressing the target antigen.

[0193] Further, by example, the disease / symptom may be a disease / symptom to be treated by nucleic acid editing, and the polypeptide of interest may be a constituent protein of a suitable site-specific nuclease nucleic acid editing system.

[0194] This disclosure provides articles of this disclosure for use, uses of articles of this disclosure, and methods comprising administering to a subject (e.g., a subject requiring treatment) polynucleotides, carriers, cells, and compositions according to this disclosure.

[0195] The articles of this disclosure are preferably administered in a “therapeuticly effective” or “preventively effective” amount, which is sufficient to demonstrate a therapeutic or preventative benefit to the subject. The actual amount administered, as well as the rate and timing of administration, will depend on the nature and severity of the disease / symptom and the specific article administered. Prescribing treatment, such as determining the dosage, is within the responsibility of a general practitioner and other physicians, and generally takes into account the disease / symptom to be treated, the individual subject’s condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the above techniques and protocols can be found in Remington’s “The Science and Practice of Pharmacy” ((A)Adejare, ed.), p. 23.Published (2020), Academic Press.

[0196] The articles of this disclosure can be administered parenterally, systemically, intravenously, intra-arterially, intramuscularly, intracavitarily, intrathecally, intraocularly, intravitreally, intraconjunctivally, subretinally, suprachoroidally, subcutaneously, intradermally, orally, nasally, topically, or percutaneously. Administration can be performed by injection or infusion.

[0197] Multiple doses of the articles of this disclosure can be provided. The multiple doses can be separated by predetermined time intervals, which can be selected as 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 one of 1, 2, 3, 4, 5, or 6 months.

[0198] Other Methods

[0199] This disclosure also provides a method for modifying cells to express therapeutic molecules, the method comprising introducing an expression system or vector according to this disclosure into the cells. This disclosure also provides a method for modifying cells to express a protein of interest according to this disclosure, the method comprising introducing an expression system or vector according to this disclosure into the cells.

[0200] In some embodiments, introducing an expression system or vector according to this disclosure into the cells comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0201] Transfection involves the process of introducing nucleic acids into cells using a method other than viral infection, and is therefore a non-viral method. Transfection can be performed by physical / mechanical methods (including electroporation, acoustic perforation, magnetic transfection, gene microinjection, and laser irradiation) or chemical methods (liposome-based or non-liposome-based). Liposome-based transfection reagents are chemicals that enable the formation of positively charged lipid aggregates, which can then fuse with the phospholipid bilayer of the cell to facilitate the entry of foreign genetic material. Examples of liposome-based transfection reagents include, but are not limited to, Olig of ecstasy®, Lipofectamine®, and DharmaFECT®. Non-liposome-based transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendritic molecules, and non-liposomal lipids. One example of a non-liposome-based transfection reagent is polyethyleneimine (PEI).

[0202] Electroporation can be performed, for example as described in Koh et al., Molecular Therapy – Nucleic Acids (2013) 2, e114, which is hereby incorporated herein by reference in its entirety.

[0203] Transduction is the process of introducing nucleic acids into cells via viruses or viral vectors. Thus, in some embodiments, polynucleotides are or are contained in viral vectors, or the vector is a viral vector. Transduction of immune cells with viral vectors is, for example, in Simmons andThe methods described in Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, the entire contents of which are incorporated herein by reference. Reagents may be used in the methods of this disclosure to enhance transduction efficiency. Hexadimethrine bromide (polygluconine) is a cationic polymer commonly used to improve transduction by neutralizing the charge repulsion between viral particles and sialic acid residues expressed on the cell surface. Other reagents commonly used to enhance transduction include, for example, poloxamer-based reagents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), SureENTRY (Qiagen), and ViraDuctin (Cell Biolabs).

[0204] In some embodiments, the method includes centrifuging (referred to in the art as “spinfection”) cells into which the expression system or vector according to the present disclosure is desired to be introduced in the presence of a cell culture medium containing a viral vector containing an expression system.

[0205] In some embodiments, the method includes culturing cells under conditions suitable for the expression of the polypeptide of interest by the cells. In some embodiments, the method includes culturing cells under conditions suitable for transcription of polynucleotides. In some embodiments, the method includes culturing cells under conditions suitable for post-transcriptional processing (e.g., splicing) of polynucleotides. In some embodiments, the method includes culturing cells under conditions suitable for translation of polypeptides from polynucleotides.

[0206] Methods for in vitro / in vitro culture (including generation and / or expansion) of cell populations—including suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, gaseous atmosphere), cell numbers, culture cycles, etc.—are well known to those skilled in the art. Conveniently, cell cultures according to the present disclosure can be maintained at 37 °C in a humid atmosphere containing 5% CO2.

[0207] This disclosure also provides a method for modifying cells to express a polypeptide of interest, the method comprising introducing an expression system or vector according to this disclosure into the cells. When the polynucleotide is an OFF-switch or wherein the vector contains / encodes an OFF-switch, the cells may express the polypeptide of interest after the expression system / vector is introduced into the cells.

[0208] Subject

[0209] According to various aspects of this disclosure, the subject may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use).

[0210] Subjects to whom the articles of this disclosure are administered (e.g., according to a therapeutic or preventative intervention) may be subjects who require such intervention. Subjects are preferably mammals, more preferably humans. Subjects may be non-human mammals, but more preferably humans. Subjects may be male or female. Subjects may be patients.

[0211] Subjects may have (e.g., may have been diagnosed with) the diseases or conditions described herein, may be suspected of having such diseases / conditions, or may be at risk of developing / infecting such diseases / conditions. In embodiments according to this disclosure, subjects may be selected for treatment based on a method of characterizing one or more markers of such diseases / conditions.

[0212] Sequence Identity

[0213] The “sequence identity” between a given nucleotide sequence (e.g., a nucleotide sequence of a polynucleotide) and a reference nucleotide sequence is calculated by determining the percentage of nucleotides in the given nucleotide sequence that are identical to 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, the “sequence identity” between a given amino acid sequence (e.g., the amino acid sequence of a polypeptide) and a reference amino acid sequence is calculated by determining the percentage of amino acids in the given amino acid sequence that are identical to those in 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.

