Hybrid promoters for muscle expression

文档序号：1926228 发布日期：2021-12-03 浏览：11次中文

阅读说明：本技术 用于肌肉表达的杂合启动子 (Hybrid promoters for muscle expression ) 是由基斯配·罗兹提帕特里斯·维达尔费德里克·敏果兹于 2020-04-07 设计创作，主要内容包括：本发明涉及在肌肉中驱动基因表达的杂合启动子。(The present invention relates to hybrid promoters that drive gene expression in muscle.)

1. A nucleic acid molecule comprising one or more liver-selective enhancers operably linked to a muscle-selective promoter, wherein:

-the liver selectivity enhancer comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 1. 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: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 32 and SEQ ID NO: 33, and a sequence selected from SEQ ID NO: 1. 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: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 32 and SEQ ID NO: 33, and functional fragments thereof, having 80% identity to any one of said sequences of seq id no; or

-the plurality of liver selectivity enhancers comprises at least one liver selectivity enhancer comprising or consisting of a sequence selected from the group consisting of: SEQ ID NO: 1. 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: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 32 and SEQ ID NO: 33, and a sequence selected from SEQ ID NO: 1. 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: 29. SEQ ID NO: 30. SEQ ID NO: 31. SEQ ID NO: 32 and SEQ ID NO: 33, and functional fragments thereof, having 80% identity to any one of said sequences of 33.

2. The nucleic acid molecule of claim 1, wherein all liver selective enhancers of the plurality have the same sequence.

3. The nucleic acid molecule of claim 1, wherein at least two of the plurality of liver selectivity enhancers have different sequences.

4. The nucleic acid molecule of any one of claims 1-3, wherein the plurality of liver selective enhancers comprises at least two liver selective enhancers.

5. The nucleic acid molecule of any one of claims 1-4, wherein the plurality of liver selectivity enhancers comprises three liver selectivity enhancers.

6. The nucleic acid molecule of any one of claims 1 to 5, wherein the sequence of the liver selectivity enhancer consists of SEQ ID NO: 1, or a polypeptide having an amino acid sequence substantially identical to SEQ ID NO: 1 is a functional variant of a sequence which is at least 80% identical.

7. The nucleic acid molecule of any one of claims 1 to 5, wherein the sequence of the liver selectivity enhancer consists of SEQ ID NO: 30, or a polypeptide having an amino acid sequence substantially identical to SEQ ID NO: 30 is a functional variant of at least 80% of the sequence.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the promoter is an spC5-12 promoter, in particular wherein the spC5-12 promoter consists of the amino acid sequence of SEQ ID NO: 2. 3 or 4, or a sequence having a sequence identical to that shown in SEQ ID NO: 2. 3 or 4 is at least 80% sequence.

9. The nucleic acid molecule according to any one of claims 1 to 7, wherein the promoter is a CK6 promoter, in particular wherein the CK6 promoter consists of the sequence of SEQ ID NO: 6, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 6 is at least 80% sequence identity.

10. The nucleic acid molecule according to any one of claims 1 to 7, wherein the promoter is a CK8 promoter, in particular wherein the CK8 promoter consists of the sequence of SEQ ID NO: 7, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 7 is at least 80% sequence identity.

11. The nucleic acid molecule of any one of claims 1 to 7, wherein the promoter is the ACTA1 promoter, particularly wherein the ACTA1 promoter consists of the sequence of SEQ ID NO: 8, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 8 is at least 80% sequence identity.

12. The nucleic acid molecule of any one of claims 1 to 11, further comprising a muscle selective enhancer located between the liver selective enhancer or the plurality of liver selective enhancers and the muscle selective promoter.

13. An expression cassette comprising the nucleic acid molecule of any one of claims 1 to 11 operably linked to a transgene of interest.

14. A vector comprising the expression cassette of claim 13, in particular wherein the vector is a plasmid or a viral vector.

15. The vector of claim 14, wherein the viral vector is an adeno-associated virus (AAV) vector.

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

17. The expression cassette of claim 13, the vector of claim 14 or 15 or the cell of claim 16 for use as a medicament.

18. The expression cassette of claim 13, the vector of claim 14 or 15, or the cell of claim 16 for use in treating a neuromuscular disorder.

