Hybrid promoters for gene expression in muscle and the CNS

Abstract

The present invention relates to novel hybrid promoters. The present invention further relates to expression cassettes and vectors containing said hybrid promoters. Methods of implementing these hybrid promoters, in particular methods of gene therapy, are also disclosed herein.

Description

[Technical field]

[0001] The present invention relates to hybrid promoters for driving gene expression in muscle and CNS. The present invention further relates to expression cassettes and vectors containing said hybrid promoters. Methods for implementing these hybrid promoters, particularly gene therapy methods, are also disclosed herein. [Background technology]

[0002] Neuromuscular disorders that require simultaneous targeting of muscle and the central nervous system (CNS) represent one of the main challenges for in vivo-based gene therapy. In particular, the high doses of vectors required to efficiently transduce them into the two tissues are likely to induce toxicity in the liver. However, poor transgene expression and anti-transgene immunity in the desired target tissues remain major hurdles in achieving successful gene therapy for many diseases. Thus, there remains a need to provide robust expression of transgenes in cells of interest, but at low doses of vector to prevent both potential toxicity of the vector and immune responses against the vector. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2020 / 208032 [Patent Document 2] WO2009 / 130208 [Patent Document 3] Patent application EP17306448.6 [Patent Document 4] Patent application EP17306447.8 [Patent Document 5] EP18305399 [Patent Document 6] WO2019 / 193119 [Patent Document 7] WO2020 / 200499 [Patent Document 8] WO2022053630 [Patent Document 9] WO2020 / 216861 [Patent Document 10] WO2022 / 003211 [Patent Document 11] WO2021 / 219762 [Patent Document 12] WO2005 / 118792 [Patent Document 13] Application PCT / 2017 / 072942 [Patent Document 14] Application PCT / EP2017 / 072945 [Patent Document 15] Application PCT / EP2017 / 072944 [Patent Document 16] EP18306088 [Patent Document 17] WO2018162748 [Patent Document 18] WO2018046772 [Patent Document 19] WO2018046774 [Patent Document 20] WO2018046775 [Patent Document 21] WO2015158924 [Non-patent literature]

[0004] [Non-Patent Document 1] Carrieri et al., 2012, Nature 491: 454-7 [Non-Patent Document 2] Zucchelli et al., 2015, RNA Biol, 12(8):771-9 [Non-Patent Document 3] Indrieri et al., 2016, Sci Rep, 6: 27315 [Non-Patent Document 4] Chuah et al., Molecular Therapy, 2014, Vol. 22, No. 9, pp. 1605-1613 [Non-Patent Document 5] Wang et al., Gene Therapy, 15, 1489-1499 (2008) [Non-Patent Document 6] Ling et al., July 18, 2016, Hum Gene Ther Methods. [Non-Patent Document 7] Vercauteren et al., 2016, Mol. Ther., vol. 24(6), pp. 1042 [Non-Patent Document 8] Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, 16026 pages [Non-Patent Document 9] McCarty et al., Gene Therapy, 2003 [Non-Patent Document 10] Wu Z. et al., Mol Ther., 2010, Vol. 18(1): 80-86. [Non-Patent Document 11] Lai Y. et al., Mol Ther., 2010, Vol. 18(1): 75-79 [Non-Patent Document 12] Wang Y. et al., Hum Gene Ther Methods, 2012, vol. 23(4): pp. 225-33 [Non-Patent Document 13] "Remington's Pharmaceutical Sciences", E.W. Martin Summary of the Invention [Problem to be solved by the invention]

[0005] Adeno-associated viral vectors (AAV) are the vectors of choice for in vivo gene therapy. The transgene expression cassettes used in AAV gene therapy may contain different elements, such as enhancers and promoters, which allow the modulation of the efficiency and specificity of the expression of the gene of interest in the target cell. Constitutive promoters, such as CMV or CAG, induce strong expression, but lack tissue specificity and are more likely to drive immune responses against the transgene.

[0006] Here, we describe the identification of hybrid promoters that allow specific and strong expression in muscle and CNS, with reduced targeting to the liver, thereby reducing the risk of immune / toxic responses. The hybrid promoters of the invention thus allow either to reduce the dose of the administered vector, or to obtain stronger expression at a comparable dose. [Means for solving the problem]

[0007] The present invention provides a genetic engineering strategy to implement novel hybrid promoters with muscle / CNS specificity without targeting the liver. These hybrid promoters can be used for gene therapy of neuromuscular diseases. These novel hybrid promoters are based on the use of one or more liver-selective enhancers in combination with two muscle-selective promoters. In a particular embodiment, the novel hybrid promoters are based on the combination of: (i) one or more liver-selective enhancers, (ii) a first muscle-specific enhancer which is a CK6 promoter or a functional variant thereof, and (iii) a second muscle-selective promoter, which is selected in the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Actal promoter, desmin promoter, and a functional variant thereof, and the second muscle-selective promoter is preferably a spC5-12 promoter or a functional variant thereof.

[0008] Surprisingly, it is shown herein that such hybrid promoters confer robust expression of transgenes in muscle and spinal cord, without targeting to the liver.

[0009] WO2020 / 208032 describes improved transgene expression of muscle-selective promoters when fused with one or more liver-selective enhancers. Surprisingly, it is shown herein that the combination of a liver-selective enhancer with a first muscle-selective promoter and a second muscle-selective promoter in the same expression cassette results in a synergistic effect when compared to: - expression produced from a combination of a liver-selective enhancer and a first muscle-selective promoter; and - Expression produced from a liver-selective enhancer in combination with a second muscle-selective promoter.

[0010] Thus, a first aspect of the present invention relates to a nucleic acid molecule comprising the following transcriptional regulatory elements operably linked to each other: (i) one or more liver-selective enhancers; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, which is selected in the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, Acta1 promoter, MCK promoter, desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter, CK6 promoter, CK8 promoter, Acta1 promoter or a functional variant thereof, wherein the second muscle-selective promoter is more preferably spC5-12 promoter, CK6 promoter or a functional variant thereof, and wherein the second muscle-selective promoter is even more preferably spC5-12 promoter or a functional variant thereof.

[0011] In certain embodiments, the nucleic acid molecule comprises, in this order from 5' to 3': - one or more liver-selective enhancers, a first muscle-selective promoter, and a second muscle-selective promoter; or - one or more liver-selective enhancers, a second muscle-selective promoter, and a first muscle-selective promoter.

[0012] In a particular embodiment, the first muscle-selective promoter (i.e., CK6 or a functional variant thereof) is located at the upstream 5' end of the second muscle-selective promoter. In a more particular embodiment, the CK6 promoter or a functional variant thereof is located at the upstream 5' end of the spc5.12 promoter or a functional variant thereof. In a more particular embodiment, the CK6 promoter or a functional variant thereof is located at the upstream 5' end of the CK8 promoter or a functional variant thereof. In a more particular embodiment, the CK6 promoter or a functional variant thereof is located at the upstream 5' end of the second CK6 promoter or a functional variant thereof. In a more particular embodiment, the CK6 promoter or a functional variant thereof is located at the upstream 5' end of the Actal promoter or a functional variant thereof.

[0013] In certain embodiments, the nucleic acid molecule comprises, in this order from 5' to 3': - one or more liver-selective enhancers, - the CK6 promoter or a functional variant thereof, and - the spC5-12 promoter or a functional variant thereof; the CK8 promoter or a functional variant thereof; the CK6 promoter or a functional variant thereof; or the Acta1 promoter or a functional variant thereof.

[0014] In certain embodiments, the nucleic acid molecule comprises, in this order from 5' to 3': - one or more liver-selective enhancers - the CK6 promoter or a functional variant thereof, and - the spC5-12 promoter or a functional variant thereof.

[0015] In a further particular embodiment, the CK6 promoter consists of a functional variant having a sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO: 7, or a sequence that is at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 35, preferably at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO: 7.

[0016] In a further particular embodiment, the spC5-12 promoter consists of a functional variant having a sequence as set forth in SEQ ID NO: 4, 5 or 6, or a sequence at least 80% identical to SEQ ID NO: 4, 5 or 6, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 4, 5 or 6. Preferably, the spC5-12 promoter consists of a functional variant having a sequence as set forth in SEQ ID NO: 6, or a sequence at least 80% identical to SEQ ID NO: 6, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 6.

[0017] In a further particular embodiment, the CK8 promoter consists of a functional variant having a sequence as set forth in SEQ ID NO: 33 or 34, or a sequence at least 80% identical to SEQ ID NO: 33 or 34, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 33 or 34. Preferably, the CK8 promoter consists of a functional variant having a sequence as set forth in SEQ ID NO: 33, or a sequence at least 80% identical to SEQ ID NO: 33, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 33.

[0018] In a further particular embodiment, the Acta1 promoter consists of a functional variant having a sequence as shown in SEQ ID NO: 37 or a sequence at least 80% identical to SEQ ID NO: 37, such as at least 85% identical to SEQ ID NO: 37, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0019] In a further particular embodiment, the nucleic acid molecule comprises one liver-selective enhancer operably linked to the muscle-selective promoter. In another embodiment, the nucleic acid molecule comprises a plurality of liver-selective enhancers operably linked to the muscle-selective promoter. In a particular embodiment, the plurality of liver-selective enhancers comprises at least two liver-selective enhancers. In a further embodiment, the plurality of liver-selective enhancers comprises three liver-selective enhancers. In a particular embodiment, the nucleic acid molecule comprises one, two or three liver-selective enhancers, more particularly three liver-selective enhancers. In a particular embodiment, all liver-selective enhancers of the plurality of liver-selective enhancers have the same sequence. In a particular embodiment, the plurality of liver-selective enhancers comprises three liver-selective enhancers having the same sequence. In a further particular embodiment, the plurality of liver-selective enhancers comprises three liver-selective enhancers having the same sequence, said sequence comprising or consisting of SEQ ID NO:1.

[0020] Preferably, the liver-selective enhancer is a sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, or SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: or comprising or consisting of a functional variant having 80% identity, such as at least 85%, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity, to any one of the sequences selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43; or - the plurality of liver-selective enhancers are sequences selected in the group consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, or 3, comprising at least one liver-selective enhancer comprising or consisting of a functional variant having 80% identity, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity, to any one of the sequences selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.

[0021] In a preferred embodiment, the sequence of the liver-selective enhancer comprises or consists of SEQ ID NO: 1 or a functional variant having a sequence at least 80% identical to SEQ ID NO: 1, such as at least 85% identical, in particular at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 1. In a more preferred embodiment, the plurality of liver-selective enhancers consists of three repeats of SEQ ID NO: 1 or three repeats of a functional variant having a sequence at least 80% identical to SEQ ID NO: 1, such as at least 85% identical, in particular at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 1.

[0022] In a particular embodiment, the nucleic acid molecule of the invention consists of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:46 or is a functional variant having a sequence at least 80% identical, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:46.

[0023] The hybrid promoters of the present invention may be operably linked to a transgene of interest.Thus, the present invention further relates to an expression cassette comprising a nucleic acid molecule as described herein, operably linked to a transgene of interest.

[0024] The present invention further relates to a vector comprising the above expression cassette. In a particular embodiment, the vector is a plasmid vector. In another embodiment, the vector is a viral vector. Representative viral vectors include, without limitation, adenoviral vectors, retroviral vectors, lentiviral vectors and parvoviral vectors, such as AAV vectors. In a particular embodiment, the viral vector is an AAV vector, such as an AAV vector comprising AAV8 or AAV9 capsid.

[0025] The present invention also relates to an isolated recombinant cell comprising a nucleic acid construct or an expression cassette or a vector according to the present invention.

[0026] The present invention further relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, a vector or an isolated cell of the invention.

[0027] Furthermore, the present invention also relates to an expression cassette, a vector or an isolated cell as disclosed herein for use as a medicament. In this aspect, the transgene of interest contained in the expression cassette, vector or isolated cell is a therapeutic transgene.

[0028] The present invention further relates to an expression cassette, a vector or an isolated cell disclosed herein for use in gene therapy.

[0029] In another aspect, the invention relates to an expression cassette, vector, or isolated cell disclosed herein for use in the treatment of a neuromuscular disorder.

[0030] In particular, neuromuscular disorders include muscular dystrophies (e.g., myotonic dystrophy (Steiner's disease), Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophies, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, motor neuron diseases (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy ( Infantile progressive spinal muscular atrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular atrophy (type 2), juvenile spinal muscular atrophy (type 3, Kugelberg-Welander disease), adult spinal muscular atrophy (type 4), spinal-bulbar muscular atrophy (Kennedy disease), inflammatory myopathies (e.g., polymyositis, dermatomyositis, inclusion body myositis), diseases of the neuromuscular junction (e.g., myasthenia gravis, Lambert-Eaton (myasthenic) syndrome, congenital myasthenic syndrome), peripheral nerve The myopathy may be selected from the group consisting of myotonia congenita, myotonia congenita, myotonia congenita, myotonia nervosa ...