[0214] Pairwise and multiple sequence alignments performed for the purpose of assessing the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, 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). When using such software, it is preferable to use default parameters, such as for gap penalties and extension penalties. Specification page 19 / 86, 22 CN 122396772 A

[0215] Exemplary sequence

[0216] Specification page 20 / 86, 23 CN 122396772 A Specification page 21 / 86, 24 CN 122396772 A DescriptionBook 22 / 86 pages 25 CN 122396772 A Instruction Manual 23 / 86 pages 26 CN 122396772 A Instruction Manual 24 / 86 pages 27 CN 122396772 A Instruction Manual 25 / 86 pages 28 CN 122396772 A Instruction Manual 26 / 86 pages 29 CN 122396772 A Instruction Manual 27 / 86 pages 30 CN 122396772 A Instruction Manual 28 / 86 pages 31 CN 122396772 A Instruction Manual 29 / 86 pages 32 CN 122396772 A Instruction Manual 30 / 86 pages 33 CN 122396772 A Instruction Manual 31 / 86 pages 34 CN 122396772 A Instruction Manual 32 / 86 pages 35 CN 122396772 A Instruction manual, page 33 / 86, 36 CN 122396772 A; page 34 / 86, 37 CN 122396772 A; page 35 / 86, 38 CN 122396772 A; page 36 / 86, 39 CN 122396772 A; page 37 / 86, 40 CN 122396772 A; page 38 / 86, 41 CN 122396772 A; page 39 / 86, 42 CN 122396772 A; page 40 / 86, 43 CN 122396772 A; page 41 / 86, 44 CN 122396772 A; page 42 / 86, 45 CN 122396772 A; page 43 / 86, 46 CN 122396772 A Instruction manual pages 44 / 86, 47 CN 122396772 A; Instruction manual pages 45 / 86, 48 CN 122396772 A; Instruction manual pages 46 / 86, 49 CN 122396772 A; Instruction manual pages 47 / 86, 50 CN 122396772 A; Instruction manual pages 48 / 86, 51 CN 122396772 A; Instruction manual pages 49 / 86, 52 CN 122396772 A; Instruction manual pages 50 / 86, 53 CN 122396772 A; Instruction manual pages 51 / 86, 54 CN 122396772 A; Instruction manual pages 52 / 86, 55 CN 122396772 A; Instruction manual pages 53 / 86.Page 56 CN 122396772 A Instruction Manual 54 / 86 Page 57 CN 122396772 A Instruction Manual 55 / 86 Page 58 CN 122396772 A Instruction Manual 56 / 86 Page 59 CN 122396772 A Instruction Manual 57 / 86 Page 60 CN 122396772 A Instruction Manual 58 / 86 Page 61 CN 122396772 A Instruction Manual 59 / 86 Page 62 CN 122396772 A Instruction Manual 60 / 86 Page 63 CN 122396772 A Instruction Manual 61 / 86 Page 64 CN 122396772 A Instruction Manual 62 / 86 Page 65 CN 122396772 A Instruction Manual 63 / 86 Page 66 CN 122396772 A Instruction Manual 64 / 86 Page 67 CN 122396772 A Specification 65 / 86 pages 68 CN 122396772 A Specification 66 / 86 pages 69 CN 122396772 A Specification 67 / 86 pages 70 CN 122396772 A

[0217] Table 1

[0218] Specification 68 / 86 pages 71 CN 122396772 A Specification 69 / 86 pages 72 CN 122396772 A Specification 70 / 86 pages 73 CN 122396772 A Specification 71 / 86 pages 74 CN 122396772 A Specification 72 / 86 pages 75 CN 122396772 A

[0219] Numbered Paragraphs

[0220] The following numbered paragraphs describe specific aspects and embodiments of this disclosure:

[0221] 1 1. A synthetic skeletal muscle-specific promoter comprising at least six MyoG elements operatively connected to a promoter element.

[0222] 2. The synthetic skeletal muscle-specific promoter according to paragraph 1, comprising at least eight MyoG elements operatively connected to a promoter element.

[0223] 3. The synthetic skeletal muscle-specific promoter according to paragraph 1 or 2, comprising six or eight MyoG elements operatively connected to a promoter element.

[0224] 4. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 3, wherein the synthetic skeletal muscle-specific promoter has eight MyoG elements.

[0225] 5. According to paragraphs 1 to 46. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 5, wherein the synthetic skeletal muscle-specific promoter comprises 8 MyoG elements.

[0226] 7. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 5, wherein the MyoG elements individually comprise or consist of the polynucleotide sequence AGCAGCTGC (SEQ ID NO:24).

[0227] 8. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 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 with SEQ ID NO:25 or the specification page 73 / 86 76 CN 122396772 A.

[0228] 9. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 7, wherein the MyoG elements are linked by at least one linker.

[0229] 9. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 8, wherein the MyoG elements are connected via a connector.

[0230] 10. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 9, wherein the MyoG elements are connected via connectors of equal length.

[0231] 11. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 10, wherein at least one connector has a length of 4 bp to 14 bp, preferably 4 bp to 6 bp, most preferably 4 bp.

[0232] 12. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 11, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% sequence identity with 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.

[0233] 13. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 12, wherein the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:49 or SEQ ID NO:50.

[0234] 14. According to paragraphs 1 to 14...13. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 15, wherein the promoter element comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0235] 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 with SEQ ID NO:27 or SEQ ID NO:28.

[0236] 16. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 15, wherein the synthetic skeletal muscle-specific promoter comprises or is composed of the sequence of SEQ ID NO:27 or SEQ ID NO:28.

[0237] 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 with 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: 100%. NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0238] 18. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 17, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.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.

[0239] 19. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 18, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similar to the sequence on page 77 of this specification, CN 122396772 A, 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.

[0240] 20. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 19, comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0241] 21. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 20, wherein the synthetic skeletal muscle-specific promoter comprises 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.

[0242] 22. A synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 21, wherein i) the ratio of promoter activity in HSMM cells 5 days after transduction with serotype 2 AAV at an MOI of 20,000 to ii) the ratio of promoter activity in Huh7 cells 3 days after transduction with the same AAV at an MOI of 20,000 is 10 or more.

[0243] 23. According to paragraph 22The synthetic skeletal muscle-specific promoter, wherein the ratio is 100 or more, 1000 or more, 10,000 or more, or 20,000 or more.

[0244] 24. The synthetic skeletal muscle-specific promoter according to any one of paragraphs 22 or 23, wherein promoter activity is determined as the fluorescence intensity of the expression of a fluorescent protein operably linked to the synthetic promoter, wherein the fluorescent protein and the synthetic promoter are contained in a transgene of AAV.

[0245] 25. The synthetic skeletal muscle-specific promoter according to paragraph 24, wherein the fluorescent protein is mGreenLantern.

[0246] 26. An expression system comprising a synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 25 operably linked to a polynucleotide encoding a polypeptide of interest.

[0247] 27. A vector comprising a synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 25 operably linked to a polynucleotide encoding a polypeptide of interest, optionally wherein the vector is an adeno-associated virus (AAV) vector.

[0248] 28. The expression system according to paragraph 26 or the vector according to paragraph 27, wherein the polynucleotide encoding the polypeptide of interest contains a start codon located at the 3' of a synthetic skeletal muscle-specific promoter.

[0249] 29. The expression system or vector according to any one of paragraphs 26 to 28, further comprising a polyadenylated sequence at the 3' of the polynucleotide encoding the polypeptide of interest.

[0250] 30. The expression system or vector according to any one of paragraphs 26 to 29, further comprising a 5' UTR sequence operatively linked to a synthetic skeletal muscle-specific promoter, wherein the 5' UTR sequence is located at the 3' of the synthetic skeletal muscle-specific promoter, optionally wherein the 5' UTR contains an intron.

[0251] 31. The expression system or vector according to paragraph 30, wherein the intron is not a complete, naturally occurring promoter intron, or wherein the intron is not a complete, naturally occurring intron.