19. The expression cassette according to claim 13, the vector according to claim 14 or 15 or the cell according to claim 16 for use according to claim 18, wherein the neuromuscular disorder is selected from the group consisting of muscular dystrophy (e.g. myotonic dystrophy (Steinert's disease), duchenne muscular dystrophy, becker muscular dystrophy, limb-girdle muscular dystrophy, shoulder-brachial muscular dystrophy, congenital muscular dystrophy, 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's disease), intermediate spinal muscular atrophy (type 2), juvenile spinal muscular atrophy (type 3, Kugelberg-Welander disease), adult-type spinal muscular atrophy (type 4)), spinal bulbar muscular atrophy (Kennedy's disease), inflammatory myopathies (e.g., polymyositis dermatomyositis, inclusion body myositis), neuromuscular junction disorders (e.g., myasthenia gravis, Lambert-Eaton syndrome, congenital myasthenia syndrome), peripheral neurological disorders (e.g., Charcot-Marie-Tooth disease, Friedel's ataxia, Dejerine-Sottas disease), muscle metabolic disorders (e.g., phosphorylase deficiency (Mackade's disease), acid maltase deficiency (Pompe's disease), phosphofructokinase deficiency (Tarui's disease), debranching enzyme deficiency (Coriolis disease or Forbes disease), mitochondrial myopathies, carnitine deficiency, carnitine palmitoyl transferase deficiency, phosphokinase deficiency, phosphoglyceromutase deficiency (Taruit's disease), Lactate dehydrogenase deficiency, myoadenosine deaminase deficiency), myopathies caused by endocrine abnormalities (e.g., hyperthyroid myopathy, hypothyroid myopathy) and other myopathies (e.g., myotonia congenita, paramyotonia congenita, central axonopathy, linear myopathy, myotubular myopathy, periodic paralysis).

Technical Field

The present invention relates to hybrid promoters that drive gene expression in muscle. The invention also relates to expression cassettes and vectors containing said hybrid promoters. Also disclosed herein are methods of using these hybrid promoters, particularly for gene therapy.

Background

Neuromuscular disorders represent one of the major challenges for in vivo-based gene therapy. For many diseases, under-expression of the transgene in the desired target tissue and immunity against the transgene remain important obstacles to successful gene therapy. Thus, there remains a need to provide strong expression of transgenes in cells of interest, but to use low doses of the vector to prevent both the potential toxicity of the vector and the immune response against the vector.

In theory, this goal can be solved by selecting promoters that provide strong expression in the target cell. However, their use can create several problems. In particular, in designing constructs for gene therapy, one must keep in mind the size limitations specific to the vector used to deliver the therapeutic transgene. For example, elements introduced into an AAV vector should have a reduced size due to the limitation imposed by the maximum envelope size of the AAV vector, i.e., about 5 kb.

Here we describe the identification of enhancer/promoter combinations of a size compatible with gene therapy vectors such as AAV vectors that allow for efficient expression of proteins in muscle.

Disclosure of Invention

The present invention provides genetic engineering strategies to generate novel hybrid promoters with muscle specificity. These hybrid promoters are useful in gene therapy for neuromuscular diseases. These novel hybrid promoters are based on a combination of one or more liver-selective enhancers operably linked to a muscle-selective promoter. Surprisingly, it is shown herein that expression of a transgene in muscle cells is increased when placed under the control of such a hybrid promoter comprising a liver-selective enhancer.

Accordingly, a first aspect of the invention relates to a nucleic acid molecule comprising one or more liver-selective enhancers operably linked to a muscle-selective promoter.

In another specific embodiment, the nucleic acid molecule comprises a liver-selective enhancer operably linked to a muscle-selective promoter. In another embodiment, the nucleic acid molecule comprises a plurality of liver selective enhancers operably linked to a muscle selective promoter. In certain embodiments, the plurality of liver selectivity enhancers comprises at least two liver selectivity enhancers. In yet another embodiment, the plurality of liver selective enhancers comprises two liver selective enhancers. In another embodiment, the plurality of liver selective enhancers comprises three liver selective enhancers. In another 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 particular 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 of the plurality of liver selectivity enhancers have the same sequence, or at least two of the plurality of liver selectivity enhancers have different sequences. In certain embodiments, all liver selective enhancers of the plurality have the same sequence.

In particular embodiments of the nucleic acid of the invention, the enhancer may be a short-size liver selective enhancer, or the plurality of liver selective enhancers may be a plurality of short-size liver selective enhancers. In particular, the liver selective enhancer used in the present invention may be composed of 10 to 175 nucleotides, for example 40 to 100 nucleotides, particularly 50 to 80 nucleotides. In a particular embodiment, the liver selective enhancer used in the present invention may consist of 70 to 75 nucleotides. In certain embodiments, the liver selective enhancer is a polypeptide consisting of SEQ ID NO: 1 or the 72 nucleotide HS-CRM8 enhancer with liver selective enhancer activity of SEQ ID NO: 1. In another embodiment, the liver selective enhancer is a functional variant of the 72 nucleotide HS-CRM8 enhancer that is identical to SEQ ID NO: 1, such as at least 80% identical to SEQ ID NO: 1, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%, wherein the functional variant has liver selective enhancer activity. In another embodiment, the liver selective enhancer is a enhancer consisting of a nucleotide sequence identical to SEQ ID NO: 1, such as at least 80% identical to SEQ ID NO: 1, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%, wherein said functional fragment has liver selective enhancer activity.