[0031] In a more particular embodiment, the disease is Coli disease and the transgene of interest is a GDE, such as a truncated form of a GDE, hi another particular embodiment, the disease is Pompe disease. [Brief description of the drawings]

[0032] [Figure 1] (A) Schematic representation of enhancer / promoter combinations (P1-P5). [Diagram 2] Scheme of the in vivo protocol. Mice were injected with AAV vectors encoding murine secreted alkaline phosphatase (mSeAP) under the transcriptional control of P1, P3, or P4 on day 0 and sacrificed 1 month after vector injection. [Diagram 3] AAV vectors carrying different combinations of enhancer / promoter (P1, P3 or P4) have similar transduction efficacies in vivo. DNA was extracted from injected mouse tissues and transduction efficiency was quantified by qPCR in liver and quadriceps. Data are expressed as vector genome copies per cell (VGCN). [Figure 4] Expression of mSeAP in A) liver, B) muscles (heart, diaphragm, quadriceps and triceps) and C) spinal cord. Data are expressed as the ratio of mSeAP to total protein quantified in tissues. Statistical analysis was performed by ANOVA. *=p<0.05 vs. PBS. [Diagram 5] Scheme of the in vivo protocol. Mice were injected with an AAV vector encoding acid-α-glucosidase (GAA) under the transcriptional control of P1–P5 on day 0 and sacrificed 1 month after vector injection. [Figure 6] AAV vectors carrying different enhancer / promoter (P1-P5) combinations have similar transduction efficacy in vivo. DNA was extracted from injected mouse tissues and transduction efficiency was quantified by qPCR in the heart. Data are expressed as vector genome copies per cell (VGCN). [Figure 7] Expression of GAA in the heart A), quantification B), Western blot C). Data are expressed as the ratio of GAA to total protein quantified in the heart. Statistical analysis was performed by ANOVA. *=p<0.05 vs. PBS. [Figure 8] Expression of GAA in quadriceps muscle A), quantification B), and Western blotting C). Data are expressed as the ratio of GAA to total protein quantified in quadriceps muscle. Statistical analysis was performed by ANOVA. *=p<0.05 vs. PBS. [Figure 9] (A) Schematic representation of enhancer / promoter combinations (P1–P3). (B) Scheme of the in vivo protocol. Mice were injected with an AAV vector encoding acid-α-glucosidase (GAA) under the transcriptional control of P1–P3 on day 0 and sacrificed 1 month after vector injection. [Figure 10] (A) Vector genome copy number (VGCN) in heart. GAA expression quantified by Western blot in heart (B-C) and quadriceps (D-E). Data are expressed as the ratio of GAA to vinculin, quantified in heart and quadriceps. Statistical analysis was performed by ANOVA. *=p<0.05 vs. PBS. [Figure 11] (A) Schematic representation of enhancer / promoter combinations (P1–P4). (B) Scheme of the in vivo protocol. Mice were injected with an AAV vector encoding acid-α-glucosidase (GAA) under the transcriptional control of P1–P4 on day 0 and sacrificed 1 month after vector injection. [Figure 12] (A) Vector genome copy number (VGCN) in the heart and (B) GAA expression in quadriceps muscle quantified by Western blot. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] definition In the context of the present invention, a "transcriptional regulatory element" is a DNA sequence that is capable of driving or enhancing expression of a transgene in a tissue or cell.

[0034] In the context of the present invention, the expression "liver-selective enhancer" includes natural or synthetic liver-selective enhancers. Additionally, the expression "muscle-selective promoter" includes natural or synthetic muscle-selective promoters.

[0035] According to the present invention, tissue selectivity means that a transcriptional regulatory element preferentially promotes (in the case of a promoter) or enhances (in the case of an enhancer) the expression of a gene operably linked to said transcriptional regulatory element in a given tissue or set of tissues compared to expression in another tissue. This definition of "tissue selectivity" does not exclude the possibility that a tissue-selective transcriptional regulatory element (e.g., a muscle-selective promoter) may leak to some extent. "Leaking", "leaking" or deviations therefrom refer to the possibility that a muscle-selective promoter may promote or increase the expression of a transgene operably linked to said promoter in another tissue, albeit at a lower expression level. For example, a muscle-selective promoter may leak in liver tissue, meaning that the expression driven by this promoter is higher in muscle tissue than in liver tissue. Alternatively, a tissue-selective transcriptional regulatory element may be a "tissue-specific" transcriptional regulatory element, meaning that not only does the transcriptional regulatory element promote or enhance expression in a given tissue or set of tissues in a preferential manner, but that the regulatory element does not promote or enhance expression in other tissues, or only slightly.

[0036] The expression "liver-selective enhancer" refers to an enhancer that is particularly effective in enhancing transgene expression in the liver. For example, Chua et al. describe a genome-wide in silico method that allows the identification of modules of liver-selective transcription (Chua et al., 2014 Molecular Therapy, Vol. 22, No. 9, pp. 1605-1613). In particular, liver-specific enhancers are as defined in Chua et al. In particular, liver-selective enhancers are cis-regulatory modules that are associated with highly expressed liver-specific promoters. In particular, liver-specific enhancers are cis-regulatory modules that contain a cluster of evolutionarily conserved transcription factor binding site motifs that are associated with strong hepatocyte-specific expression.

[0037] According to the present invention, a "transgene of interest" refers to a polynucleotide sequence that encodes an RNA or protein product and that can be introduced into a cell for the purpose sought and expressed under appropriate conditions. A transgene of interest can encode a product of interest, for example a therapeutic or diagnostic product of interest. In a particular embodiment, the transgene of interest is a therapeutic transgene, i.e. a transgene that encodes a therapeutic product of interest. A therapeutic transgene is selected and used to achieve expression of said therapeutic transgene in cells, tissues or organs where expression of said therapeutic transgene is required, in particular to achieve the desired therapeutic outcome. Therapy can be achieved in several ways, including by expressing a protein in cells that do not express said protein, by expressing a protein in cells that express a mutant form of the protein, by expressing a protein that is toxic to the target cells in which the protein is expressed (e.g. a strategy used to kill unwanted cells such as cancer cells), by expressing an antisense RNA that induces gene silencing or exon skipping, or by expressing a silencing RNA, such as an shRNA or a microRNA, whose purpose is to suppress the expression of a protein. The transgene of interest can also encode a nuclease for targeted genome manipulation, such as a CRISPR-associated protein 9 (Cas9) endonuclease, a meganuclease, or a transcription activator-like effector nuclease (TALEN). The transgene of interest can also be a guide RNA or a series of guide RNAs for use with the CRISPR / Cas9 system, or a correction matrix for use in targeted genome manipulation strategies with nucleases as previously described. Other transgenes of interest include, without limitation, synthetic long non-coding RNAs (SINEUP; Carrieri et al., 2012, Nature 491: 454-7; Zucchelli et al., 2015, RNA Biol, 12(8): 771-9; Indrieri et al., 2016, Sci Rep, 6: 27315) and artificial microRNAs.Other specific transgenes of interest useful in the practice of the present invention are described below.

[0038] Generally, "operably linked" means that a nucleic acid sequence is placed into a functional relationship with another nucleic acid sequence so that each nucleic acid sequence can perform its intended function. Two operably linked sequences can be fused directly to each other or joined through a linker sequence.

[0039] The term "functional variant" refers to a "functional derivative," "fragment," "analog," or "homolog" of a nucleic acid molecule of interest that at least partially retains the biological activity of said nucleic acid molecule of interest. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the nucleic acid molecule of interest. For example, a functional variant of a muscle-selective promoter of interest is a variant that has at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of said muscle-selective promoter, which activity corresponds to the ability to enhance transcription of a particular transgene in muscle.

[0040] The term "identical" and its derivatives refer to the sequence identity between two nucleic acid molecules. If a position in both of the two sequences compared is occupied by the same base, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are identical, the two sequences are 60% identical. Generally, the comparison is performed when the two sequences are aligned to obtain maximum identity. Nucleic acid sequences can be aligned using various bioinformatics tools known to those skilled in the art, such as BLAST or FASTA.

[0041] According to the present invention, the term "treatment" includes curative, alleviating or prophylactic effects. Thus, therapeutic and prophylactic treatments include ameliorating the symptoms of a disorder or preventing or otherwise reducing the risk of developing a particular disorder. Treatments can be administered to delay, slow or reverse the progression of a disease and / or one or more of its symptoms. The term "prophylactic" can be considered to reduce the severity or onset of a particular condition. "Prophylactic" also includes preventing the recurrence of a particular condition in a patient previously diagnosed with that condition. "Therapeutic" can also refer to a reduction in the severity of an existing condition. "Therapeutic amount" means an amount sufficient to cause a qualitative or quantitative reduction in the symptoms of the disorder when administered to a patient suffering from the disorder.

[0042] The subject to be treated in the context of the present invention is an animal, in particular a mammal, more particularly a human subject. In certain embodiments, said mammal may be an infant or adult subject, such as a human infant or human adult as described herein.

[0043] By "cell of therapeutic interest" or "tissue of therapeutic interest" herein is meant the primary cell or tissue in which expression of a therapeutic transgene is useful for the treatment of a disorder. In the present invention, the tissue of interest is muscle tissue and / or CNS tissue.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] Hybrid Promoter The inventors of the present invention have designed a combination of transcriptional regulatory elements, also referred to herein as a "hybrid promoter," to increase the efficacy of gene therapy in muscle and the CNS while reducing liver targeting and fitting within the size constraints of gene therapy vectors, e.g., the size constraints of AAV vectors.

[0046] The nucleic acid molecule of the present invention comprises the following transcriptional regulatory elements operably linked to each other: one or more liver-selective enhancers and two muscle-specific promoters.

[0047] In certain embodiments, the nucleic acid molecules of the invention comprise the following transcriptional regulatory elements operably linked to each other: (i) one or more liver-selective enhancers; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter or the CK6 promoter, or a functional variant thereof, and wherein the second muscle-selective promoter is more preferably the spC5-12 promoter or a functional variant thereof.

[0048] The liver-selective enhancer or the plurality of liver-selective enhancers can be selected from liver-selective enhancers known to those skilled in the art. In a particular embodiment, the nucleic acid molecule of the present invention comprises one and only one liver-selective enhancer. In this embodiment, the size of the liver-selective enhancer may be 10 to 500 nucleotides, for example 10 to 175 nucleotides, particularly 40 to 100 nucleotides, particularly 50 to 80 nucleotides, more particularly 70 to 75 nucleotides. In another embodiment, when multiple liver-selective enhancers are introduced, the size of the combination of the multiple liver-selective enhancers may be 10 to 500 nucleotides, for example 40 to 400 nucleotides, particularly 70 to 250 nucleotides. In a preferred embodiment, the size of the sequence corresponding to the liver-selective enhancer or the plurality of liver-selective enhancers has a length of 50 to 450 pb. In certain embodiments, the size of the sequence corresponding to the liver-selective enhancer or liver-selective enhancers is at least 50 pb, for example at least 100 pb, at least 150 pb, at least 200 pb or at least 250 pb in length. In certain embodiments, the liver-selective enhancer is a naturally occurring enhancer located in cis with a gene selectively expressed in hepatocytes. In further particular embodiments, the liver-selective enhancer may be an artificial liver-selective enhancer.

[0049] Exemplary artificial liver-selective enhancers useful in the practice of the present invention include, without limitation, those disclosed in Chuah et al., Molecule Therapy, 2014, Vol. 22, No. 9, p. 1605, in particular HS-CRM1 (SEQ ID NO: 16), HS-CRM2 (SEQ ID NO: 17), HS-CRM3 (SEQ ID NO: 18), HS-CRM4 (SEQ ID NO: 19), HS-CRM5 (SEQ ID NO: 20), HS-CRM6 (SEQ ID NO: 21), HS-CRM7 (SEQ ID NO: 22), HS-CRM8 (SEQ ID NO: 1), HS-CRM9 (SEQ ID NO: 23), HS-CRM10 (SEQ ID NO: 24), HS-CRM11 (SEQ ID NO: 25), HS-CRM12 (SEQ ID NO: 26), HS-CRM13 (SEQ ID NO: 27), and HS-CRM14 (SEQ ID NO: 28). In certain embodiments, the liver-selective enhancer may be selected in the group consisting of HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14. In further particular embodiments, the liver-selective enhancer may be selected in the group consisting of HS-CRM2, HS-CRM7, HS-CRM8, HS-CRM11, HS-CRM13 and HS-CRM14.

[0050] Other exemplary liver-selective enhancers useful in the practice of the present invention include the apolipoprotein E enhancer (ApoE-enhancer sequence shown in SEQ ID NO:39).

[0051] In a particular embodiment, the liver-selective enhancer is an Apo-E enhancer consisting of SEQ ID NO: 39, or a functional variant of SEQ ID NO: 39 that has liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of an Apo-E enhancer that is at least 80% identical to SEQ ID NO: 39, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 39, wherein said functional variant has liver-selective enhancer activity.

[0052] Other exemplary liver-selective enhancers useful in the practice of the present invention include enhancer A3 (SEQ ID NO:40), enhancer F (SEQ ID NO:41), enhancer S1 (SEQ ID NO:42) and enhancer S2 (SEQ ID NO:43), which are described in WO2009 / 130208 (see Table III on page 30 of WO2009 / 130208).

[0053] In a particular embodiment, the liver-selective enhancer is enhancer A3, which regulates expression of the ApoH gene (genomic location sequence: chr17:61597650-61598200). In a particular embodiment, the liver-selective enhancer is enhancer A3 consisting of SEQ ID NO: 40, or a functional variant of SEQ ID NO: 40 having liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of enhancer A3 that is at least 80% identical to SEQ ID NO: 40, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 40, wherein said functional variant has liver-selective enhancer activity.