[0252] 32. The expression system or vector according to paragraph 30 or 31, wherein the introns are truncated natural or synthetic introns.

[0253] 33. The vector according to any one of paragraphs 27 to 32, further comprising an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0254] 34. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome is truncated at its 5' end to its 3' end.The sequence comprises:

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

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

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

[0258] (iv) a polynucleotide encoding a therapeutic molecule;

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

[0260] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0261] 35. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises, in a 5' to 3' order:

[0262] (i) a 5' inverted terminal repeat (ITR) sequence;

[0263] (ii) a synthetic skeletal muscle-specific promoter according to any one of paragraphs 1 to 25;

[0264] (iii) optionally, a 5' UTR sequence;

[0265] (iv) a polynucleotide encoding the protein of interest;

[0266] (v) a 3' UTR sequence; and

[0267] (vi) a 3' inverted terminal repeat (ITR) sequence.

[0268] 36. The AAV vector according to paragraph 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 with the nucleotide sequence according to SEQ ID NO:1.

[0269] 37. The AAV vector according to any one of paragraphs 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 with 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.

[0270] 38. The AAV vector according to any one of paragraphs 34 to 37, wherein the polynucleotide encoding the protein of interest comprises or is composed of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence according to SEQ ID NO:36 or SEQ ID NO:37.

[0271] 39. The AAV vector according to any one of paragraphs 34 to 38, wherein the 3' UTR sequence comprises a polynucleotide sequence.Glycation sequence.

[0272] 40. The AAV vector according to any one of paragraphs 34 to 39, wherein the 3' UTR sequence comprises or is composed of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence according to SEQ ID NO:38 or SEQ ID NO:39.

[0273] 41. The AAV vector according to any one of paragraphs 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 with the nucleotide sequence according to SEQ ID NO:2.

[0274] 42. A cell comprising an expression system according to any one of paragraphs 26 to 32, or a vector according to any one of paragraphs 27 to 33, or an AAV vector according to any one of paragraphs 34 to 41.

[0275] 43. A pharmaceutical composition comprising an expression system according to any one of paragraphs 26 to 32, or a vector according to any one of paragraphs 27 to 33, or an AAV vector according to any one of paragraphs 34 to 41, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0276] 44. An expression system according to any one of paragraphs 26 to 32, or a vector according to any one of paragraphs 27 to 33, or an AAV vector according to any one of paragraphs 34 to 41, or a pharmaceutical composition according to paragraph 43, for treating or preventing a disease or condition from which therapeutic or preventive benefits are derived from an increase in the level of the polypeptide of interest expressed in skeletal muscle cells.

[0277] 45. The use of an expression system according to any one of paragraphs 26 to 32, or a vector according to any one of paragraphs 27 to 33, or an AAV vector according to any one of paragraphs 34 to 41, or a pharmaceutical composition according to paragraph 43, in the manufacture of a medicament for treating or preventing a disease or condition from which therapeutic or preventive benefits will be derived by an increase in the level of the expressed polypeptide of interest.

[0278] 46. A method of treating or preventing a disease or condition from which therapeutic or preventive benefits will be derived by an increase in the level of the expressed polypeptide of interest, comprising administering to a subject an expression system according to any one of paragraphs 26 to 32, or a vector according to any one of paragraphs 27 to 33, or an AAV vector according to paragraph 34 to 41.The AAV carrier described in any one of paragraphs 41, or the pharmaceutical composition described in paragraph 43.

[0279] 47. The invention as described above with reference to the examples and drawings.

[0280] ***

[0281] This disclosure includes combinations of the described aspects and preferred features unless such combination is obviously not permitted or explicitly avoided.

[0282] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0283] Aspects and embodiments of this disclosure will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0284] Throughout the specification (including the following claims), unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” should be understood to imply inclusion of the stated integers or steps or groups of steps, but not to exclude any other integers or steps or groups of steps.

[0285] As used herein, “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length of more than about 50 amino acids. The peptides, polypeptides and proteins mentioned herein also include glycopeptides / glycopeptides / glycoproteins, lipopeptides / lipopeptides / lipoproteins, nucleopeptides / nucleopeptides / nucleoproteins, etc.

[0286] As used herein, an amino acid sequence or polypeptide region “corresponding to” a specified reference amino acid sequence or polypeptide region has at least 60% (e.g., at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the amino acid sequence / polypeptide / region. The amino acid sequence / region / position of the polypeptide / amino acid sequence “corresponding” to the specified reference amino acid sequence / region / position can be identified by sequence alignment of the test sequence with the reference sequence, for example using sequence alignment software such as Clustal Omega (Söding, J. 2005, Bioinformatics 21, 951-960).

[0287] Similarly, the nucleotide sequence or polynucleotide region “corresponding” to the specified reference nucleotide sequence or polynucleotide region has at least 60% (e.g., at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%) of the amino acid sequence of that nucleotide sequence / polynucleotide / region.Sequence identity of ≥99% or 100%. The polynucleotide / nucleotide sequence "corresponding" to the specified reference nucleotide sequence / region / position can be identified by sequence alignment of the test sequence with the reference sequence, for example using sequence alignment software such as ClustalOmega (Söding, J. Instruction manual 77 / 86 pages 80 CN 122396772 A 2005, Bioinformatics 21, 951-960).

[0288] As used herein, an amino acid sequence “derived from” a reference amino acid sequence (e.g., an amino acid sequence of a reference peptide / peptide / domain / region) comprises, or is composed of, an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the reference amino acid sequence. Similarly, a nucleotide sequence “derived from” a reference nucleotide sequence (e.g., a nucleotide sequence of a polynucleotide) comprises, or is composed of, a nucleotide sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the reference nucleotide sequence.

[0289] It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. A range 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 one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, the particular value will be understood to form another embodiment by using the antecedent “about.”

[0290] When nucleic acid sequences are disclosed herein, their reverse complementary sequences are also explicitly considered.

[0291] The methods described herein may preferably be performed in vitro. The term “in vitro” is intended to cover procedures performed in cultures using cells, while the term “in vivo” is intended to cover procedures performed using / on intact multicellular organisms. Brief Description of the Drawings

[0292] Embodiments and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings.

[0293] Figure 1: Reporter gene expression of a first-generation AAV vector in human skeletal muscle myocytes (HSMM). Report AAV was transduced into HSMMs at MOIs ranging from 100 to 400,000, and eGFP levels were measured using cytometry.Cells transduced with a promoterless reporter construct (using mGL instead of eGFP) were used as controls. The mean fluorescence index (MFI) was plotted.

[0294] 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 (using mGL instead of eGFP) were used as controls. The mean fluorescence index (MFI) was plotted.

[0295] Figure 3: Atlas of synthetic promoters containing muscle-specific transcription factor motifs and minimal promoter elements. The location of transcription factor binding motifs (black arrows) and minimal promoters (gray arrows) is indicated below the nucleotide sequence.

[0296] Figure 4: Reporter gene expression of second- and third-generation AAV vectors in human skeletal muscle myocytes (HSMM). The reporter AAV was transduced into HSMM cells at an MOI of 400,000, and mGL levels were measured using cytometry. Cells transduced with a promoterless reporter construct were used as controls. The mean fluorescence index (MFI) was plotted.