Preferably, the promoter is a short-size muscle-selective promoter. In particular embodiments, the promoter is the CK6 promoter, the CK8 promoter, the Acta1 promoter, or the synthetic promoter C5.12(spc5.12, also referred to herein as "C5.12"). In a particular embodiment, the muscle-selective promoter is the spc5.12 promoter. In another specific embodiment, the spC5-12 promoter is selected from the group consisting of:

-a polypeptide consisting of SEQ ID NO: 2. 3 or 4, in particular the sequence shown in SEQ ID NO: 2, or SEQ ID NO: 2. 3 or 4, in particular SEQ ID NO: 2, wherein said fragment has muscle-selective promoter activity;

-a polypeptide consisting of a sequence identical to SEQ ID NO: 2. 3 and 4, e.g., at least 80% identical to any one of SEQ ID NOs: 2. 3 and 4, in particular to SEQ ID NO: 2, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity; and

Optionally, the nucleic acid molecules described herein may also comprise other enhancers, such as muscle selective enhancers, for example SEQ ID NO: 34 or the SA195 enhancer of SEQ ID NO: 5 or a functional variant of these enhancers, said functional variant having a sequence identical to SEQ ID NO: 34 or SEQ ID NO: 5, e.g., at least 80% identical to SEQ ID NO: 34 or SEQ ID NO: 5 is a sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%. In certain embodiments, the additional enhancer is located between the liver selective enhancer or enhancers and the muscle selective promoter. In certain embodiments, no other enhancer is provided between the liver selective enhancer or enhancers and the muscle selective promoter.

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

The invention also relates to a vector containing the 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, but are not limited to, adenoviral vectors, retroviral vectors, lentiviral vectors, and parvoviral vectors, such as AAV vectors. In particular embodiments, the viral vector is an AAV vector, e.g., an AAV vector comprising an AAV8 or AAV9 capsid.

The invention also relates to an isolated recombinant cell comprising a nucleic acid construct according to the invention.

The invention also relates to a pharmaceutical composition comprising a vector or isolated cell of the invention in a pharmaceutically acceptable carrier.

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

The invention also relates to an expression cassette, vector or cell as disclosed herein for use in gene therapy.

In another aspect, the invention relates to an expression cassette, vector or cell as disclosed herein for use in the treatment of a neuromuscular disorder. In particular, the neuromuscular disorder may be selected from the group consisting of muscular dystrophy (e.g. myotonic dystrophy (Steinert'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 disease (e.g. Amyotrophic Lateral Sclerosis (ALS), spinal muscular dystrophy (infantile progressive spinal muscular dystrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular dystrophy (type 2), juvenile spinal muscular dystrophy (type 3, Kugelberg-welader disease), adult spinal muscular dystrophy (type 4)), spinal muscular atrophy (kennedy's disease)), inflammatory myopathy (e.g. polymyositis dermatomyositis, dermatomyositis polymyositis, leyanosis, duchenne's disease), and the like, Inclusion body myositis), neuromuscular junction diseases (e.g. myasthenia gravis, Lambert-Eaton syndrome, myasthenia congenital syndrome), peripheral neurological diseases (e.g. Charcot-Marie-Tooth disease, Friedel's ataxia, Dejerine-Sottas disease), metabolic muscle diseases (e.g. phosphorylase deficiency (Mackade's disease), acid maltase deficiency (Pompe disease), phosphofructokinase deficiency (Tarui disease), debranching enzyme deficiency (Cori disease or Forbes disease), mitochondrial myopathy, carnitine deficiency, carnitine palmitoyl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, adenylate deaminase deficiency), endocrine abnormality-induced myopathies (e.g. hyperthyroid myopathy, hypothyroidism) and other myopathies (e.g. myotonia congenital myopathy, myotonia), Myotonia congenita, central axonopathy, linear myopathy, myotubular myopathy, periodic paralysis).

In another specific embodiment, the disease is coriolis disease and the transgene of interest is GDE, e.g., a truncated form of GDE.

Drawings

FIG. 1 schematic representation of promoter/enhancer associations. The dimensions of each element are noted.

FIG. 2 expression of mSeAP protein in muscle. 2X 10 mice were used for C57BL/6 mice¹¹Vg/mouse injection of AAV9 vector expressing murine secreted alkaline phosphatase (mSEAP) reporter gene under transcriptional control of spC5-12 promoter (spC5-12) or fusions of the same promoter with MCK (MCK-spC5-12), H1 and MCK (H1-MCK-spC5-12) or H3 and MCK (H3-MCK-spC5-12) enhancer. Mice injected with PBS were used as control (PBS). One month after injection, the mSeAP activity in different muscles was measured and reported as fold change compared to the levels measured in the PBS group. Statistical analysis was performed by ANOVA (═ p)<0.05, n ═ 5 per group).