[0054] In a particular embodiment, the liver-selective enhancer is enhancer F that regulates expression of the FGA gene (genomic location sequence: chr4:155869502-155869575). In a particular embodiment, the liver-selective enhancer is enhancer F consisting of SEQ ID NO: 41, or a functional variant of SEQ ID NO: 41 having liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of enhancer F that is at least 80% identical to SEQ ID NO: 41, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 41, wherein said functional variant has liver-selective enhancer activity.

[0055] In a particular embodiment, the liver-selective enhancer is enhancer S1 regulating expression of the serpinA1 gene (genomic location sequence: chr14:93891375-93891462). In a particular embodiment, the liver-selective enhancer is enhancer S1 consisting of SEQ ID NO: 42, or a functional variant of SEQ ID NO: 42 having liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of enhancer S1 that is at least 80% identical to SEQ ID NO: 42, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 42, wherein said functional variant has liver-selective enhancer activity.

[0056] In a particular embodiment, the liver-selective enhancer is enhancer S2, which regulates expression of the serpinA1 gene (genomic location sequence: chr14:93897160-93897200). In a particular embodiment, the liver-selective enhancer is enhancer S2 consisting of SEQ ID NO: 43, or a functional variant of SEQ ID NO: 43 having liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of enhancer S1 that is at least 80% identical to SEQ ID NO: 43, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 43, wherein said functional variant has liver-selective enhancer activity.

[0057] In certain embodiments, the liver-selective enhancer is selected from the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14.

[0058] In certain embodiments, the liver-selective enhancer is selected from the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), and HS-CRM8.

[0059] In certain embodiments, the liver-selective enhancer is selected in the group consisting of an ApoE enhancer and HS-CRM8.

[0060] In a particular embodiment, the liver-selective enhancer is HS-CRM8.

[0061] In a particular embodiment, the liver-selective enhancer is an HS-CRM8 enhancer consisting of SEQ ID NO: 1, or a functional variant of SEQ ID NO: 1 having liver-selective enhancer activity. In another embodiment, the liver-selective enhancer is a functional variant of an HS-CRM8 enhancer having at least 80% identity to SEQ ID NO: 1, such as at least 85% identity, particularly at least 90% identity, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity to SEQ ID NO: 1, said functional variant having liver-selective enhancer activity. In the case of multiple liver-selective enhancers, said enhancers may be directly fused or separated by a linker (same or different linker). Direct fusion means that the first nucleotide of an enhancer immediately follows the last nucleotide of the upstream enhancer. When linked via a linker, a nucleotide sequence is present between the last nucleotide of the upstream enhancer and the first nucleotide of the following downstream enhancer. For example, the length of the linker may be comprised between 1 and 50 nucleotides, for example, 1 to 40 nucleotides, for example, 1 to 30 nucleotides, for example, 1 to 20 nucleotides, for example, 1 to 10 nucleotides. In the present invention, the design of the nucleic acid molecule can take into account the size constraints mentioned above, and therefore such linkers, if any, are preferably short. Representative short linkers include nucleic acid sequences that consist of linkers of less than 15 nucleotides, in particular less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 nucleotides, for example, 1 nucleotide. In a particular embodiment, the linker is a restriction enzyme recognition site. In a particular embodiment, the linker is AAGCTT.

[0062] In certain embodiments, the nucleic acid molecule comprises a plurality of liver-selective enhancers, i.e. at least two liver-selective enhancers or at least three liver-selective enhancers. The number of liver-selective enhancers can be determined by the skilled artisan depending on the size of the transgene whose expression is controlled by the nucleic acid molecule of the present invention. In certain embodiments, the plurality of liver-selective enhancers comprises at least two liver-selective enhancers and at most ten liver-selective enhancers. In certain embodiments, the plurality of liver-selective enhancers comprises at least two liver-selective enhancers and at most six liver-selective enhancers. In yet another embodiment, the plurality of liver-selective enhancers comprises two liver-selective enhancers. In a further embodiment, the plurality of liver-selective enhancers comprises three liver-selective enhancers. In a further embodiment, the plurality of liver-selective enhancers comprises four liver-selective enhancers. In yet another embodiment, the plurality of liver-selective enhancers comprises five liver-selective enhancers. In certain embodiments, the nucleic acid molecule comprises one, two or three liver-selective enhancers, more particularly one or three liver-selective enhancers.In certain embodiments, all liver-selective enhancers of the multiple liver-selective enhancers have the same sequence.In certain embodiments, at least two liver-selective enhancers of the multiple liver-selective enhancers have different sequences.

[0063] In certain embodiments, the nucleic acid molecule comprises 1, 2, 3, 4 or 5 repeats of the S1 enhancer consisting of SEQ ID NO:42, or a functional variant of SEQ ID NO:42 that has liver-selective enhancer activity, as described above.

[0064] In certain embodiments, the nucleic acid molecule comprises 1, 2, 3, 4 or 5 repeats of the S2 enhancer consisting of SEQ ID NO:43, or a functional variant of SEQ ID NO:43 that has liver-selective enhancer activity, as described above.

[0065] In certain embodiments, the nucleic acid molecule comprises one, two, three, four or five repeats of enhancer F consisting of SEQ ID NO: 41, or a functional variant of SEQ ID NO: 41 having liver-selective enhancer activity, as described above. In certain embodiments, the nucleic acid molecule comprises three repeats of enhancer F consisting of SEQ ID NO: 41, or a functional variant of SEQ ID NO: 41 having liver-selective enhancer activity, as described above.

[0066] In certain embodiments, all of the liver-selective enhancers of the plurality of liver-selective enhancers have the same sequence. In a preferred embodiment, all of the liver-selective enhancers of the plurality of liver-selective enhancers have the same sequence, which is the sequence of SEQ ID NO: 1 or a functional variant of SEQ ID NO: 1 having liver-selective enhancer activity, as described above.

[0067] In a particular embodiment, the nucleic acid molecule comprises one, two, three, four or five repeats of the HS-CRM8 enhancer consisting of SEQ ID NO: 1 or a functional variant of SEQ ID NO: 1 having liver-selective enhancer activity, as described above. In a particular embodiment, the nucleic acid molecule of the invention comprises three repeats of the HS-CRM8 enhancer consisting of SEQ ID NO: 1 or a functional variant of SEQ ID NO: 1 having liver-selective enhancer activity. In another embodiment, the nucleic acid molecule of the invention comprises three repeats of a functional variant of the HS-CRM8 enhancer that is at least 80% identical to SEQ ID NO: 1, such as at least 85% identical to SEQ ID NO: 1, in particular at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 1, said functional variant having liver-selective enhancer activity. The liver-selective enhancer activity can be determined as described in Chua et al. (Chua et al., 2014 Molecular Therapy, Vol. 22, No. 9, pp. 1605-1613).

[0068] In certain embodiments, the sequence corresponding to the multiple liver-selective enhancers is SEQ ID NO:2 or SEQ ID NO:3, or a functional variant that is at least 80% identical to SEQ ID NO:2 or 3, such as at least 85% identical, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO:2 or 3. SEQ ID NO:2 and SEQ ID NO:3 contain three repeats of the HS-CRM8 enhancer of SEQ ID NO:1.

[0069] In addition, but optionally, the nucleic acid molecule may comprise an additional liver-selective enhancer or additional liver-selective enhancers. According to this embodiment, the nucleic acid molecule may comprise, from 5' to 3', in this order: - a first liver-selective enhancer or a plurality of first liver-selective enhancers, e.g., two or three liver-selective enhancers; - a first muscle-selective promoter as defined above (i.e. the CK6 promoter or a functional variant thereof); - a second liver-selective enhancer or a plurality of second liver-selective enhancers, e.g., two or three liver-selective enhancers; and A second muscle-selective promoter as defined above.

[0070] According to another variant of this embodiment, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer or a plurality of first liver-selective enhancers, e.g., two or three liver-selective enhancers; - a second muscle-selective promoter as defined above (i.e. a muscle-selective promoter selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 or a functional variant thereof); - a second liver-selective enhancer or a plurality of second liver-selective enhancers, e.g., two or three liver-selective enhancers; and - a first muscle-selective promoter as defined above (i.e. the CK6 promoter or a functional variant thereof).

[0071] According to this embodiment, the first liver-selective enhancer or multiple liver-selective enhancers and the second liver-selective enhancer or multiple liver-selective enhancers may be any of the liver-selective enhancers or multiple liver-selective enhancers as described above.

[0072] In certain embodiments, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer or a plurality of first liver-selective enhancers, which liver-selective enhancers are selected from the group consisting of ApoE enhancer (SEQ ID NO: 39), enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1 (SEQ ID NO: 16), HS-CRM2 (SEQ ID NO: 17), HS-CRM3 (SEQ ID NO: 18), HS-CRM4 (SEQ ID NO: No. 19), HS-CRM5 (SEQ ID NO: 20), HS-CRM6 (SEQ ID NO: 21), HS-CRM7 (SEQ ID NO: 22), HS-CRM8 (SEQ ID NO: 1), HS-CRM9 (SEQ ID NO: 23), HS-CRM10 (SEQ ID NO: 24), HS-CRM11 (SEQ ID NO: 25), HS-CRM12 (SEQ ID NO: 26), HS-CRM13 (SEQ ID NO: 27) and HS-CRM14 (SEQ ID NO: 28), in particular HS-CRM8 (SEQ ID NO: 1); - a muscle-selective promoter, which is the CK6 promoter or a functional variant thereof; - a second liver-selective enhancer or a plurality of second liver-selective enhancers, which liver-selective enhancers are ApoE enhancer (SEQ ID NO: 39), enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1 (SEQ ID NO: 16), HS-CRM2 (SEQ ID NO: 17), HS-CRM3 (SEQ ID NO: 18), HS-CRM4 (SEQ ID NO: 19), 9), HS-CRM5 (SEQ ID NO:20), HS-CRM6 (SEQ ID NO:21), HS-CRM7 (SEQ ID NO:22), HS-CRM8 (SEQ ID NO:1), HS-CRM9 (SEQ ID NO:23), HS-CRM10 (SEQ ID NO:24), HS-CRM11 (SEQ ID NO:25), HS-CRM12 (SEQ ID NO:26), HS-CRM13 (SEQ ID NO:27) and HS-CRM14 (SEQ ID NO:28), in particular HS-CRM8 (SEQ ID NO:1); and a muscle-selective promoter selected from the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 or a functional variant thereof.

[0073] According to another variant of this embodiment, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer or a plurality of first liver-selective enhancers, which liver-selective enhancers are ApoE enhancer (SEQ ID NO: 39), enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1 (SEQ ID NO: 16), HS-CRM2 (SEQ ID NO: 17), HS-CRM3 (SEQ ID NO: 18), HS-CRM4 (SEQ ID NO: 19), No. 19), HS-CRM5 (SEQ ID NO: 20), HS-CRM6 (SEQ ID NO: 21), HS-CRM7 (SEQ ID NO: 22), HS-CRM8 (SEQ ID NO: 1), HS-CRM9 (SEQ ID NO: 23), HS-CRM10 (SEQ ID NO: 24), HS-CRM11 (SEQ ID NO: 25), HS-CRM12 (SEQ ID NO: 26), HS-CRM13 (SEQ ID NO: 27) and HS-CRM14 (SEQ ID NO: 28), in particular HS-CRM8 (SEQ ID NO: 1); - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 or a functional variant thereof; - a second liver-selective enhancer or a plurality of second liver-selective enhancers, which liver-selective enhancers are selected from the group consisting of ApoE enhancer (SEQ ID NO: 39), enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1 (SEQ ID NO: 16), HS-CRM2 (SEQ ID NO: 17), HS-CRM3 (SEQ ID NO: 18), HS-CRM4 (SEQ ID NO: 19), HS-CRM5 (SEQ ID NO:20), HS-CRM6 (SEQ ID NO:21), HS-CRM7 (SEQ ID NO:22), HS-CRM8 (SEQ ID NO:1), HS-CRM9 (SEQ ID NO:23), HS-CRM10 (SEQ ID NO:24), HS-CRM11 (SEQ ID NO:25), HS-CRM12 (SEQ ID NO:26), HS-CRM13 (SEQ ID NO:27) and HS-CRM14 (SEQ ID NO:28), in particular HS-CRM8 (SEQ ID NO:1); and a muscle-selective promoter, which is the CK6 promoter or a functional variant thereof.

[0074] In certain embodiments, the nucleic acid molecules of the invention comprise the following transcriptional regulatory elements operably linked to each other: (i) the Apo-E enhancer of SEQ ID NO: 39 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter, the CK6 promoter or a functional variant thereof, and wherein the second muscle-selective promoter is more preferably the spC5-12 promoter or a functional variant thereof.

[0075] In certain embodiments, a nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other in this order from 5' to 3': (i) the Apo-E enhancer of SEQ ID NO: 39 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter, the CK6 promoter or a functional variant thereof, and wherein the second muscle-selective promoter is more preferably the spC5-12 promoter or a functional variant thereof.

[0076] In certain embodiments, a nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other in this order from 5' to 3': (i) the Apo-E enhancer of SEQ ID NO: 39 or a functional variant thereof; (ii) a first muscle-selective promoter, which is the CK6 promoter of SEQ ID NO: 7 or a functional variant thereof; and (iii) a second muscle-selective promoter, which is the spC5-12 promoter of the sequence shown in SEQ ID NO: 4, 5 or 6, in particular SEQ ID NO: 6, or a functional variant thereof.