[0297] Figure 5: Reporter gene expression of second- and third-generation AAV vectors in hepatocellular carcinoma cell lines. The reporter AAV was transduced into HepG2 (A) and Huh7 (B) cells at an MOI of 250,000, and mGL levels were measured using cytometry. Cells transduced with a promoterless construct were used as controls. The mean fluorescence index (MFI) was plotted.

[0298] Figure 6: Reporter gene expression of subsets of second- and third-generation AAV vectors in HSMM, HepG2, and Huh7 cells. The reporter AAV was transduced into HSMM, HepG2, and Huh7 cells at an MOI of 20,000, and mGL levels were measured using cytometry. Cells transduced with a promoterless reporter construct were used as controls. (6A) Mean fluorescence index (MFI); (6B) Ratio of reporter gene expression in HSMM and Huh7 cells. For each construct, the MFI in the HSMM was divided by the MFI in Huh7. The MFI of the promoterless control was subtracted from the MFI of the test construct before calculating the ratio.

[0299] Figure 7: Relative gene expression of the fourth-generation design of the optimized 8x MyoG motif CMV2 reporter gene. Adherent HEK293 cells were co-transfected with the fourth-generation promoter construct and MyoG transcription factor or control plasmid, respectively. The nonameric MyoG was studied.The effect of the number of binding motifs (0x, 2x, 4x, 6x, 8x, 12x, 14x, 16x) on gene expression was investigated (7A). 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 around the core binding motif (7B) did not result in a corresponding increase in gene expression. The effect of the distance between motifs was measured at 4 bp, 6 bp (8xMoG CMV2), 8 bp, 10 bp, 12 bp, and 14 bp (7C). The highest geometric mean fluorescence index (MFI) (a 1.39-fold increase) was achieved at a 4 bp distance between motifs, with decreased expression observed at longer distances. Reducing the spacing between the last motif and the minimal promoter from 44 bp to 0 bp resulted in a 2.10-fold increase in expression (7D). Finally, the performance of the additional minimal promoter tested was not superior to the original minimal CMV promoter 2 (7E). Data represent mean ± SD of three biological replicates, each containing three technical replicates.

[0300] Figure 8: Evaluation of improved fifth-generation reporter gene design relative to the 8x MyoG motif CMV2 reporter gene. Reducing the distance between the last MyoG binding motif and the minimum promoter had the most significant effect 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 motif: 1.72-fold increase, 6x MyoG motif: 2.09-fold increase). The optimal configuration, combining 6 MyoG binding motifs without spacers, resulted in a 2.09-fold increase in expression. Data represent mean ± SD of two biological replicates.

[0301] Examples

[0302] Example 1: General Method

[0303] 1.1 Recombinant DNA Technology

[0304] DNA was manipulated using standard methods, as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. The desired gene fragment was synthesized from synthetic oligonucleotides and PCR products by automated gene synthesis at Geneart AG (Regensburg, Germany). The gene fragment flanked by a single restriction endonuclease cleavage site was cloned into a standard cloning / sequencing plasmid. The plasmid was purified from transformed bacteria.DNA was used, and its concentration was determined by ultraviolet spectroscopy. The DNA sequence of the cloned gene fragment was confirmed by DNA sequencing. The gene fragment was designed with suitable restriction sites to allow transfer into the corresponding reporter vector.

[0305] 1.2 Production of rAAV by triple transfection

[0306] Recombinant AAV was produced by triple transfection of Expi293F (Thermo-Fisher Scientific) cells using PEI MAX (Polysciences) as the transfection reagent, as recommended by the manufacturer. Briefly, Expi293F cells were grown to a density of 3 x 10⁶ cells / mL in 500 mL shake flasks and 125 mL culture volumes. For transfection, equimolar amounts of the first plasmid (pAAV2 rep / cap) carrying the AAV2 rep-cap gene, the second plasmid (pHelper) carrying the adenovirus helper genes E4, E2a, and VA, and the third plasmid (pTransgene) carrying the reporter gene of interest flanked by AAV2 ITR were combined and diluted in 2.5 mL of Expi293 expression medium. A total of 0.6 µg of DNA was used per 10⁶ cells. PEI MAX was mixed separately with 2.5 mL of Expi293 expression medium and combined with the previously prepared DNA mixture at a ratio of 1:2.5 (DNA:PEI). After incubation, the DNA-PEI complex was added dropwise to the cells. Finally, the transfected cell culture was incubated at 37 °C, 125 rpm, and 8% CO₂ for 72 hours.

[0307] To harvest recombinant AAV, 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, cells were incubated at 37°C and 180 rpm with agitation for 1.5 h. To remove cell debris after lysis, the suspension was centrifuged at 4,000 x g for 60 min. Finally, the supernatant was passed through a 0.8 / 0.2 µm double filter and collected. The sterile and clear cell lysate was stored at -80°C until iodixanol gradient purification was performed.

[0308] 1.3 Purification of rAAV by Iodixanol Gradient Ultracentrifugation

[0309] To purify and isolate the desired intact rAAV capsid, iodixanol density gradient ultracentrifugation was applied using 60%, 40%, 25%, and 15% concentrations.Iodixanol layer. Thaw the filtered viral lysate and transfer it to an ultracentrifuge tube. Then, a gradient of different concentrations of iodixanol was added to the top. The tubes were equilibrated, sealed, and centrifuged at 230,000 xg for 2 hours at 4°C in a Beckman 50.2 Ti rotor. After centrifugation, the tubes were firmly secured and punctured at 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 was collected from the 40% phase. The resulting vector stock solution was stored at -80°C or further processed to exchange iodixanol with PBS and increase viral concentration. This was achieved by ultrafiltration using an Amicon Ultra-15 centrifuge filter. The concentrated sample was stored at -80°C.

[0310] 1.4 Analysis of rAAV transduction and reporter gene expression

[0311] Primary human skeletal muscle myoblasts (HSMMs) were used as a cell model to measure promoter activity in human skeletal muscle. Two hepatocellular carcinoma cell lines, HepG2 and Huh7, were used as control cell lines to assess non-muscle-specific promoter activity.

[0312] For HSMM transduction, cells were seeded at 3 to 4.5 x 10⁴ cells / cm² (usually in 96-well format) in growth medium (Lonza SkBM-2 or PromoCell skeletal muscle growth medium with appropriate supplements provided by the supplier) and checked for correct morphology. On the second day, the cells were transduced with constructs diluted in MEM (L-glutamine, Pen / Strep, 10 µM etoposide, phenol red-free). 4–5 hours after transduction, the cells were supplemented with fetal bovine serum (cf = 2.5% v / v). On the third day, etoposide was removed by changing the growth medium. On day 5, the culture medium was changed to differentiation medium (Lonza DMEM / BioWhittaker F-12 [1:1], L-glutamine, heat-inactivated horse serum plus Pen / Strep). Between d7 and d9, the expression of fluorophores (e.g., eGFP, mGreenLantern) of cells was usually analyzed on Incucyte (Sartorius) or by flow cytometry. For flow cytometry, cells were washed twice with PBS and separated with 0.05% trypsin / EDTA. Separation was stopped by adding growth medium or PBS / 10% FBS. Cells were then centrifuged, washed and co-stained with viability (7AAD) for 15 minutes.