FIG. 3 mSEAP expression in non-muscle tissue. Liver, brain and kidney mSEPA activity from C57BL/6 mice treated as described in FIG. 2 was analyzed. The measured mSeAP activity is reported as fold change compared to the level measured in PBS-injected mice. Statistical analysis was performed by ANOVA (═ p <0.05, n ═ 5 per group).

FIG. 4. the presence of the MCK enhancer is not necessary in order to increase transgene expression in muscle. C57BL/6 mice were used 4X 10¹¹vg/mouse AAV9 vector injection, expressing a murine secreted alkaline phosphatase (mSEAP) reporter gene under transcriptional control of the spC5-12 promoter (spC5-12) or fusions of the same promoter with H3(H3-spC5-12) or H3 and MCK (H3-MCK-spC5-12) enhancers. Mice injected with PBS were used as control (PBS). One month after injection, the mSeAP activity in heart, diaphragm, quadriceps and triceps was measured and reported as fold change compared to the levels measured in the PBS group. Statistical analysis was performed by ANOVA (═ p as indicated)<0.05, n — 4 per group).

FIG. 5 the H3 enhancer increases muscle expression when fused to different muscle specific promoters. C57BL/6 mice were used 5X 10¹¹vg/mouse AAV9 vector injection expressing a murine secreted alkaline phosphatase (mSeAP) reporter gene under the transcriptional control of the CK6 or CK8 promoter or an enhancer-promoter combination consisting of the H3 enhancer fused to CK6(H3-CK6) or CK8(H3-CK 8). Mice injected with PBS were used as control (PBS). The mSeAP activity in heart, quadriceps and triceps was measured 15 days after vector injection and reported as fold change compared to levels measured in mice injected with mSeAP under the control of the CK6 promoter. Statistical analysis was performed by ANOVA (═ p as indicated)<0.05, n — 4 per group).

FIG. 6 the H3 enhancer increased muscle expression when fused to the ACTA1 muscle specific promoter. C57BL/6 mice were used 4X 10¹¹vg/mouse AAV9 vector, expressing a murine secreted alkaline phosphatase (mSeAP) reporter gene under the transcriptional control of an enhancer-promoter combination consisting of the H3 enhancer fused to either the spC5-12(H3-spC5-12) or Acta1(H3-Acta1) promoter. Mice injected with PBS were used as control (PBS). One month after injection, the mSeAP activity in heart, diaphragm, quadriceps and triceps was measured and reported as fold change compared to the levels measured in the PBS group. Statistical analysis was performed by ANOVA (═ p compared to PBS)<0.05, n — 4 per group).

Figure 7F enhancer increases muscle expression when fused to muscle specific promoter. C57BL/6 mice were used 4X 10¹¹vg/mouse AAV9 vector injection expressing a murine secreted alkaline phosphatase (mSEAP) reporter gene under the transcriptional control of either the spC5-12 promoter or the combination of the F enhancer and the spC5-12 promoter (F-spC 5-12). Mice injected with PBS were used as control (PBS). At 15 days post vector injection, the mSeAP activity in the quadriceps muscle was measured and reported as fold change compared to the levels measured in the PBS group. Statistical analysis was performed by ANOVA (═ p compared to spC5-12<0.05, n-3-4 per group).

Detailed Description

Definition of

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

In the context of the present invention, the expression "muscle-selective promoter" includes natural or synthetic muscle-selective promoters. Furthermore, the expression "hepatic selectivity enhancer" includes natural or synthetic hepatic selectivity enhancers.

According to the invention, tissue-selective means that a transcriptional regulatory element preferentially drives (in the case of a promoter) or enhances (in the case of an enhancer) the expression of a gene operably linked to the transcriptional regulatory element in a given tissue or group of tissues compared to the expression in another tissue. This definition of "tissue-selective" does not exclude the possibility that tissue-selective transcriptional regulatory elements (e.g., muscle-selective promoters) are to some extent leaky. "leakage" or its derivatives mean the possibility of a muscle-selective promoter driving or increasing expression of a transgene operably linked to the promoter in another tissue, albeit at a lower level. For example, a muscle-selective promoter may leak in liver tissue, meaning that expression driven by the 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 the transcriptional regulatory element not only drives or enhances expression in a given tissue or group of tissues in a preferential manner, but that the regulatory element does not drive or enhance or only drives or enhances expression in other tissues in a minor amount.