[0077] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer A3 of SEQ ID NO: 40 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter or a functional variant thereof.

[0078] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer A3 of SEQ ID NO: 40 or a functional variant thereof; (ii) a first muscle-selective promoter, which is the CK6 promoter of SEQ ID NO: 7 or a functional variant thereof; and (iii) a second muscle-selective promoter, which is the spC5-12 promoter of the sequence shown in SEQ ID NO: 4, 5 or 6, in particular SEQ ID NO: 6, or a functional variant thereof.

[0079] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer F of SEQ ID NO: 41 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter or a functional variant thereof.

[0080] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer F of SEQ ID NO: 41 or a functional variant thereof; (ii) a first muscle-selective promoter, which is the CK6 promoter of SEQ ID NO: 7 or a functional variant thereof; and (iii) a second muscle-selective promoter, which is the spC5-12 promoter of the sequence shown in SEQ ID NO: 4, 5 or 6, in particular SEQ ID NO: 6, or a functional variant thereof.

[0081] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': - a first liver-selective enhancer consisting of one, two, three, four or five repeats of enhancer F of SEQ ID NO: 41, or an active and functional variant liver-selective enhancer thereof, as described above; - (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and - (iii) a second muscle-selective promoter, which is selected in the group consisting of the spC5-12 promoter, the CK6 promoter, the CK8 promoter, the MCK promoter, the Acta1 promoter, the desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter or a functional variant thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0082] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': - a first liver-selective enhancer consisting of three repeats of enhancer F of SEQ ID NO: 41, or a functional variant thereof having liver-selective enhancer activity, as described above; - (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and - (iii) a second muscle-selective promoter, which is selected in the group consisting of the spC5-12 promoter, the CK6 promoter, the CK8 promoter, the MCK promoter, the Acta1 promoter, the desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter or a functional variant thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0083] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer S1 of SEQ ID NO: 42 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, Acta1 promoter, MCK promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter or a functional variant thereof.

[0084] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer S1 of SEQ ID NO: 42 or a functional variant thereof; (ii) a first muscle-selective promoter, which is the CK6 promoter of SEQ ID NO: 7 or a functional variant thereof; and (iii) a second muscle-selective promoter, which is the spC5-12 promoter of the sequence shown in SEQ ID NO: 4, 5 or 6, in particular SEQ ID NO: 6, or a functional variant thereof.

[0085] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': - a first liver-selective enhancer consisting of 1, 2, 3, 4 or 5 repeats of enhancer S1 of SEQ ID NO: 42 or a functional variant thereof having liver-selective enhancer activity, as described above; - (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and - (iii) a second muscle-selective promoter, which is selected in the group consisting of the spC5-12 promoter, the CK6 promoter, the CK8 promoter, the MCK promoter, the Acta1 promoter, the desmin promoter, and a functional variant thereof, wherein the second muscle-selective promoter is preferably the spC5-12 promoter or a functional variant thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0086] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer S2 of SEQ ID NO: 43 or a functional variant thereof; (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter, selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter or a functional variant thereof.

[0087] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': (i) enhancer S2 of SEQ ID NO: 43 or a functional variant thereof; (ii) a first muscle-selective promoter, which is the CK6 promoter of SEQ ID NO: 7 or a functional variant thereof; and (iii) a second muscle-selective promoter, which is the spC5-12 promoter of the sequence shown in SEQ ID NO: 4, 5 or 6, in particular SEQ ID NO: 6, or a functional variant thereof.

[0088] In another specific embodiment, the nucleic acid molecule of the invention comprises the following transcriptional regulatory elements operably linked to each other, preferably in this order from 5' to 3': - a first liver-selective enhancer consisting of 1, 2, 3, 4 or 5 repeats of enhancer S2 of SEQ ID NO: 43, or an active and functional variant liver-selective enhancer thereof, as described above; - (ii) a first muscle-selective promoter, which is a CK6 promoter or a functional variant thereof; and - (iii) a second muscle-selective promoter, which is selected in the group consisting of the spC5-12 promoter, the CK6 promoter, the CK8 promoter, the MCK promoter, the Acta1 promoter, the desmin promoter, and a functional variant thereof, wherein this second muscle-selective promoter is preferably the spC5-12 promoter or a functional variant thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0089] In certain embodiments, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer consisting of one, two, three, four or five repeats of the HS-CRM8 enhancer as set forth in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; - a first muscle-selective promoter, which is the CK6 promoter or a functional variant thereof; - a second liver-selective enhancer consisting of one, two, three, four or five repeats of the HS-CRM8 enhancer as set forth in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; and a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0090] In certain embodiments, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer consisting of one, two, three, four or five repeats of the HS-CRM8 enhancer as set forth in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof; - a second liver-selective enhancer consisting of one, two, three, four or five repeats of the HS-CRM8 enhancer as set forth in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; and a muscle-selective promoter, which is the CK6 promoter or a functional variant thereof.

[0091] In more specific embodiments, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer consisting of three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; - a muscle-selective promoter, which is the CK6 promoter or a functional variant thereof; - a second liver-selective enhancer consisting of three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; and a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0092] In certain embodiments, the nucleic acid molecule may comprise, in this order from 5' to 3': - a first liver-selective enhancer consisting of three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof; - a second liver-selective enhancer consisting of three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof having liver-selective enhancer activity, as described above; and a muscle-selective promoter, which is the CK6 promoter or a functional variant thereof.

[0093] In certain embodiments, the sequence of the CK6 promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence shown in SEQ ID NO: 7; - a functional fragment having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 7, said fragment having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence shown in SEQ ID NO: 7, which consists of a sequence which is at least 80% identical to SEQ ID NO: 7, such as at least 85% identical to SEQ ID NO: 7, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0094] A "functional variant" of CK6 refers to a variant that at least partially retains the biological activity of the CK6 promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the CK6 promoter. For example, a functional variant of a CK6 promoter is a variant that has an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the CK6 promoter, which activity corresponds to the ability of CK6 to enhance the transcription of a particular transgene in muscle. In a particular embodiment, the sequence of the CK6 promoter consists of SEQ ID NO: 7 or SEQ ID NO: 35, or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 35, such as at least 85% identical to SEQ ID NO: 7 or SEQ ID NO: 35, in particular at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0095] In certain embodiments, the second muscle-selective promoter is a synthetic promoter C5.12 (spC5.12, alternatively referred to herein as "C5.12"), such as the spC5.12 set forth in SEQ ID NO: 4, 5, or 6, or the spC5.12 promoter disclosed in Wang et al., Gene Therapy, 15, 1489-1499 (2008). In certain embodiments, the sequence of the spC5-12 promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence as shown in SEQ ID NO: 4, 5 or 6, in particular the sequence as shown in SEQ ID NO: 6; - functional fragments having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 4, 5 or 6, in particular compared to SEQ ID NO: 6, said fragments having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence as set out in SEQ ID NO: 4, 5 or 6, in particular the sequence as set out in SEQ ID NO: 6, which consists of a sequence which is at least 80% identical to any one of SEQ ID NO: 4, 5 or 6, in particular at least 85% identical to any one of SEQ ID NO: 4, 5 or 6, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to any one of SEQ ID NO: 4, 5 or 6, in particular at least 85% identical to any one of SEQ ID NO: 6, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0096] A "functional variant" of spC5-12 refers to a variant that at least partially retains the biological activity of the spC5-12 promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the spC5-12 promoter. For example, a functional variant of the spC5-12 promoter is a variant that has at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the spC5-12 promoter, which activity corresponds to the ability of spC5-12 to enhance transcription of a particular transgene in muscle.

[0097] In certain embodiments, the second muscle-selective promoter is a CK8 promoter. In certain embodiments, the sequence of the CK8 promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence shown in SEQ ID NO: 33; - a functional fragment having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 33, said fragment having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence shown in SEQ ID NO: 33, which consists of a sequence which is at least 80% identical to SEQ ID NO: 33, such as at least 85% identical to SEQ ID NO: 33, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 33.

[0098] A "functional variant" of CK8 refers to a variant that at least partially retains the biological activity of the CK8 promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the CK8 promoter. For example, a functional variant of a CK8 promoter is a variant that has an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the CK8 promoter, which activity corresponds to the ability of CK8 to enhance the transcription of a particular transgene in muscle. In a particular embodiment, the sequence of the CK8 promoter consists of SEQ ID NO: 33 or SEQ ID NO: 34, or a functional variant thereof having a sequence that is at least 80% identical to SEQ ID NO: 33 or SEQ ID NO: 34, such as at least 85% identical to SEQ ID NO: 33 or SEQ ID NO: 34, in particular at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0099] In certain embodiments, the second muscle-selective promoter is an MCK promoter. In certain embodiments, the sequence of the MCK promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence shown in SEQ ID NO: 36; - a functional fragment having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 36, said fragment having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence shown in SEQ ID NO: 36, which consists of a sequence which is at least 80% identical to SEQ ID NO: 36, such as at least 85% identical to SEQ ID NO: 36, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 36.

[0100] A "functional variant" of MCK refers to a variant that at least partially retains the biological activity of the MCK promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the MCK promoter. For example, a functional variant of an MCK promoter is a variant that has an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the MCK promoter, which activity corresponds to the ability of MCK to enhance the transcription of a particular transgene in muscle.

[0101] In certain embodiments, the second muscle-selective promoter is the Actal promoter. In certain embodiments, the sequence of the Actal promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence shown in SEQ ID NO: 37; - a functional fragment having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 37, said fragment having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence shown in SEQ ID NO: 37, which consists of a sequence which is at least 80% identical to SEQ ID NO: 37, such as at least 85% identical to SEQ ID NO: 37, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0102] A "functional variant" of Acta1 refers to a variant that at least partially retains the biological activity of the Acta1 promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the Acta1 promoter. For example, a functional variant of the Acta1 promoter is a variant that has an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the Acta1 promoter, which activity corresponds to the ability of Acta1 to enhance the transcription of a particular transgene in muscle.

[0103] In certain embodiments, the second muscle-selective promoter is a desmin promoter. In certain embodiments, the sequence of the desmin promoter or a functional variant thereof is selected from the following: - a sequence consisting of the sequence shown in SEQ ID NO: 38; - a functional fragment having at most 10 extra nucleotides or at most 10 missing nucleotides compared to SEQ ID NO: 38, said fragment having muscle-selective promoter activity; - a sequence which is a functional variant of the sequence shown in SEQ ID NO: 38, which consists of a sequence which is at least 80% identical to SEQ ID NO: 38, such as at least 85% identical to SEQ ID NO: 38, in particular at least 90% identical, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical.

[0104] A "functional variant" of desmin refers to a variant that at least partially retains the biological activity of the desmin promoter. A functional variant can have an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the desmin promoter. For example, a functional variant of the desmin promoter is a variant that has an activity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the desmin promoter, which activity corresponds to the ability of desmin to enhance the transcription of a particular transgene in muscle.

[0105] In the context of the present invention, the transcriptional regulatory elements introduced into the nucleic acid molecule of the present invention (i.e. (i) the liver-selective enhancer or enhancers; (ii) the optional further liver-selective enhancer or enhancers; (iv) the first muscle-selective promoter (i.e. the CK6 promoter); and (v) the second muscle-selective promoter, preferably the spC5-12 promoter) may be directly fused or linked via a linker.

[0106] In a particular embodiment, the nucleic acid molecule of the invention comprises: (i) one or more liver-selective enhancers as described above linked via a linker, in particular via a linker of the sequence ACTAGT or CGCGCC; (ii) a CK6 promoter or a functional variant thereof; said CK6 promoter linked via a linker, in particular via a linker of the sequence TTAATGACCC (SEQ ID NO: 8) or TTCC; (iii) a second promoter, said second promoter being selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof.

[0107] Preferably, the nucleic acid molecule of the invention comprises: (i) one or more liver-selective enhancers as described above linked via a linker, in particular via a linker of the sequence CGCGCC; (ii) the CK6 promoter or a functional variant thereof; said CK6 promoter linked via a linker, in particular via a linker of the sequence TTCC; (iii) a second promoter, said second promoter being selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof.

[0108] For example, in the case of one liver-selective enhancer directly fused to the CK6 promoter, direct fusion means that the first nucleotide of the CK6 promoter immediately follows the last nucleotide of the liver-selective enhancer. Additionally, in the case of a design with multiple liver-selective enhancers directly fused to the CK6 promoter, direct fusion means that the first nucleotide of the CK6 promoter immediately follows the last nucleotide of the 3'-most liver-selective enhancer.

[0109] For example, in the case of a single liver-selective enhancer directly fused to the spC5-12 promoter, direct fusion means that the first nucleotide of the spC5-12 promoter immediately follows the last nucleotide of the liver-selective enhancer. Additionally, in the case of a design with multiple liver-selective enhancers directly fused to the spC5-12 promoter, direct fusion means that the first nucleotide of the spC5-12 promoter immediately follows the last nucleotide of the 3'-most liver-selective enhancer.

[0110] In the case of linking two transcriptional regulatory elements via a linker, the nucleotide sequence is - the last nucleotide of the first transcriptional regulatory element; - the first nucleotide of the second transcriptional regulatory element It exists between.

[0111] For example, in the case of linking a liver-selective enhancer to a CK6 promoter via a linker, the nucleotide sequence is the last nucleotide of a liver-selective enhancer; - the first nucleotide of the CK6 promoter It exists between.