[0313] For transduction of HepG2 or Huh7 cells, cells were seeded per cm2 (96-well format).4.5 x 10⁴ (HepG2) or 6 x 10⁴ (Huh7) cells. Cells were transduced at different MOIs (see illustration) by aspiration of culture medium, and then 100 µL of fresh culture medium was added with 100 µL of the corresponding AAV dilution in the culture medium. Transgenic expression in cells was analyzed on day 3.

[0314] Example 2

[0315] 2.1 Database search for skeletal muscle-specific promoters and construction of first-generation reporter constructs

[0316] Potential skeletal muscle-specific promoters were identified in a three-step process by combining information from different genomic databases. First, skeletal muscle-specific genes were screened in the GTEx tissue expression database (Nat Genet. 2013 Jun; 45(6): 580–585.). Second, annotated promoter regions were identified using the FANTOM5 sequence set (Nature. 2014 Mar 27; 507(7493):462–70). Third, to narrow down potential promoter boundaries, the binding sites of known muscle-specific transcription factors (MYOD, MYOG, MYF5, MYF6) were mapped. In this way, eight candidate promoters of 1.9, 1.2, or 0.5 kb in length were defined. These promoter elements were combined in the 5' to 3' directions with the 5' UTR of the ACTA1 gene (with or without the first intron), the open reading frame encoding eGFP, and the 3' UTR consisting of bovine growth hormone polyadenylation signal (bGH polyadenylation; J Biol Chem. 15 Aug 1992; 267(23): 16330-4) and human gastrin terminator element (HGT; Mol Cell Biol 14 Apr 1986; 6(4): 1032-1043). These reporter genes were inserted between the AAV2 inverted terminal repeat sequences of the rAAV transgenic plasmid. Two corresponding plasmids were generated for comparison, containing either the well-characterized muscle-specific promoter MHCK7 (Mol Ther. Feb. 2007; 15(2): 320–9) or the strongly ubiquitous promoter CAG (Gene. Dec. 15. 1991; 108(2): 193–9).

[0317] 2.2 Evaluation of the first-generation reporter constructs

[0318] rAAV particles were generated and purified as described above and then transduced into HSMM cells. Constructs carrying eGFP under the control of potential skeletal muscle-specific promoters were transduced at MOIs between 100 and 400,000.Transduction was performed to assess the dynamic range of the assay. rAAV carrying the strongly ubiquitous CAG promoter was transduced at a single intermediate MOI of 20,000, while for the promoterless construct, a maximum MOI of 400,000 was applied. Reporter gene expression was measured by flow cytometry.

[0319] As expected, the measured fluorescence increased with increasing MOI (Fig. 1). However, the signal tended to plateau at MOIs of 100,000 or higher. Constructs driven solely by the ACTA1 or MYH2 promoters, except for the control constructs based on MHCK7 and CAG, showed significantly higher eGFP expression than the promoterless control. CAG-driven reporter rAAV expression was significantly higher than any other construct.

[0320] When the same constructs were transduced into HepG2 or Huh7, strong reporter gene expression was observed only when using the CAG promoter (Fig. 2). The ACTA1 promoter construct was expressed slightly higher than the control without a promoter, while all other constructs were expressed at background levels.

[0321] Based on these results, the ACTA1 and MYH2 promoters were selected for further engineering to improve the strength and specificity of skeletal muscle-specific expression.

[0322] Example 3

[0323] 3.1 Construction of the second-generation reporter construct

[0324] Three additional versions of the MYH2 promoter were created. In one version (MYH2 0.7 kb), the 5' end 500 bp was deleted from the 1.2 kb fragment, resulting in a 0.7 kb fragment. In the second version (MYH2 0.7 kb double), this 0.7 kb fragment was repeated. In the third version (MYH2 1.2 kb min CMV5'), the DNA fragment 31 bp upstream of the TATA box was replaced with the corresponding sequence of the human CMV major immediate early promoter. These fragments were combined at the 5' to 3' direction with a short synthetic 5' UTR, a short human β-globin / human Ig heavy chain chimeric intron, an open reading frame encoding mGreenLantern (mGL; PNAS. Dec 1, 2020; 117(48): 30710–30721), a marmot hepatitis virus posttranscriptional regulatory element (WPRE; Hum Gene Ther. Sep 20, 1999; 10(14): 2295–305), and a human growth hormone polyadenylate signal (hGH polyadenylate, GenBank accession number NG_011676). These reporter genes were then inserted into the AAV2 ITR of the rAAV transgenic plasmid.Between. For comparison, five corresponding plasmids were generated, containing a promoterless, a 1.9 kb ACTA1 promoter, a 1.2 kb MYH2 promoter, an MHCK7 promoter, or a ubiquitous strong CBA promoter (a variant of the CAG promoter lacking the first 78 bp and differing at the other four nucleotide positions). An additional MHCK7-based construct was created containing the SV40 late 16s minimal intron (as described in European patent application EP3442600), instead of the human β-globin / human Ig heavy chain chimeric intron.

[0325] To compensate for the different promoter lengths and keep the size of the recombinant AAV genome approximately constant, 500, 1000, or 1481 bp of non-coding filler DNA was inserted upstream of the 3' ITR sequence.

[0326] Furthermore, a comparative promoter occupancy analysis was performed based on transcriptomic data from HepG2 and HSMM cells using the first-generation promoter (Nat Rev Genet. July 2009; 10(7): 443-456). The goal was to enhance the activation of transcription factors in skeletal muscle cells and attenuate the activity of repressive transcription factors, while in other cells (especially hepatocytes), the aim was to produce the opposite effect. GTEx tissue expression data for various transcription factors were also considered.

[0327] Based on these results, four additional ACTA1 promoter variants were designed. First, the 1.9 kb ACTA1 promoter was shortened to 800 bp at the 3' end (ACTA1 Δ 400 bp). Second, in this 800 bp ACTA1 promoter, three MYOG binding motifs were enhanced (ACTA1 3xMYOG). Third, the binding site of the transcriptional repressor ZNF619 in ACTA1 Δ 400bp (G3 (Bethesda). Jan 8, 2018; 8(1): 219–229) was replaced with the binding site of ZNF121 (Genome Res. Dec 2016; 26(12): 1742–1752), which is not expressed in HSMM and skeletal muscle. This modification produced the promoter ACTA1 ZNF619 / ZNF121. Fourth, the proximal SRF site in ACTA1 ZNF619 / ZNF121 (Cell. Jan 17, 2013; 152(1–2): 327–39) was removed to produce the ACTA1 Δ SRF site.