According to the present invention, "transgene of interest" refers to a polynucleotide sequence encoding an RNA or protein product, which can be introduced into a cell for a desired purpose, and which is capable of being expressed under suitable conditions. The transgene of interest may encode a product of interest, e.g., a therapeutic or diagnostic product of interest. A "therapeutic transgene" is selected and used to elicit a desired therapeutic result, particularly for effecting expression of the therapeutic transgene in a cell, tissue or organ in which expression of the therapeutic transgene is desired. Therapy can be achieved in a variety of ways, including by expressing the protein in cells that do not express the protein, by expressing the protein in cells that express mutant versions of the protein, by expressing a protein that is toxic to the target cells in which it is expressed (a strategy used, for example, to kill unwanted cells such as cancer cells), by expressing antisense RNA to induce gene repression or exon skipping, or by expressing silencing RNA such as shRNA that is intended to inhibit protein expression. The transgene of interest may also encode nucleases for targeted genome engineering, such as CRISPR-associated protein 9(Cas9) endonuclease, meganuclease or transcription activator-like effector nuclease (TALEN). The transgene of interest can also be a guide RNA or a set of guide RNAs for use with the CRISPR/Cas9 system, or a calibration matrix for use with the previously described nucleases for targeted genome engineering strategies. Other transgenes of interest include, but are not limited to, synthetic long non-coding RNAs (SINEUP; Carrieri et al, 2012, Nature 491: 454-7; Zuccheli et al, 2015, RNA Biol 12(8): 771-9; Indriri et al, 2016, Sci Rep 6:27315) and artificial microRNAs. Other specific transgenes of interest useful in the practice of the invention are described below.

According to the present invention, the term "treatment" includes curative, palliative or prophylactic effects. Thus, therapeutic and prophylactic treatment includes ameliorating the symptoms of a disorder or preventing or otherwise reducing the risk of developing a particular disorder. Treatment may be provided to delay, slow or reverse the progression of the disease and/or one or more symptoms thereof. The term "prophylactic" can be considered to reduce the severity or onset of a particular disorder. "prophylactic" also includes preventing recurrence of a particular disorder in a patient previously diagnosed as having the disorder. "therapeutic" may also refer to reducing the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that may benefit an animal, particularly a mammal, more particularly a human subject. In particular embodiments, the mammal may be an infant or adult subject, such as a human infant or adult.

By "cells of therapeutic interest" or "tissues of therapeutic interest" is meant herein the primary cells or tissues in which expression of the therapeutic transgene will be useful for the treatment of the disorder. In the present invention, the tissue of interest is muscle tissue.

Hybrid promoters

The present inventors designed transcriptional regulatory elements, also referred to herein as "hybrid promoters", for increasing the efficacy of gene therapy while meeting the size limitations of gene therapy vectors, such as the size limitations of AAV vectors.

The nucleic acid molecule of the invention comprises (i) one or more liver selective enhancers operably linked to (ii) a muscle selective promoter.

The liver selective enhancer or enhancers may be selected from liver selective enhancers known to those skilled in the art. In a particular embodiment, the nucleic acid molecule of the invention comprises one and only one liver selective enhancer. In this embodiment, the liver selective enhancer may be 10 to 500 nucleotides in size, 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, wherein a plurality of liver selectivity enhancers is used, the size of the combination of the plurality of liver selectivity enhancers may be 10 to 500 nucleotides, such as 40 to 400 nucleotides, in particular 70 to 250 nucleotides. In certain embodiments, the liver selective enhancer is a naturally occurring enhancer located in the cis position of a gene selectively expressed in hepatocytes. In another particular embodiment, the liver selectivity enhancer may be an artificial liver selectivity enhancer. Illustrative artificial liver selectivity enhancers useful in the practice of the present invention include, but are not limited to, those disclosed in Chuah et al, molecular Therapy,2014, Vol.22, p.9, p.1605, in particular selected from HS-CRM1(SEQ ID NO: 21), HS-CRM2(SEQ ID NO: 22), HS-CRM3(SEQ ID NO: 23), HS-CRM4(SEQ ID NO: 24), HS-CRM5(SEQ ID NO: 25), HS-CRM6(SEQ ID NO: 26), HS-CRM7(SEQ ID NO: 27), HS-CRM8(SEQ ID NO: 1), HS-CRM9(SEQ ID NO: 28), HS-CRM10(SEQ ID NO: 29), HS-CRM11(SEQ ID NO: 30), HS-CRM12(SEQ ID NO: 31), HS-CRM13(SEQ ID NO: 32) and HS-CRM14(SEQ ID NO: 33). In particular embodiments, the liver selective enhancer may be selected from HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM13, and HS-CRM 14. In another specific embodiment, the liver selective enhancer may be selected from the group consisting of HS-CRM2, HS-CRM7, HS-CRM8, HS-CRM11, HS-CRM13, and HS-CRM 14. In certain embodiments, the liver selective enhancer is a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 1. In another embodiment, the liver selective enhancer is a nucleic acid sequence identical to SEQ ID NO: 1, such as at least 80% identical to SEQ ID NO: 1, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity, wherein the functional variant has liver selective enhancer activity. In another embodiment, the liver selective enhancer is a enhancer consisting of a nucleotide sequence identical to SEQ ID NO: 1, such as at least 80% identical to SEQ ID NO: 1, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%, wherein said functional fragment has liver selective enhancer activity. In the case of multiple liver selective enhancers, the enhancers may be fused directly or separated by a linker. Direct fusion means that the first nucleotide of the enhancer immediately follows the last nucleotide of the upstream enhancer. In the case of ligation by a linker, there is a nucleotide sequence between the last nucleotide of the upstream enhancer and the first nucleotide of the subsequent downstream enhancer. For example, the linker may be between 1 to 50 nucleotides, such as 1 to 40 nucleotides, for example 1 to 30 nucleotides, such as 1 to 20 nucleotides, for example 1 to 10 nucleotides in length. In the present invention, the design of the nucleic acid molecule may take into account the above-mentioned size limitations, and therefore such linkers, if present, are preferably short. Representative short linkers include nucleic acid sequences consisting of less than 15 nucleotides, particularly less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 nucleotides, e.g., 1 nucleotide linker.