[0112] For example, in the case of linking a liver-selective enhancer to an spC5-12 promoter via a linker, the nucleotide sequence is the last nucleotide of a liver-selective enhancer; - the first nucleotide of the spC5-12 promoter It exists between.

[0113] According to another example, in the case of linking multiple liver-selective enhancers to the CK6 promoter via a linker, the nucleotide sequence is the last nucleotide of the 3'-most liver-selective enhancer; - the first nucleotide of the CK6 promoter It exists between.

[0114] According to another example, in the case of linking multiple liver-selective enhancers to the spC5-12 promoter via a linker, the nucleotide sequence is the last nucleotide of the 3'-most liver-selective enhancer; - the first nucleotide of the spC5-12 promoter It exists between.

[0115] The length of the linker between the enhancer or enhancers and the first promoter may be comprised between 1 nucleotide and 1500 nucleotides, for example, 1 nucleotide to 1000 nucleotides (e.g., 101, 300, 500 or 1000 nucleotides), for example, 1 nucleotide to 500 nucleotides, for example, 1 nucleotide to 300 nucleotides, for example, 1 nucleotide to 100 nucleotides, for example, 1 nucleotide to 50 nucleotides, for example, 1 nucleotide to 40 nucleotides, for example, 1 nucleotide to 30 nucleotides, for example, 1 nucleotide to 20 nucleotides, for example, 1 nucleotide to 10 nucleotides. In the present invention, the design of the core molecule can take into account the size constraints of the vector, in particular the AAV vector, and therefore such linkers, if any, are preferably short. Exemplary short linkers include nucleic acid sequences consisting of less than 15 nucleotides, in particular less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 nucleotides or less than 2 nucleotides, for example, a linker of 1 nucleotide.

[0116] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - the CK6 promoter or a functional variant thereof; and a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably the spC5-12 promoter or a functional variant thereof.

[0117] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof; and - the CK6 promoter or a functional variant thereof.

[0118] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a muscle-selective promoter selected from the group consisting of: CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof; and - the CK6 promoter or a functional variant thereof.

[0119] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - the CK6 promoter or a functional variant thereof; - one or more liver-selective enhancers as described above; and a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof.

[0120] According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of a sequence as shown in SEQ ID NO: 31, or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 31, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 31.

[0121] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a muscle-selective promoter (c) selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof; - one or more liver-selective enhancers as described above; and - the CK6 promoter or a functional variant thereof.

[0122] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - the CK6 promoter or a functional variant thereof; - one or more liver-selective enhancers as described above; and - a muscle-selective promoter (c) selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof.

[0123] According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of a sequence as shown in SEQ ID NO: 32, or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 32, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 32.

[0124] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter or a functional variant thereof; - one or more liver-selective enhancers as described above; and - the CK6 promoter or a functional variant thereof.

[0125] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one liver-selective enhancer, in particular the HS-CRM8 enhancer as shown in SEQ ID NO: 1, or a functional variant thereof; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - a muscle-selective promoter selected in the group consisting of: spC5-12 CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0126] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one liver-selective enhancer, in particular the HS-CRM8 enhancer as shown in SEQ ID NO: 1, or a functional variant thereof; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0127] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one liver-selective enhancer, in particular the HS-CRM8 enhancer as shown in SEQ ID NO: 1, or a functional variant thereof; - a muscle-selective promoter selected from the group consisting of: CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0128] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - two liver-selective enhancers, in particular two repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - the CK6 promoter, in particular the CK6 promoter as shown in SEQ ID NO: 7 or a functional variant; and - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0129] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - two liver-selective enhancers, in particular two repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0130] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - two liver-selective enhancers, in particular two repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a muscle-selective promoter selected from the group consisting of: CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0131] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - the CK6 promoter, in particular the CK6 promoter as shown in SEQ ID NO: 7 or a functional variant thereof; and - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof, preferably spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0132] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0133] According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of a sequence as shown in SEQ ID NO:29, or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO:29, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO:29.

[0134] According to another particular variant, the nucleic acid molecule of the invention consists of a sequence as shown in SEQ ID NO:30, or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO:30, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO:30.

[0135] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a muscle-selective promoter selected in the group consisting of: spC5-12 promoter, CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter and functional variants thereof, preferably spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0136] In certain embodiments, a nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a muscle-selective promoter selected from the group consisting of: CK6 promoter, CK8 promoter, MCK promoter, Acta1 promoter, desmin promoter, and functional variants thereof; and - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0137] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0138] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, which liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14, preferably the liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43) and HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0139] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, wherein the liver-selective enhancer is HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0140] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the spC5-12 promoter, in particular as shown in SEQ ID NO: 4, 5 or 6, in particular as shown in SEQ ID NO: 6, or a functional variant thereof.

[0141] According to a particular variant, the nucleic acid molecule of the invention consists of the sequence shown in SEQ ID NO: 30 or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 30, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 30. The sequence of SEQ ID NO: 30 comprises, operably linked to each other: three repeats of the HS-CRM8 enhancer, the CK6 promoter of SEQ ID NO: 7, and the spC5-12 promoter of SEQ ID NO: 6.

[0142] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - a CK8 promoter, in particular a CK8 promoter as shown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional variant thereof.

[0143] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, which are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14, preferably the liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43) and HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - a CK8 promoter, in particular a CK8 promoter as shown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional variant thereof.

[0144] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, wherein the liver-selective enhancer is HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - a CK8 promoter, in particular a CK8 promoter as shown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional variant thereof.

[0145] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - a CK8 promoter, in particular a CK8 promoter as shown in SEQ ID NO: 33 or SEQ ID NO: 34 or a functional variant thereof.

[0146] According to a particular variant, the nucleic acid molecule of the invention consists of the sequence shown in SEQ ID NO: 44 or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 44, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 44. The sequence of SEQ ID NO: 44 comprises, operably linked to each other: three repeats of the HS-CRM8 enhancer, the CK6 promoter of SEQ ID NO: 7 and the CK8 promoter of SEQ ID NO: 33.

[0147] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a first CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and a second CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0148] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, which liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14, preferably the liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43) and HS-CRM8; - a first CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and a second CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0149] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, wherein the liver-selective enhancer is HS-CRM8; - a first CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and a second CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0150] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a first CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and a second CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof.

[0151] According to a particular variant, the nucleic acid molecule of the invention consists of the sequence shown in SEQ ID NO: 45 or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 45, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 45. The sequence of SEQ ID NO: 45 comprises, operably linked to each other: three repeats of the HS-CRM8 enhancer, a first CK6 promoter of SEQ ID NO: 7 and a second CK6 promoter of SEQ ID NO: 7.

[0152] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers as described above; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the Acta1 promoter, in particular the Acta1 promoter as shown in SEQ ID NO: 37, or a functional variant thereof.

[0153] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, which are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43), HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13 and HS-CRM14, preferably the liver-selective enhancers are selected in the group consisting of ApoE enhancer, enhancer A3 (SEQ ID NO: 40), enhancer F (SEQ ID NO: 41), enhancer S1 (SEQ ID NO: 42), enhancer S2 (SEQ ID NO: 43) and HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the Acta1 promoter, in particular the Acta1 promoter as shown in SEQ ID NO: 37, or a functional variant thereof.

[0154] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - one or more liver-selective enhancers, which liver-selective enhancer is HS-CRM8; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the Acta1 promoter, in particular the Acta1 promoter as shown in SEQ ID NO: 37, or a functional variant thereof.

[0155] In another particular embodiment, the nucleic acid molecule of the invention comprises, specifically from 5' to 3' in this order: - three liver-selective enhancers, in particular three repeats of the HS-CRM8 enhancer as shown in SEQ ID NO:1, or a functional variant thereof; - a CK6 promoter, in particular a CK6 promoter as shown in SEQ ID NO: 7, or a functional variant thereof; and - the Acta1 promoter, in particular the Acta1 promoter as shown in SEQ ID NO: 37, or a functional variant thereof.

[0156] According to a particular variant, the nucleic acid molecule of the invention consists of the sequence shown in SEQ ID NO: 46 or a functional variant thereof having a sequence at least 80% identical to SEQ ID NO: 46, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identical to SEQ ID NO: 46. The sequence of SEQ ID NO: 46 comprises, operably linked to each other: three repeats of the HS-CRM8 enhancer, the CK6 promoter of SEQ ID NO: 7 and the Actal promoter of SEQ ID NO: 37.

[0157] In all embodiments of the nucleic acid molecules of the invention specifically disclosed herein, said nucleic acid molecules may comprise a linker located between the two transcriptional regulatory elements.

[0158] Furthermore, in all embodiments of the nucleic acid molecule of the present invention specifically disclosed herein, the nucleic acid molecule may include a linker located between two liver-selective enhancers in the plurality of liver-selective enhancers. For example, in one embodiment including a plurality of liver-selective enhancers made of two liver-selective enhancers, a linker may or may not be located between the two liver-selective enhancers. Additionally, in one embodiment including a plurality of liver-selective enhancers including three liver-selective enhancers, a linker may be included between the first and second liver-selective enhancers and / or between the second and third liver-selective enhancers. For example, in one embodiment including a plurality of liver-selective enhancers including three liver-selective enhancers, a linker is located between the first and second liver-selective enhancers, and no linker is located between the second and third liver-selective enhancers. In another variation, in one embodiment using three liver-selective enhancers, no linker is located between the first and second liver-selective enhancer, and a linker is located between the second liver-selective enhancer and the third liver-selective enhancer.

[0159] Expression cassette The nucleic acid molecules of the invention can be introduced into expression cassettes designed to direct expression of the transgene of interest to a tissue of interest.

[0160] Thus, an expression cassette of the present invention comprises a nucleic acid molecule as described above and a transgene of interest.

[0161] The expression cassette may also include at least one additional regulatory sequence capable of further controlling expression of the therapeutic transgene of interest by stabilizing the mRNA coding for the protein of interest, e.g., the therapeutic protein encoded by the transgene of interest, by reducing or suppressing its expression in certain tissues other than the tissue of interest. These sequences include, for example, silencers (e.g., tissue-specific silencers), microRNA target sequences, introns, and polyadenylation signals.

[0162] In certain embodiments, an expression cassette of the invention comprises, in this order from 5' to 3': - a nucleic acid molecule of the invention; - a transgene of interest; and - polyadenylation signal.

[0163] In a particular variation of this embodiment, an intron can be introduced between the nucleic acid molecule of the invention and the transgene of interest. Alternatively, the intron can be located within the transgene of interest. In a particular embodiment, the intron can be an SV40 intron, for example, the SV40 intron of SEQ ID NO: 9. In a particular embodiment, the nucleic acid construct comprises a human beta globin b2 (or HBB2) intron, for example, the HBB2 intron of SEQ ID NO: 10 or SEQ ID NO: 11; a coagulation factor IX (FIX) intron, for example, the FIX intron of SEQ ID NO: 12 or SEQ ID NO: 13; or a chicken beta-globin intron, for example, the chicken beta globin intron of SEQ ID NO: 14 or SEQ ID NO: 15.

[0164] Naturally, from the teachings disclosed herein and general knowledge in the field of molecular biology and gene therapy, a person skilled in the art can select and adapt the number of enhancers, the size of the enhancers, the size of the promoter, the size of the linkers, and any other elements, such as further enhancers and introns, according to the size of the transgene of interest incorporated in the expression cassette.

[0165] The transgene of interest may be any transgene as described in the section "Definitions" above. In addition, specific exemplary transgenes of interest are provided in the table below, in which the transgenes are rearranged by the family of neuromuscular disorders they can treat:

[0166] Muscular dystrophies

[0167] [Table 1A]

[0168] [Table 1B]

[0169] Congenital muscular dystrophies

[0170] [Table 2]

[0171] Congenital myopathies

[0172] [Table 3]

[0173] Distal myopathy

[0174] [Table 4]

[0175] Other myopathies

[0176] [Table 5]

[0177] muscle tension syndrome

[0178] [Table 6]

[0179] Ion channel muscle disease

[0180] [Table 7]

[0181] Malignant hyperthermia

[0182] [Table 8]

[0183] Metabolic myopathy

[0184] [Table 9]

[0185] Hereditary cardiac myopathies

[0186] [Table 10A]

[0187] [Table 10B]

[0188] [Table 10C]

[0189] Congenital myasthenic syndrome

[0190] [Table 11]

[0191] Motor neuron disease

[0192] [Table 12A]

[0193] [Table 12B]

[0194] Hereditary motor and sensory neuropathies

[0195] [Table 13A]

[0196] [Table 13B]

[0197] Hereditary paraplegia

[0198] [Table 14A]

[0199] [Table 14B]

[0200] Other neuromuscular disorders

[0201] [Table 15]

[0202] In certain embodiments, the transgene of interest is: alpha-L-iduronidase, acid-alpha-glucosidase (GAA), glycogen debranching enzyme (GDE) or a truncated form of GDE, G6P, alpha-sarcoglycan (SGCA), dystrophin or a truncated form thereof; or SMN1.

[0203] In certain embodiments, the transgene has a length of at most 3500 bp.

[0204] Vectors, cells and pharmaceutical compositions The expression cassette of the present invention can be introduced into a vector. Thus, the present invention also relates to a vector comprising the expression cassette described above. The vector used in the present invention is a vector suitable for RNA / protein expression, particularly suitable for gene therapy.