[0328] In addition, four additional MYH2 were designed.Promoter variants. First, the MYH2 promoter was shortened to 500 bp at the 3' end, resulting in MYH2 0.5 kb. Second, the MYOG binding site within the 500 bp fragment was enhanced, resulting in MYH2 enhanced MYOG. Third, the binding site of the transcriptional repressor ZNF136 (G3 (Bethesda). Jan 8, 2018; 8(1): 219–229) in the latter version was replaced with the binding site of the transcriptional repressor ZNF439 (G3 (Bethesda). Jan 8, 2018; 8(1): 219–229), which is highly expressed in most human tissues but not in skeletal muscle or HSMM. This produced the version MYH2 ZNF136 / ZNF439. Finally, the weak EGR1 site around position 240 of MYH2 ZNF136 / ZNF439 (Cell. January 17, 2013; 152(1-2): 327-39) was replaced with the moderately strong MEF2D site (Cell. January 17, 2013; 152(1-2): 327-39) to generate MYH2 EGR1 / MEF2D.

[0329] 3.2 Construction of the third-generation reporter construct

[0330] Four synthetic promoters were designed by combining the binding motif of skeletal muscle-specific transcription factors with minimal promoters (Figure 3).

[0331] In one version, four nonameric binding motif copies of MYOG separated from each other by a hexameric random sequence (AGCCTT) (AGCAGCTGC; Nucleic Acids Res. Jan 7, 2022; 50(D1): D165–D173) are combined with a short spacer sequence and a minimal promoter sequence containing a TATA box and an Inr promoter element to produce Pmin 4xMyoG. In the same manner, an additional version with eight MYOG binding sites is created and named Pmin 8xMyoG. In a third synthetic promoter called CMV2 8xMyoG, the same fragment containing eight MYOG binding motifs and spacers is combined with a minimal CMV MIE promoter sequence extending from position -54 to position +66. By using the four MYOG binding motifs in CMV2 8xMyoG with two octamer binding motifs targeting the muscle-specific transcription factor MYOD (GCACCTGT; Nucleic Acids Res. 2022 Jan 7; 50(D1): D165–D173) and the muscle-specific transcription activator MEF2D, respectively, the four MYOG binding motifs in CMV2 8xMyoG were compared with those targeting the muscle-specific transcription factor MYOD.The two decameric binding motifs (CTATAAATAG; Nucleic Acids Res. 2022 Jan 7; 50(D1): D165–D173) were replaced to obtain a fourth promoter called CMV2 MyoG&MyoD&Mef2D. These promoters were then combined with a short synthetic 5' UTR, a short human β-globin / human Ig heavy chain chimeric intron, an open reading frame encoding the mGreenLantern (page 82 / 86 of the specification), a marmot hepatitis virus posttranscriptional regulatory element, and a human growth hormone polyadenylate signaling sequence in the 5'–3' direction, in the same manner as the second-generation reporter constructs. These reporter genes were inserted between the AAV2 ITRs of the rAAV transgenic plasmid. In these constructs, a 1481 bp filler sequence was placed upstream of the 3' ITR.

[0332] 3.3 Evaluation of Second- and Third-Generation Reporter Constructs

[0333] rAAV particles carrying mGL reporter genes under the control of second- and third-generation promoters were generated and purified as described above and transduced into HSMM cells at an MOI of 400,000. Reporter gene expression was measured by flow cytometry. Corresponding rAAVs with promoter-free mGL reporter genes were also transduced to assess the measured background signal. In the first experiment, no corresponding construct containing the CBA promoter was transduced because the reporter signal was expected to be out of range (Fig. 4).

[0334] As for the new MYH2 promoter variants, none of them showed higher reporter gene expression than the first-generation 1.2 kb version. This is contrary to expectations, as the modification was intended to increase expression in HSMMs. Modification applied to the 1.9 kB ACTA1 promoter, except for the ACTA1 Δ SRF site, also did not significantly alter reporter gene expression. This one inadvertently showed much lower expression than all other versions. Both the MHCK7 promoter construct and the 1.9 kb ACTA1 promoter showed similar expression levels, which were higher than those of the MYH2 construct.

[0335] All synthetic promoters were active in HSMM cells and drove higher reporter gene expression than the promoter-free control. In particular, the two promoters Pmin 8xMyoG and CMV2 8xMyoG, which contain eight MyoG binding motifs (referred to herein as MyoG elements), were more active than all other constructs tested. Surprisingly, replacing four MYOG motifs in CMV2 8xMyoG with two MYOD and two MEFD2 motifs (producing CMV2 MyoG&MyoD & Mef2D significantly reduced reporter gene expression in HSMM cells.

[0336] The same constructs were transduced into HepG2 and Huh7 cells at an MOI of 250,000 (Fig. 5). In HepG2 cells, reporter gene expression was very high in the promoter-free control, and most constructs showed similar or only slightly higher expression. Relatively strong reporter gene expression was observed only when the ubiquitous CBA promoter was used. In Huh7, the CBA promoter constructs also showed strong expression. In addition, increased expression of ACTA1 1.9 kb, ACTA1 Δ 400 bp, and ACTA1 3MYOG was observed compared to the promoter-free control. Interestingly, ACTA1 ZNF619 / ZNF121-driven expression was reduced compared to ACTA1 Δ 400 bp, suggesting that replacing the ZNF619 binding motif with the ZNF121 motif reduced undesirable non-muscle expression.

[0337] In summary, we observed significant muscle cell-specific expression using the synthetic promoters Pmin 8xMyoG and CMV2 8xMyoG, and a reduction in non-muscle expression using ACTA1 ZNF619 / ZNF121. In contrast, any modification to MYH2 1.2kb did not show the expected effect. Therefore, we decided to further investigate the ability of Pmin 8xMyoG, CMV2 8xMyoG, ACTA1 ZNF619 / 121, and MYH2 1.2 kb, as well as the corresponding control constructs, to drive muscle-specific gene expression. To this end, the corresponding AAV vector carrying the mGL reporter gene was transduced into HSMM, HepG2, and Huh7 cells at an MOI of 20,000, and mGL expression was determined by cytometry (Figure 6A).

[0338] 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 approximately 2-fold (Pmin 8xMyoG) or 6-fold (CMV2 8xMyoG) higher than the muscle-specific benchmark promoter MHCK7 SV40 intron. Importantly, Pmin 8xMyoG and CMV2 8xMyoG showed almost no reporter gene expression in either of the two hepatocellular carcinoma cell lines.

[0339] Similar to previous experiments, expression in HepG2 was generally lower than in the promoter-free control, even when using the CBA promoter. Therefore, we focused our analysis on HSMM and Huh7 cells.Cells. As a quantitative measure specific to muscle, we calculated the ratio of expression levels in HSMM cells and Huh7 cells (Figure 6B). The expression ratio of Pmin 8xMyoG to HSMM / Huh7 was about four times that of using the MHCK7 SV40 intron and even ten times higher than that of using the CBA promoter. When using CMV2 8xMyoG, the expression ratio shifted even more towards HSMM, six times higher than that of using the MHCK7 SV40 intron and 15 times higher than that of using CBA.