The second transcription regulatory element present in the nucleic acid molecule of the invention is a muscle-selective promoter, for example a natural or synthetic muscle-selective promoter. The muscle-selective promoter is a short-size muscle-selective promoter. In the context of the present invention, a "short-size promoter" has a length of 2600 nucleotides or less, in particular 2000 nucleotides or less, and has muscle-selective promoter activity when operably linked to a transgene. In particular embodiments, the muscle-selective promoter has a length of 1500 nucleotides or less, 1100 nucleotides or less, 600 nucleotides or less, 500 nucleotides or less, 400 nucleotides or less, 300 nucleotides or less, or 200 nucleotides or less. Illustrative muscle promoters useful in the practice of the present invention include, but are not limited to, the CK6 promoter (SEQ ID NO: 6), the CK8 promoter (SEQ ID NO: 7), the Acta1 promoter (SEQ ID NO: 8), or the synthetic promoter C5.12. In a particular embodiment, the muscle-selective promoter is a synthetic promoter C5.12(spc5.12, also referred to herein as "C5.12"), such as the promoter described in SEQ ID NO: 2. 3 or 4 or the spC5.12 promoter disclosed in Wang et al, Gene Therapy, 15 th, page 1489-1499 (2008). Functional fragments and functional variants of muscle-selective promoters may also be used in the present invention. In particular, muscle-selective promoters useful in the practice of the present invention may be selected from, but are not limited to:

-a polypeptide consisting of SEQ ID NO: 2. 3, 4, 6, 7 or 8, in particular SEQ ID NO: 2. 3 or 4, more particularly the sequence shown in SEQ ID NO: 2, or SEQ ID NO: 2. 3, 4, 6, 7 or 8, in particular SEQ ID NO: 2. 3 or 4, more particularly SEQ ID NO: 2, wherein said fragment has muscle-selective promoter activity;

-a polypeptide consisting of a sequence identical to SEQ ID NO: 2. 3, 4, 6, 7, or 8, e.g., at least 80% identical to any one of SEQ ID NOs: 2. 3, 4, 6, 7 or 8, in particular SEQ ID NO: 2. 3 and 4, in particular SEQ ID NO: 2, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity; and

Other muscle-selective promoters include, but are not limited to, the MCK promoter (SEQ ID NO: 14), the myostatin promoter (SEQ ID NO: 15), and the unc45b promoter (SEQ ID NO: 16) or functional fragments of these promoters having muscle-selective promoter activity. In another embodiment, the muscle-selective promoter consists of a sequence identical to SEQ ID NO: 14. 15 or 16, e.g., at least 80% identical to any one of SEQ ID NOs: 14. 15 or 16, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%. In another embodiment, the muscle-selective promoter is a promoter consisting of a sequence identical to SEQ ID NO: 14. 15 or 16, e.g., at least 80% identical to any one of SEQ ID NOs: 14. 15 or 16, in particular at least 90%, more in particular at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99%, wherein said functional fragment has muscle-selective promoter activity.

Additionally but optionally, the nucleic acid molecules described herein may also comprise other enhancers, such as muscle selective enhancers, for example SEQ ID NO: 5 or a fragment thereof having an amino acid sequence identical to SEQ ID NO: 5, e.g., at least 80% identical to SEQ ID NO: 5 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% of the sequence. In particular embodiments, the additional enhancer, in particular SEQ ID NO: 5 MCK enhancer or functional variant thereof, in

The liver selectivity enhancer or enhancers, and

among the muscle-selective promoters.