[0205] In one embodiment, the vector is a plasmid vector.

[0206] In another embodiment, the vector is a non-viral vector, such as a nanoparticle, lipid nanoparticle (LNP) or liposome, containing an expression cassette of the invention.

[0207] In another embodiment, the vector is a system based on a transposon, such as the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al., 2009), that allows integration of the expression cassette of the invention in the genome of the target cell.

[0208] In a further embodiment, the transgene of interest is a repair matrix useful for targeted genome engineering, e.g., a repair matrix suitable for gene correction together with an endonuclease as described above. More particularly, the vector comprises a repair matrix containing arms of homology to the gene of interest for homology-driven integration.

[0209] In another embodiment, the vector is a viral vector suitable for gene therapy targeted to muscle and / or CNS. In this case, further sequences suitable for generating efficient viral vectors, as known in the art, are added to the expression cassette of the invention. In a particular embodiment, the viral vector is derived from an integrating virus. In particular, the viral vector may be derived from an adenovirus, a retrovirus or a lentivirus (e.g. an integration-defective lentivirus). In a particular embodiment, the lentivirus is a pseudotyped lentivirus with an envelope that allows targeting the cells / tissues of interest, e.g. muscle cells (described in patent applications EP17306448.6 and EP17306447.8). When the viral vector is derived from a retrovirus or lentivirus, the further sequences are retroviral or lentiviral LTR sequences flanking the expression cassette. In another particular embodiment, the viral vector is a parvoviral vector, e.g. an AAV vector, e.g. an AAV vector suitable for transduction of muscle and / or CNS. In this embodiment, the further sequences are AAV ITR sequences flanking the expression cassette.

[0210] In a preferred embodiment, the vector is an AAV vector.The human parvovirus adeno-associated virus (AAV) is a naturally replication-defective dependovirus that can integrate into the genome of infected cells to establish latent infection.The last property appears to be unique among mammalian viruses, since integration occurs at a specific site located on chromosome 19 (19q13.3-qter) in the human genome, called AAVS1.

[0211] AAV vectors have therefore attracted considerable interest as potential vectors for human gene therapy. Among the favorable properties of the virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines derived from different tissues that can be infected.

[0212] Among the AAV serotypes isolated and well characterized from humans or non-human primates (NHPs), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other AAV serotypes currently in use include AAV-1, AAV-2 variants (e.g., quadruple mutant capsid-optimized AAV-2, comprising an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., 2016, July 18, Hum Gene Ther Methods.), -3 and AAV-3 variants (e.g., AAV3-ST variant, comprising an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol. Ther., 24(6), 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026), -7, -8, -9, -2G9, -10, e.g., cy10 and -rh10, -rh74, -rh74-9, etc., as disclosed in EP18305399 (e.g., Hybrid Cap rh74-9 serotype as described in the Examples of EP18305399; rh74-9 serotype is also referred to herein as "-rh74-9", "AAVrh74-9" or "AAV-rh74-9"); -9-rh74 as disclosed in EP18305399 (e.g., Hybrid Cap AAVs include the 9-rh74 serotype (-9-rh74 serotype is also referred to herein as "-9-rh74", "AAV9-rh74", "AAV-9-rh74", or "rh74-AAV9"), -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, such as AAVpo4 and AAVpo6, as well as tyrosine, lysine and serine capsid mutants of AAV serotypes, etc. In addition, other non-naturally occurring, engineered mutants and chimeric AAVs may also be useful.

[0213] AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tailoring stability and particle longevity, for efficient degradation, and for precise delivery to the nucleus.

[0214] Desirable AAV fragments for assembly into vectors include the cap proteins, including vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells.

[0215] AAV-based recombinant vectors that lack Rep proteins integrate into the host genome with low efficiency and exist primarily as stable circular episomes that can persist for several years in target cells.

[0216] Instead of using AAV natural serotypes, in the context of the present invention, artificial AAV serotypes can be used, including, without limitation, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences that can be obtained from different selected AAV serotypes, non-contiguous parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotypes can be, without limitation, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0217] In the context of the present invention, an AAV vector comprises an AAV capsid capable of transducing a target cell of interest, i.e., muscle cells and CNS cells. By "CNS" is meant all cells and tissues of the brain and spinal cord. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc.

[0218] According to certain embodiments, the AAV vectors include AAV-1, -2, AAV-2 variants (e.g., quadruple mutant capsid-optimized AAV-2, comprising an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., HumGene Ther Methods, July 18, 2016 [Epub ahead of print]), -3 and AAV-3 variants (e.g., AAV3-ST variant, comprising an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol. Ther., 24(6):1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., Rosario et al., 2016, Mol. Ther. Methods Clin. Dev. 3, p. 16026), -7, -8, -9, -2G9, -10, e.g., -cy10 and -rh10, -rh39, -rh43, -rh74, -rh74-9, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAVs, e.g., AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes, and the like. In certain embodiments, the AAV vector is a vector of AAV8, AAV9, AAVrh74, AAVrh74-9, or AAV2i8 serotype (i.e., the AAV vector has a capsid of AAV8, AAV9, AAVrh74, AAVrh74-9, or AAV2i8 serotype). In further particular embodiments, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are derived from AAV of different serotypes. For example, the pseudotyped AAV vector may be a vector whose genome is derived from one of the above-mentioned AAV serotypes, in particular the AAV2 serotype, and whose capsid is derived from another serotype. For example, the genome of the pseudotyped vector may have a capsid derived from AAV8, AAV9, AAVrh74, AAVrh74-9, or AAV2i8 serotype, and whose genome may be derived from a different serotype.In certain embodiments, the AAV vector has a capsid of the AAV8, AAV9, AAVrh74 or AAVrh74-9 serotype, particularly a capsid of the AAV8 or AAV9 serotype, more particularly a capsid of the AAV8 serotype.

[0219] In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more mutant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0220] In certain embodiments, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins derived from at least two different AAV serotypes, or comprises at least one chimeric VP protein that combines VP protein regions or domains derived from at least two AAV serotypes. For example, a chimeric AAV vector can be derived from a combination of an AAV8 capsid sequence and a set of AAV serotypes different from the AAV8 serotype, such as any of those specifically mentioned above.

[0221] In another embodiment, the modified capsid can also be derived from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In addition, the capsid mutant can include a single amino acid change, such as a tyrosine mutation (e.g., as described in Zhong et al., 2008). In a particular embodiment, the capsid of the AAV vector is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, such as an AAV9-rh74 hybrid capsid or an AAVrh74-9 hybrid capsid modified with a P1 peptide, as described in WO2019 / 193119 or WO2020 / 200499 or WO2022053630. In certain embodiments, the AAV capsid is an AAV9-rh74 capsid as described in WO2019 / 193119, an AAV9-rh74-P1 capsid as described in WO2020 / 200499, or an AAV9-rh74-HB-P1 capsid as described in WO2022053630.

[0222] In further embodiments, the AAV vector is an AAV vector as described in WO2020 / 216861 or an AAV vector as described in WO2022 / 003211. In particular, the AAV vector may have a mutant AAV2 capsid as described in WO2020 / 216861, or a hybrid capsid between AAV8 and AAV2 / 13 as described in WO2022 / 003211.

[0223] In a further embodiment, the AAV vector comprises a porcine AAV serotype 1 (AAVpo1) capsid wild-type or modified with the A1 peptide as described in WO2021 / 219762 (AAVpo1-A1).

[0224] In addition, the genome of the AAV vector can be either single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal release site from one of the AAV terminal repeats. These modified vectors, whose replicative genomes are half the length of the wild-type AAV genome, tend to package DNA dimers. In a preferred embodiment, the AAV vector introduced into the practice of the present invention has a single-stranded genome, and more preferably comprises an AAV8, AAV9, AAVrh74, AAVrh74-9, or AAV2i8 capsid, particularly an AAV8, AAV9, AAVrh74, or AAVrh74-9 capsid, such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. As known in the art, additional appropriate sequences can be introduced into the nucleic acid construct of the present invention to obtain a functional viral vector. Suitable sequences include the AAV ITRs.

[0225] Of course, in designing the nucleic acid sequence of the present invention and the expression cassette of the present invention, the skilled artisan will take into consideration the size limit of the vector used to deliver said construct to a cell or organ. In particular, as noted above, when the vector is an AAV vector, the skilled artisan will be aware that the main limitation of an AAV vector is its payload capacity, which may vary with each AAV serotype, but which is considered to be limited to the vicinity of the size of the parental viral genome. For example, 5 kb is the maximum size that is considered to be normally packaged in an AAV8 capsid. (Wu Z. et al., Mol Ther., 2010, vol. 18(1): pp. 80-86; Lai Y. et al., Mol Ther., 2010, vol. 18(1): pp. 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, vol. 23(4): pp. 225-33). Thus, in practicing the invention, the skilled artisan will be mindful to select the components of the nucleic acid construct of the invention such that the resulting nucleic acid sequence, including sequences encoding the AAV 5'- and 3'-ITRs, preferably does not exceed 110% of the payload capacity of the AAV vector into which it is introduced, and particularly preferably does not exceed 5.5 kb.

[0226] The present invention also relates to isolated cells, such as muscle cells or CNS cells, transformed with the nucleic acid sequence of the present invention or the expression cassette of the present invention. The isolated cells of the present invention can be delivered to a subject in need thereof via injection into the tissue of interest or into the bloodstream of said subject. In a particular embodiment, the present invention comprises the step of introducing the nucleic acid molecule or expression cassette of the present invention into isolated cells of the subject to be treated, and returning said cells into which the nucleic acid or expression cassette has been introduced, to this subject.

[0227] The present invention also provides pharmaceutical compositions comprising the nucleic acid molecules, vectors or isolated cells of the present invention. Such compositions comprise a therapeutically effective amount of the nucleic acid sequences, vectors or isolated cells of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory federal agency or state government, or listed in the United States or European Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0228] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject. In certain embodiments, the nucleic acid sequences, expression cassettes, vectors, or isolated cells of the invention are formulated into a composition comprising phosphate buffered saline and supplemented with 0.25% human serum albumin. In another particular embodiment, the vector of the invention is formulated in a composition comprising Ringer's lactate and a non-ionic surfactant, such as Pluronic F68, at a final concentration of 0.01-0.0001% by weight of the total composition, for example at a concentration of 0.001% by weight. The formulation may further comprise serum albumin, in particular human serum albumin, for example human serum albumin at 0.25%. Other suitable formulations for either storage or administration are known in the art, in particular from WO2005 / 118792 or Allay et al., 2011.

[0229] In a preferred embodiment, the composition is formulated according to a routine procedure as a pharmaceutical composition adapted for intravenous or intramuscular administration to humans, preferably intravenous administration.Usually, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site.

[0230] In one embodiment, the nucleic acid sequence, expression cassette or vector of the invention can be delivered in a vesicle, in particular a liposome, hi yet another embodiment, the nucleic acid sequence, expression cassette or vector of the invention can be delivered in a controlled release system.

[0231] How to use By virtue of the present invention, transgenes of interest can be expressed in muscle and CNS cells.

[0232] The nucleic acid molecules, expression cassettes or vectors of the invention can be used to express genes in muscle and / or in CNS cells. Thus, the invention provides a method for expressing a transgene of interest in a muscle or CNS cell, where an expression cassette of the invention is introduced into the cell and the transgene of interest is expressed. The method may be an in vitro, ex vivo or in vivo method for expressing a transgene of interest in a muscle or CNS cell.

[0233] In a particular aspect, the present invention relates to a nucleic acid molecule, expression cassette or vector of the invention for use in an ex-vivo method for expressing a transgene of interest in a cell, wherein the expression cassette of the invention is introduced into a cell and the transgene of interest is expressed.

[0234] The nucleic acid molecule, expression cassette or vector of the present invention can also be used for gene therapy.Accordingly, in one aspect, the present invention relates to a nucleic acid molecule, expression cassette, vector, isolated cell or pharmaceutical composition as described above for use as a medicament.Accordingly, in one aspect, the present invention relates to a nucleic acid molecule, expression cassette or vector disclosed herein for use in therapy, particularly gene therapy.Similarly, the isolated cell of the present invention can be used in therapy, particularly cell therapy.

[0235] In another aspect, the invention relates to a nucleic acid molecule, an expression cassette, a vector, an isolated cell or a pharmaceutical composition as described above for use in a method for treating a neuromuscular disorder.

[0236] In a further aspect, the invention relates to the use of a nucleic acid molecule, expression cassette, vector, isolated cell or pharmaceutical composition as described above for the manufacture of a medicament for use in the treatment of a neuromuscular disorder.

[0237] In another aspect, the invention relates to a method for the treatment of a neuromuscular disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule, expression cassette, vector, isolated cell or pharmaceutical composition described herein.

[0238] The neuromuscular disorder is in particular an inherited or acquired disorder, such as an inherited or acquired neuromuscular disease. Naturally, the therapeutic transgene and promoter driving expression to the tissue of therapeutic interest will be selected having regard to the disorder to be treated.