[0340] Example 4

[0341] 4.1 Construction of the fourth generation reporter construct

[0342] To further optimize, additional constructs were designed based on the most successful third generation reporter construct (the highly specific CMV2 8xMyoG reporter gene). This design was carefully chosen, taking into account not only its success in Example 3 but also its unique specificity to skeletal muscle. This specificity is particularly important because certain AAV capsids can transduce not only skeletal muscle cells but also cardiomyocytes and smooth muscle cells, as well as non-target tissues such as the liver, lymphocytes, and testes. By utilizing skeletal muscle-specific promoters, gene expression is restricted to muscle cells, thereby minimizing off-target effects and significantly enhancing the precision and efficacy of the treatment. Several aspects were investigated, such as i) the number of nonameric MyoG binding motifs (AGCAGCTGC; Nucleic Acids Res. January 7, 2022; 50(D1): D165–D173; SEQ ID NO:28, 50, 52–57), ii) the directionality and localization of such motifs (SEQ ID NO:58–61), iii) the length of the sequence separating MyoG binding motifs (SEQ ID NO:28, 62–66), iv) the length of the spacer sequence between the last binding motif and the minimum promoter (SEQ ID NO:28, 67–77), and v) the design of the minimum promoter (SEQ ID NO:28, 50, 78–85). Synthesized 5' UTR (px1000, SEQ ID NO:90), a short human β-globin / human Ig heavy chain chimeric intron, an open reading frame encoding mGreenLantern (mGL; PNAS. Dec 1, 2020; 117 (48): 30710–30721), and a marmot hepatitis virus posttranscriptional regulatory element (WPRE; Hum Gene Ther. Sep 20, 1999; 10(14):2295-305) and human growth hormone polyadenylation signal (hGH polyadenylation, GenBank accession number NG_011676) were unchanged.

[0343] 4.2 Transfection of adherent HEK293

[0344] To improve the CMV2 8xMyoG reporter construct, adherent HEK293 cells were co-transfected with the reporter gene variant and transcription factor MyoG or a negative control using Lipofectamine™ 3000 transfection reagent (Invitrogen™) according to the manufacturer's protocol. Briefly, adherent HEK293 cells were seeded into 96-well plates to achieve 70-90% confluence at transfection. Each sample included transcription factor MyoG or a negative control, one of the aforementioned reporter gene variants (3.5 fmol), and a transfection control. The total DNA in each well was 93 ng and 70.6 ng, respectively. DNA was diluted in Opti-MEM™ serum-depleted medium, and 0.2 µL of P3000™ reagent was added to the DNA solution. Additionally, Lipofectamine™ 3000 reagent was diluted to 0.2 µL per well in Opti-MEM™ serum-depleted medium. The diluted DNA / P3000™ mixture was combined with the diluted Lipofectamine™ 3000 reagent and incubated at room temperature for 15 minutes to form a complex. The DNA-Lipofectamine™ 3000 complex was then added to HEK293 cells in 96-well plates, and the plate was gently shaken to ensure uniform distribution. Cells were incubated in a CO2 incubator at 37°C for 72 hours. After incubation, transfection efficiency and gene expression were analyzed using appropriate assays such as fluorescence microscopy and flow cytometry.

[0345] Each biological replicate included three technical replicates, and a total of three biological replicates were performed to ensure robustness and reproducibility.

[0346] 4.3 Assessment of Reporter Gene Expression: Determination of Key Parameters

[0347] In this embodiment of the invention, the effects of different numbers of nonameric MyoG binding motifs (0x, 2x, 4x, 6x, 8x, 12x, 14x, 16x) on gene expression were investigated (Figure 7A). The starting construct contained 8 motifs. It was observed that the construct containing 6 motif repeats (SEQ ID NO:54) achieved the highest expression level, showing a 1.73-fold increase in expression compared to the original CMV2 8xMyoG reporter construct (8-motif construct, SEQ ID NO:28) on pages 84 / 86 of the specification. SurprisinglyYes, this finding shows that reducing the number of motifs to six can enhance gene expression, highlighting the importance of optimizing the number of binding motifs. In contrast, no significant changes in expression were observed by designing bidirectional clusters or changing the nucleotides around the core binding sequence (JASPAR: agCAGCTGc, HOCOMOCO: cgCAGCTGcc; Fig. 7B).

[0348] Next, the distance between motifs was examined (Fig. 7C). A distance of 4 bp (SEQ ID NO: 62) was observed to result in the highest expression, with almost equal expression levels observed for distances of 6 bp and 8 bp between motifs. In contrast, expression levels decreased as the distance between motifs (such as 10, 12, and 14 bp) increased, suggesting that shorter distances between motifs are more conducive to optimal gene expression.

[0349] A thorough study of the distance between motifs and promoter sequences revealed some noteworthy results (Fig. 7D). The initial reporter construct had a 44 bp spacer sequence. Starting from this point, the distance was systematically shortened. While distances of 42bp, 36bp, and 20bp maintained expression levels similar to the original construct, distances of 40bp, 28bp, 24bp, and 16bp resulted in decreased expression. Notably, shorter distances led to increased expression: a 12bp spacer sequence resulted in a 1.23-fold increase, an 8bp spacer sequence resulted in a 1.75-fold increase, and a 0bp spacer sequence achieved a 2.10-fold increase. These findings highlight the significant effect of reducing the distance between the motif and the promoter sequence on enhancing gene expression.

[0350] Furthermore, the efficacy of several additional promoters was evaluated to identify candidates that might be superior to the initially selected minimal promoter (minimal promoter 2, SEQ ID NO: 50). Five pairs of minimal promoters were tested, including shorter versions of synthetic promoters (SEQ ID NO: 28, 50, 78-81) and endogenous promoters (SEQ ID NO: 82-85). Despite rigorous testing, none of the five promoter pairs exhibited superior performance compared to the initial minimal promoter 2.

[0351] The data suggest that careful consideration of the location and number of binding motifs is crucial for maximizing transcriptional efficiency. These findings provide a robust framework for developing efficiently synthesized cell-specific regulatory elements that can be tailored for a variety of applications in gene therapy, synthetic biology, and biotechnology. Insights gained from the design of the fourth-generation construct will inform future iterations and improvements to the previously identified CMV2 8 MyoG reporter construct.

[0352] Example 5

[0353] 5.1 Construction of the fifth-generation reporter construct

[0354] Following Example 4Based on experience, further report constructs were designed. This included a construct (SEQ ID NO: 86) with eight nonameric MyoG binding motifs (AGCAGCTGC; Nucleic Acids Res. 2022 Jan 7; 50(D1): D165–D173), wherein each motif is 4 bp apart, and there is no base pair between the last motif and the CMV2 promoter (SEQ ID NO: 50). The other three designs contained six MyoG binding motifs, each motif is 4 bp apart, and there are 8 bp, 4 bp, or 0 bp between the last motif and the CMV2 promoter (SEQ ID NO: 50). Synthesized 5' UTR, short human β-globin / human Ig heavy chain chimeric introns, open reading frames encoding mGreenLantern (mGL; PNAS. Dec 1, 2020; 117(48): 30710–30721), marmot hepatitis virus posttranscriptional regulatory elements (WPRE; Hum Gene Ther. Sep 20, 1999; 10(14): 2295–305), and human growth hormone polyadenylate signaling (hGH polyadenylate, GenBank accession number NG_011676) were used as described above.