In the context of the present invention, the transcriptional regulatory elements introduced into the nucleic acid molecules of the present invention (i.e. (i) the liver-selective enhancer or enhancers, (ii) optionally further enhancers mentioned in the preceding paragraph, and (iii) the muscle-selective promoter) may be fused directly or linked by a linker. For example, in the case of a design with a liver-selective enhancer and a muscle-selective promoter, direct fusion means that the first nucleotide of the promoter immediately follows the last nucleotide of the liver-selective enhancer. Furthermore, in the case of a design with multiple liver-selective enhancers and muscle-selective promoters, direct fusion means that the first nucleotide of the promoter immediately follows the last nucleotide of the 3' most liver-selective enhancer. In the case of ligation by a linker, there is a nucleotide sequence between the last nucleotide of the only liver selective enhancer and the first nucleotide of the promoter, or between the last nucleotide of the 3' most liver selective enhancer and the first nucleotide of the promoter. For example, the linker may be between 1 and 1500 nucleotides, such as 1 to 1000 nucleotides (e.g., 101, 300, 500 or 1000 nucleotides, such as the linkers shown in SEQ ID NOs 17, 18, 19 and 20, respectively), such as 1 to 500 nucleotides, such as 1 to 300 nucleotides, such as 1 to 100 nucleotides, such as 1 to 50 nucleotides, such as 1 to 40 nucleotides, such as 1 to 30 nucleotides, such as 1 to 20 nucleotides, such as 1 to 10 nucleotides in length. In the present invention, the design of the nucleic acid molecule may take into account the above-mentioned size limitations, and therefore such linkers, if present, are preferably short. Representative short linkers include nucleic acid sequences consisting of less than 15 nucleotides, particularly less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 nucleotides, e.g., 1 nucleotide linker.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

-a selective hepatic selective enhancer, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of SEQ ID NO: 9, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 9, such as at least 80% identical to SEQ ID NO: 9 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence and has muscle-selective promoter activity of SEQ ID NO: 9, or a functional variant of the sequence shown in fig. 9.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

-a selective hepatic selective enhancer, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof;

-muscle selective enhancers, such as MCK enhancer; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of SEQ ID NO: 10, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 10, such as at least 80% identity to SEQ ID NO: 10 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence and has muscle-selective promoter activity of SEQ ID NO: 10, or a functional variant of the sequence shown in fig. 10.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

two repeated sequences of two selective liver selective enhancers, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

two repeated sequences of two selective liver selective enhancers, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof;

-muscle selective enhancers, such as MCK enhancer; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

-three repetitive sequences of three selective liver selective enhancers, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of SEQ ID NO: 11, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 11, such as at least 80% identity to SEQ ID NO: 11 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence and has muscle-selective promoter activity of SEQ ID NO: 11, or a functional variant of the sequence shown in fig. 11.

In a particular embodiment, the nucleic acid molecule of the invention comprises, in particular in order from 5 'to 3':

-three repetitive sequences of three selective liver selective enhancers, in particular the HS-CRM8 enhancer or a functional variant or functional fragment thereof;

-muscle selective enhancers, such as MCK enhancer; and

a muscle-selective promoter, in particular the spc5.12 promoter or a functional variant or functional fragment thereof.

According to a particular variant of this embodiment, the nucleic acid molecule of the invention consists of SEQ ID NO: 12, or consists of a sequence having a sequence identical to that shown in SEQ ID NO: 12, such as at least 80% identity to SEQ ID NO: 12 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence and has muscle-selective promoter activity of SEQ ID NO: 12, or a functional variant of the sequence shown in figure 12.

In all embodiments of the nucleic acid molecules of the invention specifically disclosed herein, the nucleic acid molecule may comprise a linker between the liver-selective enhancer and the muscle-selective promoter.

Furthermore, in all embodiments of the nucleic acid molecules of the invention specifically disclosed herein, the nucleic acid molecule may comprise a linker between two liver selective enhancers. For example, in embodiments comprising two liver selective enhancers, a linker may or may not be located between the two liver selective enhancers. Furthermore, in embodiments comprising three liver selectivity enhancers, a linker may be comprised between the first and second liver selectivity enhancers and/or between the second and third liver selectivity enhancers. For example, in embodiments having three hepatic selectivity enhancers, a linker is located between the first and second hepatic selectivity enhancers, and no linker is located between the second and third hepatic selectivity enhancers. In another variation, in an embodiment with three liver selectivity enhancers, no linker is located between the first and second liver selectivity enhancers, and a linker is located between the second and third liver selectivity enhancers.

Expression cassette

The nucleic acid molecules of the invention may be introduced into expression cassettes designed to provide expression of a transgene of interest in a tissue of interest.

Thus, the expression cassette of the present invention includes the above-described nucleic acid molecule and the transgene of interest.