[0239] The term "neuromuscular disorder" includes diseases and conditions that impair muscle function, either directly (pathology of voluntary muscles) or indirectly (pathology of nerves or neuromuscular junction). Exemplary neuromuscular disorders include, without limitation, muscular dystrophies (e.g., myotonic dystrophy (Steiner's disease), Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy), motor neuron diseases (e.g., amyotrophic lateral sclerosis (AL), S), spinal muscular atrophy (infantile progressive spinal muscular atrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular atrophy (type 2), juvenile spinal muscular atrophy (type 3, Kugelberg-Welander disease), adult spinal muscular atrophy (type 4), spinal-bulbar muscular atrophy (Kennedy disease)), inflammatory myopathies (e.g., polymyositis, dermatomyositis, inclusion body myositis), diseases of the neuromuscular junction (e.g., myasthenia gravis, Lambert-Eaton (myasthenic) syndrome, congenital myasthenic syndrome, asthenic syndrome), peripheral nerve disorders (e.g., Charcot-Marie-Tooth disease, Friedreich's ataxia, Dejerine-Sottas disease), muscle metabolic disorders (e.g., phosphorylase deficiency (McArdle disease), acid maltase deficiency (Pompe disease), phosphofructokinase deficiency (Tarui disease), debranching enzyme deficiency (Cori disease or Forbes disease), mitochondrial myopathy, carnitine deficiency, carnitine palmityltransferase deficiency, myopathy (e.g., hyperthyroid myopathy, hypothyroid myopathy), and other myopathies (e.g., myotonia congenita, paramyotonia congenita, central core disease, nemaline myopathy, myotubular myopathy, periodic paralysis). In this embodiment, the nucleic acid sequence of the present invention includes a liver-selective, muscle-selective, and / or neuron-selective transcriptional regulatory element, e.g., liver-selective and muscle-selective transcriptional regulatory element, liver-selective and neuron-selective transcriptional regulatory element, and liver-selective, muscle-selective and neuron-selective transcriptional regulatory element.

[0240] In a particular embodiment, the disorder is a glycogen storage disease. The term "glycogen storage disease" refers to a group of inherited metabolic disorders involving the enzymes responsible for the synthesis and degradation of glycogen. In a more particular embodiment, the glycogen storage disease may be GSDI (von Gierke's disease), GSDII (Pompe's disease), GSDIII (Cori's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII or fatal congenital glycogen storage disease of the heart. More particularly, the glycogen storage disease is selected in the group consisting of GSDI, GSDII and GSDIII, even more particularly in the group consisting of GSDII and GSDIII. In an even more particular embodiment, the glycogen storage disease is GSDII. In particular, the nucleic acid molecules of the invention may be useful in gene therapy for treating GAA deficiency conditions or other conditions associated with glycogen accumulation, such as GSDI (von Gierke's disease), GSDII (Pompe's disease), GSDIII (Cori's disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and fatal congenital glycogen storage disease of the heart, more particularly GSDI, GSDII or GSDIII, even more particularly GSDII and GSDIII. In a further particular embodiment, the disorder is Pompe's disease and the therapeutic transgene is a gene encoding acid alpha-glucosidase (GAA) or a variant thereof. Such variants of GAA are disclosed in particular in applications PCT / 2017 / 072942, PCT / EP2017 / 072945 and PCT / EP2017 / 072944, which are hereby incorporated by reference in their entirety. In this embodiment, the nucleic acid sequence of the present invention comprises a liver-selective, muscle-selective and / or neuron-selective transcriptional regulatory element, such as a liver-selective and muscle-selective transcriptional regulatory element, a liver-selective and neuron-selective transcriptional regulatory element, a muscle-selective and neuron-selective transcriptional regulatory element, and a liver-selective, muscle-selective and neuron-selective transcriptional regulatory element. In a particular embodiment, the disorder is infantile-onset Pompe disease (IOPD) or late-onset Pompe disease (LOPD). Preferably, the disorder is IOPD.

[0241] Those skilled in the art are aware of transgenes of interest that are useful for the treatment of these and other disorders by gene therapy. For example, therapeutic transgenes are lysosomal enzyme alpha-L-iduronidase [IDUA (alpha-L-iduronidase)] for MPS I, acid-alpha-glucosidase (GAA) for Pompe disease, glycogen debranching enzyme (GDE) or truncated forms of GDE (also called truncated forms of GDE or small GDE) for Cori disease (GSDIII), G6P for GSDI, alpha-sarcoglycan (SGCA) for LGMD2D, dystrophin or truncated forms thereof for DMD, and SMN1 for SMA. Transgenes of interest may also be transgenes that provide other therapeutic properties than providing a missing protein or RNA that suppresses the expression of a given protein. For example, transgenes of interest may include, without limitation, transgenes that can increase muscle strength.

[0242] Specific examples of therapeutic transgenes of interest that can be operably linked to the hybrid promoters of the present invention for specific diseases are provided below.

[0243] In a particular embodiment, the disease is coli disease and the transgene of interest encodes a GDE or a truncated form of a GDE. A truncated form of a GDE suitable for use in the present invention may include, without limitation, those described in EP18306088. Alternatively, the present invention is used in a double AAV vector system for expressing a GDE, for example the double AAV vector system disclosed in WO2018162748. In this embodiment, the vector of the present invention may correspond to the first AAV vector of the double AAV vector system comprising the AAV ITR between 5' and 3', i.e. the first nucleic acid sequence encoding the N-terminal portion of the GDE, under the control of the nucleic acid molecule of the present invention.

[0244] In another specific embodiment, the disease is Pompe disease and the transgene of interest encodes acid-alpha-glucosidase (GAA), or modified GAA. Modified GAA suitable for use in the present invention include, without limitation, those disclosed in WO2018046772, WO2018046774, and WO2018046775.

[0245] In more particular embodiments, the disorder is selected from Duchenne muscular dystrophy, myotubular myopathy, spinal muscular atrophy, limb-girdle muscular dystrophy types 2I, 2A, 2B, 2C or 2D, and myotonic dystrophy type 1.

[0246] Methods of administration of the vectors of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, local area administration, and oral routes as described in WO2015158924. In certain embodiments, administration is via intravenous or intramuscular routes. The vectors of the present invention can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active substances. Administration can be systemic or local.

[0247] In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the present invention locally to the area requiring treatment, e.g., liver or muscle. This can be accomplished, for example, by means of an implant, which may be a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

[0248] The amount of the vector of the invention that is effective in treating the disorder to be treated can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays can be optionally used to help predict optimal dosage ranges. The exact dose to be employed in the formulation will also depend on the route of administration, and the severity of the disease, and should be determined according to the judgment of the physician and each patient's circumstances. The dose of the vector of the invention administered to a subject in need thereof will vary based on several factors, including, without limitation, the route of administration, the particular disease being treated, the age of the subject, or the level of expression required to obtain a therapeutic effect. One of skill in the art can readily determine the required dosage range based on these factors and others, based on his or her knowledge in the art. For treatments involving administration of an AAV vector to a subject, a typical dose of the vector is at least 1×10 per kilogram of body weight. 8 of vector genome (vg / kg), e.g., at least 1 × 10 9 vg / kg, at least 1 × 10 10 vg / kg, at least 1 × 10 11 vg / kg, at least 1 × 10 12 vg / kg, at least 1 × 10 13 vg / kg, at least 1 × 10 14 vg / kg or at least 1 × 10 15 vg / kg.

[0249] In certain embodiments, the vector of the present invention may be administered at a lower dose than the typical dose used in gene therapy.In particular, in the treatment comprising administering an AAV vector to a subject in need thereof, the vector may be administered at a dose of 2 times less than the typical dose described above, in particular at a dose of 3 times less, 4 times less, 5 times less, 6 times less, 7 times less, 8 times less, 9 times less, 10 times less, 11 times less, 12 times less, 13 times less, 14 times less, 15 times less, 16 times less, 17 times less, 18 times less, 19 times less, 20 times less, 21 times less, 22 times less, or less than 23 times lower, or less than 24 times lower, or less than 25 times lower, or less than 26 times lower, or less than 27 times lower, or less than 28 times lower, or less than 29 times lower, or less than 30 times lower, or less than 31 times lower, or less than 32 times lower, or less than 33 times lower, or less than 34 times lower, or less than 35 times lower, or less than 36 times lower, or less than 37 times lower, or less than 38 times lower, or less than 39 times lower, or less than 40 times lower, or less than 41 times lower, or less than 42 times lower, or less than 43 times lower, or less than 44 times lower, or less than 45 times lower, or less than 46 times lower, or less than 47 times lower, or less than 48 times lower, or less than 49 times lower, or even at least 50 times lower. EXAMPLES

[0250] Materials and Methods Generation of AAV vectors The AAV vectors used in this experiment were generated using an adenovirus-free method of transient transfection of HEK293 cells and purified by Akta. qPCR was used to determine the titer of the AAV vector stocks. All vector preparations used in this experiment were stratified against each other prior to use. Primers used for qPCR on the ITR SEQ annealed to the AAV genome or against the codon-optimized hGAA transgene sequence: forward: 5'-agatacgccggacattggactg-3'; reverse, 5'-agatacgccggacattggactg-3'.

[0251] In vivo experiments Experiments on mice were performed in accordance with French and European legislation on animal care and experimentation (2010 / 63 / EU) and approved by the local institutional ethical committee. Wild-type male C57BL / 6 mice were purchased from Charles River Laboratories. Gaa knockout mice (Gaa - / - ) were purchased from The Jackson Laboratory (B6;129-Gaatm1Rabn / J, stock number 004154, 6neo). These mice were originally generated by Raben et al. 95 Littermate male mice were used. These were affected (Gaa - / - ) or Health (Gaa + / + ) or IV. AAV vectors were delivered to adult mice via the tail vein in a volume of 0.2 mL. One month after injection, mice were sacrificed and blood and tissues were collected. Mouse experimental groups were sized at n=4 based on data generated in previous experiments. All samples and animals analyzed were included in the data and any outliers were excluded.

[0252] GAA activity assay Snap-frozen tissues were homogenized in UltraPure DNase- and RNase-free distilled water (Thermo Fisher Scientific). Tissues were weighed, homogenized, and centrifuged at 10,000 g for 10 min, and the supernatant was collected. Enzyme reactions were set up in black 96-well plates (PerkinElmer) using 10 μL of appropriately diluted sample (plasma or tissue homogenate) and 20 μL of substrate, 4-methylumbelliferone (4MU) aD-glucoside. The reaction mixtures were incubated at 37°C for 1 h, and then stopped by adding 150 μL of sodium carbonate buffer (pH 10.5). Using a calibration curve (0-2,500 pmol / mL 4MU), the fluorescence 4MU released from each reaction mixture was measured using an EnSpire Alpha plate reader (PerkinElmer) at 449 nm (emission) and 360 nm (excitation). Protein concentrations of clarified supernatants were quantified by BCA (Thermo Fisher Scientific). To calculate GAA activity in tissues, the released 4MU concentration was divided by the sample protein concentration, and activity was reported as nanomoles per hour per milligram of protein or milliliter of serum.

[0253] Western blot analysis Western blotting on mouse plasma was performed on samples diluted 1:4 with distilled water. Homogenates of mouse tissues were prepared as indicated for GAA activity. Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific). SDS-PAGE electrophoresis was performed in 4%-12% polyacrylamide gels. After transfer, membranes were blocked with Odyssey buffer (LI-COR Biosciences) and incubated with anti-GAA antibody (rabbit monoclonal, clone EPR4716(2), Abcam) and anti-Gapdh (rabbit polyclonal, PA1-988, Thermo Fisher Scientific). Membranes were washed, incubated with appropriate secondary antibodies (LI-COR Biosciences) and visualized with an Odyssey imaging system (LI-COR Biosciences). For quantification of Western blots, either ImageJ or Image Studio Lite 4.0 was used. Either Gapdh band was used to normalize the quantification of hGAA protein bands in mouse tissues, and the quantification of hGAA protein bands in plasma was normalized using a nonspecific band detected by anti-hGAA antibodies in mouse plasma (used as a loading control).

[0254] Anti-GAA and anti-capsid antibody detection Anti-hGAA IgG capture assays were performed in Maxisorp 96-well plates (Thermo Fisher Scientific) coated with rhGAA at 2 mg / mL. IgG standard curves were generated by serial 1-2 dilutions of commercially available mouse recombinant IgGs (Sigma-Aldrich) coated directly onto the wells in duplicate (from 1 mg / mL to 0.15 mg / mL). Plasma samples appropriately diluted in 10 mM PBS (pH 7.4) containing 2% BSA were analyzed in duplicate. HRP-conjugated anti-mouse IgG antibody (human ads-HRP, Southern Biotech) was used as the secondary antibody. Plates were exposed to OPD substrate (o-phenylenediamine dihydrochloride, Sigma). The reaction was stopped with a 3 M solution of H2SO4, and optical density (OD) measurements were taken at 492 nm using a microplate reader (ENSPIRE, PerkinElmer, Waltham, USA). Anti-AAV IgG concentrations were determined against a standard curve.

[0255] mSeAP quantification Snap-frozen tissues were homogenized in UltraPure DNase- and RNase-free distilled water (Thermo Fisher Scientific). Tissues were weighed, homogenized, and centrifuged at 10,000 g for 10 min, and the supernatant was collected. Enzyme reactions were set up using the Life Tech T1015 mseap kit. Briefly, 10 μL of heated sample (plasma or tissue homogenate) was incubated with 10 μL of assay buffer for 5 min, then incubated with 10 μL of reaction buffer for 20 min. Using a calibration curve (0–6 ng / μL mSEAP), the luminescence emitted from each reaction mixture was measured using an EnSpire alpha plate reader (PerkinElmer). Protein concentrations of clarified supernatants were quantified by BCA (Thermo Fisher Scientific). To calculate mSeAP expression in tissues.