[0355] 5.2 Evaluation of the novel design of the selected fourth-generation reporter construct

[0356] Co-transfection and measurements of all reporter gene variants were performed as described in 4.2. Two biological replicates were performed to ensure robust data evaluation. The fifth-generation reporter construct design supported the findings of the fourth-generation design (Figure 8).

[0357] From left to right, Figure 8 contains the following constructs: Specification 85 / 86 pages 88 CN 122396772 A

[0358] 0x motif, 44bp distance: SEQ ID NO:50

[0359] 8x motif, 8bp, 44bp distance: SEQ ID NO:63

[0360] 8x motif, 6bp, 44bp distance: SEQ ID NO:28

[0361] 8x motif, 6bp, 42bp distance: SEQ ID NO:67

[0362] 8x motif, 6bp, 8bp distance: SEQ ID NO:76

[0363] 8x motif, 6bp, 0bp distance: SEQ ID NO:77

[0364] 8x motif, 4bp, 44bp distance: SEQ ID NO:62

[0365] 6x Motif, 6bp, 44bp distance: SEQ ID NO:54

[0366] 8xMotif, 4bp, 0bp distance: SEQ ID NO:86

[0367] 6x motif, 4bp, 8bp distance: SEQ ID NO:87

[0368] 6x motif, 4bp, 4bp distance: SEQ ID NO:88

[0369] 6x motif, 4bp, 0bp distance: SEQ ID NO:89.

[0370] The reduced distance between the last MyoG binding motif and the smallest promoter resulted in the greatest impact of a single parameter (SEQ ID NO:87 (8bp, 1.52x increase) vs. SEQ ID NO:88 (6bp, 1.75x increase) vs. SEQ ID NO:89 (0bp, 2.09x increase)), followed by the ideal number of motifs (SEQ ID NO:86 (8x MyoG motif; 1.72x increase) vs. SEQ ID NO:89 (6x MyoG motif, 2.09x increase)). However, the greatest effect was observed when all the main conditions were combined.

[0371] Specifically, the optimal configuration was achieved by minimizing the distance between the MyoG binding motif and the minimum promoter (0bp) while optimizing the number of MyoG binding motifs (6x MyoG binding motifs). This combined effect (SEQ ID NO:89 (2.09-fold increase)) highlights the importance of both spatial arrangement and motif number 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 only 195 base pairs.

[0372] Example 6

[0373] In vivo validation of fifth-generation reporter constructs

[0374] In this study, a robust in vivo model was used to evaluate the efficacy and specificity of three different promoters for gene expression using the AAVrh74 capsid. C57BL / 6 mice were randomly divided into three groups (n=4 per group) and administered the AAV vector intravenously at a dose of 1 x 10^12 vector genomes (vg) per mouse (5 x 10^13 vg / mL). Promoters tested included the constitutive CAG promoter (SEQ ID NO:12), the latest MHCK7 promoter (Toscano et al., Gene Ther. 18 (2011) 117-127), and the novel fifth-generation MyoG promoter (SEQ ID NO:89), each driving reporter gene expression (e.g., mGreenLantern or luciferase). After a 4-week period, three mice from each group were sacrificed for comprehensive immunohistochemistry (IHC) of various tissues, including heart, skeletal muscle, and intestinal smooth muscle, as well as off-target tissues such as liver, spleen, and lung.In situ hybridization (ISH) analysis was performed. Remaining mice in each group underwent whole-animal clearance studies using bioluminescence or fluorescence imaging to assess the biodistribution and persistence of the AAV vector. Quantitative measurements of transgene expression levels and tissue distribution were obtained to compare promoter performance. This validation study aimed to confirm the effectiveness of the designed generation 5 promoter in driving skeletal muscle-specific gene expression, providing important insights for the development of targeted gene therapies and supporting the advancement of these promoter designs towards clinical application. Instruction manual page 86 / 86, page 89, CN 122396772 A, Figure 1; Instruction manual figure 1 / 12 page, page 90, CN 122396772 A, Figure 2; Instruction manual figure 2 / 12 page, page 91, CN 122396772 A, Figure 3; Instruction manual figure 3 / 12 page, page 92, CN 122396772 A, Figure 4; Instruction manual figure 4 / 12 page, page 93, CN 122396772 A, Figure 5; Instruction manual figure 5 / 12 page, page 94, CN 122396772 A, Figure 6A; Instruction manual figure 6 / 12 page, page 95, CN 122396772 A, Figure 6B; Instruction manual figure 7 / 12 page, page 96, CN 122396772 A, Figure 7A; Figure 7B; Instruction manual figure 8 / 12 page, page 97, CN 122396772 A, Figure 7C; Instruction manual figure 9 / 12 page, page 98, CN 122396772 A Figure 7D: Appendix to the Instruction Manual, Page 10 / 12, No. 99, CN 122396772 A Figure 7E: Appendix to the Instruction Manual, Page 11 / 12, No. 100, CN 122396772 A Figure 8: Appendix to the Instruction Manual, Page 12 / 12, No. 101, CN 122396772 A

Claims

1. A synthetic skeletal muscle-specific promoter comprising at least six MyoG elements operatively linked to promoter elements.

2. The synthetic skeletal muscle-specific promoter of claim 1, comprising at least eight MyoG elements operatively connected to the promoter elements.

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 element comprises alone or is composed 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 element is connected via a connector.

6. The synthetic skeletal muscle-specific promoter according to any one of claims 1 to 5, wherein the length of the connector is 4 to 14 bp, preferably 4 to 6 bp, and 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 with 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 with 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 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 having at least 80%, 90%, 95%, 96%, 97%, 98%, and 99% of the sequence. Or a nucleotide sequence with 100% sequence identity.

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

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

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

14. The expression system or vector according to claim 11 or 13, wherein the intron is not a complete, naturally occurring intron.

15. The expression system or 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 a 5' to 3' sequence: (i) 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) Polynucleotides encoding therapeutic molecules; (v) 3' UTR sequence; and (vi) 3' Inverted terminal repeat (ITR) sequence.

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

18. The expression system according to claims 11 to 15, or the vector according to any one of claims 12 to 15, or the AAV vector according to claim 16, or the pharmaceutical composition according to claim 17, for the treatment or prevention of a disease or condition from which therapeutic or preventive benefits will be derived by increasing the expression level of the target polypeptide in skeletal muscle cells.

19. Use of the expression system according to claims 11 to 15, or the vector according to any one of claims 12 to 15, or the AAV vector according to claim 16, or the pharmaceutical composition according to claim 17 in the manufacture of a medicament for treating or preventing a disease or condition from which therapeutic or preventive benefits will be obtained from an increase in the expression level of the target polypeptide.

20. A method for treating or preventing a disease or condition from which therapeutic or preventive benefits will be derived by increasing the expression level of a target polypeptide, the method comprising administering to a subject the expression system according to claims 11 to 15, or the carrier according to any one of claims 12 to 15, or the AAV carrier according to claim 16, or the pharmaceutical composition according to claim 17.

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