[0256] Vector genome copy number analysis DNA was extracted from tissue homogenates and quantified using Nucleo Mag Pathogen (Macherey-Nagel, France). Vector genome copy numbers were determined by qPCR using 500 ng of DNA, primers and probes annealed to the ITRs or to the codon-optimized hGAA (forward, 5'-agatacgccggacattggactg-3'; reverse, 5'-agatacgccggacattggactg-3'; probe, 5'-gtgtggtcctcttgggagc-3'), and mouse titin as a reference gene (forward: 5'-aaaacgagcagtgacgtgagc-3'; reverse: 5'-ttcagtcatgctgctagcgc-3'; probe, 5'-tgcacggaagcgtctcgtctcagt-3'). qPCR was performed using the TaqMan method.

[0257] statistical analysis All data presented in this manuscript are reported as mean ± SD. The number of units sampled, n, on the basis of which statistics are reported, is a single mouse for the in vivo experiments (one mouse n=1). Statistical analysis was performed using GraphPad Prism 7.0 software. The Shapiro-Wilk test was used to evaluate the normal distribution of data obtained from the different measurements (amount of anti-hGAA IgG in plasma, expression of hGAA protein in plasma and tissues, GAA enzyme activity in tissues). Statistical tests used unpaired Student's t-tests for comparisons of two groups and one-way ANOVA with Tukey's post-hoc for comparisons of more than two groups. Alpha = 0.05 for all data sets analyzed in parametric tests. All statistical tests were performed two-sided. p<0.05 was considered significant. The statistical analysis performed for each data set is indicated in the figure legends. For all figures, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

[0258] result In Figure 1A, a ubiquitous CAG promoter (referred to as "P1") and four different combinations are shown: (i) H3 enhancer and spC5-12 promoter (P2), (ii) H3 enhancer and CK6 promoter (P3), (iii) H3 enhancer, CK6 promoter and spC5-12 promoter (P4), and (iv) CK6 promoter and spC5-12 promoter (P5). The H3 enhancer corresponds to three repeats of the HS-CRM8 enhancer. The sequence of P4 is as shown in SEQ ID NO:30.

[0259] The in vivo protocol is described in Figure 2. Six-week-old C57BL / 6 wild-type (WT) mice were intravenously injected with 4E11 vg / mouse AAV-MT vector (AAV9-rh74-P1 vector) encoding murine secreted alkaline phosphatase (mSeAP) under the transcriptional control of P1, P3, or P4. One month after injection, mice were sacrificed and tissues were analyzed.

[0260] Figure 3 depicts the transgene copy number per cell in liver and quadriceps muscle. The data show that both tissues were transduced with similar efficiency by the three AAV vectors.

[0261] Figure 4 shows mSEAP activity measured in the liver, four different muscles (heart, diaphragm, quadriceps and triceps) and spinal cord of vector-injected mice. Improved mSEAP activity was observed after transduction with AAV-MT vectors expressing mSEAP under P1 and P4 control in different muscles and spinal cord. Only P1 (ubiquitous promoter) expressed mSEAP in the liver.

[0262] Figure 5 describes the in vivo protocol. Six-week-old C57BL / 6 wild-type (WT) mice were intravenously injected with 5E10 vg / mouse of AAV-MT (AAV9-rh74-P1) vector encoding an optimized human GAA codon (hGAAco) under the transcriptional control of P1–P5. One month after injection, mice were sacrificed and tissues were analyzed.

[0263] Figure 6 reports vector genome copy numbers (VGCN) normalized by titin quantification from DNA extracted from cardiac tissue at sacrifice. VGCN did not differ significantly between groups.

[0264] Figure 7 corresponds to GAA measured in hearts from mice injected with a vector encoding GAA under the transcriptional control of P1-P5. Figure 7A represents GAA activity measured by enzymatic assay and normalized to total protein. Figures 7B and 7C correspond to GAA quantification by Western blot assay. Figure 7B shows the bands of GAA intensity normalized by the bands of vinculin intensity obtained from Figure 7C. Using these two methods, GAA was more expressed in mice injected with vector P4 (H3-CK6-C512) compared to the other groups.

[0265] Figure 8 corresponds to GAA measured in quadriceps muscles from mice injected with a vector encoding GAA under the transcriptional control of P1-P5. Figure 8A represents GAA activity measured by enzymatic assay and normalized to total protein. Figures 8B and 8C correspond to GAA quantification by Western blot assay. Figure 8B shows the quantification of the band of GAA intensity normalized to the band of vinculin intensity obtained from Figure 8C. Using these two methods, GAA was more expressed in mice injected with vector P4 (H3-CK6-C512) compared to the other groups.

[0266] In Figure 9A, three different combinations of H3 enhancer and 575bp linker are represented (P1): used as a negative control to show the inactivity of the linker. P2 is composed of H3 enhancer; linker used for P1. P3 corresponds to H3 enhancer, CK6 and spC5-12 promoter. It was used as a positive control. The sequence of P3 is as shown in SEQ ID NO: 30. Figure 11B describes the in vivo protocol. Six-week-old C57BL / 6 wild-type (WT) mice were intravenously injected with 1E11vg / mouse AAV-MT (AAV9-rh74-P1) vector encoding an optimized human GAA codon (hGAAco) under the transcriptional control of P1-P3. One month after injection, mice were sacrificed and tissues were analyzed.

[0267] In Figure 10A, vector genome copy numbers (VGCN) normalized by titin quantification from DNA extracted from heart tissue at the time of sacrifice are reported. VGCN did not differ significantly between groups. Figures 10B-10E correspond to GAA measured by Western blotting in hearts (B-C) and quadriceps (D-E) from mice injected with vectors encoding GAA under the transcriptional control of P1-P3. Figures 10B and 10D show quantification of bands of GAA intensity normalized by bands of vinculin intensity from Figures 10C and 120. GAA was more expressed in mice injected with vector P3 (H3-CK6-C512) compared to the other groups. These results demonstrate that the synergistic effect of the H3-CK6-spC5-12 combination is due to the addition of the CK6 promoter between H3 and spC5-12, and not due to an increase in the distance between H3 and spC5-12.

[0268] In FIG. 11A, four different combinations are depicted: (i) H3 enhancer, CK6 promoter and spC5-12 promoter (P1); (ii) H3 enhancer, CK6 promoter and Actal promoter (P2); (iii) H3 enhancer, CK6 promoter and CK6 promoter (P3); (iv) H3 enhancer, CK6 promoter and CK8 promoter (P4). The H3 enhancer corresponds to three repeats of the HS-CRM8 enhancer. The sequence of P1 is as shown in SEQ ID NO: 30. FIG. 11B describes the in vivo protocol. Six-week-old C57BL / 6 wild-type (WT) mice were intravenously injected with 3.8E11vg / mouse AAV-MT (AAV9-rh74-P1) vector encoding an optimized human GAA codon (hGAAco) under the transcriptional control of P1-P4. One month after injection, the mice were sacrificed and tissues were analyzed.

[0269] In Figure 12A, vector genome copy numbers (VGCN) normalized by titin quantification from DNA extracted from cardiac tissue at the time of sacrifice are reported. VGCN was not significantly different for each group. Figure 12B corresponds to GAA measured by Western blotting in quadriceps muscles from mice injected with vectors encoding GAA under the transcriptional control of P1-P4. The results show that similar levels of GAA expression are obtained when using P1 (H3-CK6-C5.12), P2 (H3-CK6-Acta1) or P3 (H3-CK6-CK8). P4 (H3-CK6-CK6) is also very efficient in that it appears to show even stronger expression compared to P1. Thus, the results show that spC5.12 can be replaced by other muscle-specific promoters without affecting the level of transgene expression in muscle.

[0270] These data surprisingly suggest that the combination of a liver-selective enhancer (H3) with two muscle-selective promoters (CK6 plus a second muscle promoter) greatly improves protein expression in muscle and spinal cord (a strong ubiquitous promoter compared to similar expression) without targeting to the liver.

Claims

1. operably linked to each other: (i) one or more liver-selective enhancers; (ii) a first muscle-selective promoter, wherein the first muscle-selective promoter is a CK6 promoter or a functional variant thereof; and (iii) a second muscle-selective promoter selected from the group consisting of spC5-12 promoter, CK6 promoter, CK8 promoter, Acta1 promoter, MCK promoter, desmin promoter, and functional variants thereof, wherein the second muscle-selective promoter is preferably spC5-12 promoter, CK6 promoter, CK8 promoter, Acta1 promoter, or a functional variant thereof, and the second muscle-selective promoter is preferably spC5-12 promoter or a functional variant thereof. A nucleic acid molecule comprising:

2. 2. The nucleic acid molecule of claim 1, wherein the first muscle-selective promoter is located at the upstream 5' end of the second muscle-selective promoter.

3. From 5' to 3' in this order: - one or more liver-selective enhancers; - a first muscle-selective promoter that is the CK6 promoter or a functional variant thereof; and - a second muscle-selective promoter, preferably the spC5-12 promoter, the CK6 promoter, the CK8 promoter, the Acta1 promoter or a functional variant thereof, and the second muscle-selective enhancer, preferably the spC5-12 promoter or a functional variant thereof; The nucleic acid molecule of claim 1, comprising:

4. 2. The nucleic acid molecule of claim 1, wherein the CK6 promoter consists of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO: 7, or a functional variant having a sequence at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO:

7.

5. A second muscle-selective promoter - the spC5-12 promoter consisting of the sequence shown in SEQ ID NO: 4, 5 or 6, preferably SEQ ID NO: 6, or a functional variant having a sequence at least 80% identical to SEQ ID NO: 4, 5 or 6, preferably SEQ ID NO: 6; - a CK8 promoter consisting of the sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, preferably SEQ ID NO: 33, or a functional variant having a sequence at least 80% identical to SEQ ID NO: 33 or SEQ ID NO: 34, preferably SEQ ID NO: 33; - a CK6 promoter consisting of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO: 7, or a functional variant having a sequence at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 35, preferably SEQ ID NO: 7; or - an Acta1 promoter consisting of the sequence shown in SEQ ID NO: 37 or a functional variant having a sequence at least 80% identical to SEQ ID NO: 37; The nucleic acid molecule of claim 1,

6. the liver-selective enhancer comprises or consists of a sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, a functional variant having 80% identity to any one of the sequences selected from SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28; or 2. The nucleic acid molecule of claim 1, wherein the plurality of liver-selective enhancers comprises at least one liver-selective enhancer comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, a functional variant having 80% identity to any one of the sequences selected from SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:

43.

7. The nucleic acid molecule of claim 1, wherein all of the liver-selective enhancers of the multiple liver-selective enhancers have the same sequence.

8. 2. The nucleic acid molecule of claim 1, wherein the plurality of liver-selective enhancers comprises at least two liver-selective enhancers, and preferably the plurality of liver-selective enhancers comprises three liver-selective enhancers.

9. 2. The nucleic acid molecule of claim 1, wherein the sequence of the liver-selective enhancer consists of SEQ ID NO: 1 or is a functional variant having a sequence at least 80% identical to SEQ ID NO:

1.

10. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46, or is a functional variant having a sequence at least 80% identical to SEQ ID NO: 30, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO:

46.

11. An expression cassette comprising the nucleic acid molecule of claim 1 operably linked to a transgene of interest.

12. A vector comprising the expression cassette of claim 11, in particular wherein the vector is a plasmid or a viral vector, preferably an adeno-associated viral (AAV) vector.

13. 12. An isolated recombinant cell comprising the expression cassette of claim 11.

14. A composition for use as a pharmaceutical, comprising the expression cassette of claim 11, the vector of claim 12, or the cell of claim 13.

15. A composition for use in treating a neuromuscular disorder, comprising the expression cassette of claim 11, the vector of claim 12, or the cell of claim 13, wherein the neuromuscular disorder is preferably a muscular dystrophy (e.g., myotonic dystrophy (Steiner's disease), Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, or the like). , Emery-Dreifuss muscular dystrophy, motor neuron diseases (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophies (infantile progressive spinal muscular atrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular atrophy (type 2), juvenile spinal muscular atrophy (type 3, Kugelberg-Welander disease), adult spinal muscular atrophy (type 4)), spinal-bulbar muscular atrophy (Kennedy disease)), inflammatory myopathies (e.g., polymyositis, dermatomyositis, inclusion body myositis), diseases of the neuromuscular junction (e.g., myasthenia gravis ... asthenia, Lambert-Eaton (myasthenic) syndrome, congenital myasthenic syndrome), peripheral nerve disorders (e.g., Charcot-Marie-Tooth disease, Friedreich's ataxia, Dejerine-Sottas disease), muscle metabolic disorders (e.g., phosphorylase deficiency (McArdle disease), acid maltase deficiency (Pompe disease), phosphofructokinase deficiency (Tarui disease), debranching enzyme deficiency (Cori disease or Forbes disease), mitochondrial myopathy, carnitine deficiency, carnitine palmitate myotransferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, myoadenylate deaminase deficiency), myopathies due to endocrine abnormalities (e.g., hyperthyroid myopathy, hypothyroid myopathy), and other myopathies (e.g., myotonia congenita, paramyotonia congenita, central core myopathy, nemaline myopathy, myotubular myopathy, periodic paralysis).

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