The expression cassette may comprise at least one further regulatory sequence capable of further controlling the expression of the therapeutic transgene of interest by: by reducing or inhibiting its expression in certain tissues not of interest, or by stabilizing the mRNA encoding the protein of interest, e.g., a therapeutic protein encoded by the transgene of interest. These sequences include, for example, silencers (e.g., tissue-specific silencers), microRNA target sequences, introns, and polyadenylation signals.

In a particular embodiment, the expression cassette of the invention comprises, in order from 5 'to 3':

-a nucleic acid molecule of the invention;

-a transgene of interest; and

-a polyadenylation signal.

In a particular variant of this embodiment, an intron may be introduced between the nucleic acid molecule of the invention and the transgene of interest. As a result, the intron is located between the muscle-selective promoter contained in the nucleic acid molecule as described above and the transgene of interest. Alternatively, the intron can be located within the transgene of interest. In particular embodiments, the intron may be the SV40 intron, for example, the intron consisting of SEQ ID NO: 13, and the SV40 intron.

Of course, from the teachings disclosed herein and the general knowledge in the field of molecular biology and gene therapy, one skilled in the art can select and vary the number of enhancers, enhancer size, promoter size, linker size, and any other elements such as other enhancers (e.g., MCK enhancer or functional variants thereof) and introns, depending on the size of the transgene of interest incorporated into the expression cassette.

The transgene of interest may be any of the transgenes described in the "definitions" section above. In addition, specific exemplary transgenes of interest are provided in the following table, wherein the transgenes are regrouped according to the family of neuromuscular disorders they may treat:

muscular dystrophy

Congenital muscular dystrophy

Congenital myopathy

Distal myopathy

Other myopathies

Myotonic syndrome

Ion channel myopathy

Malignant hyperthermia

Gene	Protein
		RYR1	Ryanodine receptor 1 (skeletal muscle)
CACNA1S	Voltage-dependent calcium channel, L-form, alpha 1S subunit

Metabolic myopathy

Hereditary cardiomyopathy

Congenital myasthenia syndrome

Motor neuron disease

Hereditary motor and sensory neuropathy

Hereditary paraplegia

Other neuromuscular disorders

Carrier, cell and pharmaceutical composition

The expression cassette of the present invention may be introduced into a vector. The invention therefore also relates to a vector comprising an expression cassette as described above. The vectors used in the present invention are suitable for RNA/protein expression, in particular for gene therapy.

In one embodiment, the vector is a plasmid vector.

In another embodiment, the vector is a non-viral vector, such as a nanoparticle, Lipid Nanoparticle (LNP) or liposome comprising an expression cassette of the invention.

In another embodiment, the vector is a transposon-based system, allowing integration of the expression cassette of the invention into the genome of a target cell, such as the hyperactive sleeping beauty (SB100X) transposon system (Mates et al, 2009).

In another embodiment, the transgene of interest is a repair matrix useful for targeted genomic engineering, such as a repair matrix suitable for use with an endonuclease as described above for gene correction. More specifically, the vector includes a repair matrix containing arms homologous to the gene of interest for homology-driven integration.

In another embodiment, the vector is a muscle-targeting viral vector suitable for gene therapy. In this case, additional sequences suitable for the production of highly efficient viral vectors are added to the expression cassettes of the invention, as is well known in the art. In a particular embodiment, the viral vector is derived from an integrating virus. In particular, the viral vector may be derived from an adenovirus, retrovirus or lentivirus (e.g., an integration-defective lentivirus). In a particular embodiment, the lentivirus is a pseudotyped lentivirus having an envelope capable of targeting a cell/tissue of interest, such as a liver and/or muscle cell (as described in patent applications EP17306448.6 and EP 17306447.8). In the case where the viral vector is derived from a retrovirus or lentivirus, the additional sequence is a retrovirus or lentivirus LTR sequence flanking the expression cassette. In another specific embodiment, the viral vector is a parvoviral vector, such as an AAV vector, e.g., an AAV vector suitable for transducing muscle. In this embodiment, the additional sequence is an AAV ITR sequence flanked by the expression cassettes.

In a preferred embodiment, the vector is an AAV vector. Human parvovirus adeno-associated virus (AAV) is a naturally replication-defective dependent virus that is capable of integrating into the genome of infected cells to establish latent infection. This last property appears to be unique in mammalian viruses, since integration occurs at a specific site in the human genome, termed AAVS1, on chromosome 19 (19q 13.3-qter).

Thus, AAV vectors have attracted considerable interest as potential vectors for human gene therapy. Advantageous properties of the virus include its absence of association with any human disease, its ability to infect both dividing and non-dividing cells, and the possibility of infecting a wide range of cell lines derived from different tissues.

Among the serotypes of AAV isolated and well characterized from human or non-human primates (NHPs), human serotype 2 is the first AAV developed as a gene transfer vector. Other presently used AAV serotypes include AAV-1, AAV-2 variants (e.g., quadruple mutated capsid optimized AAV-2 comprising an engineered capsid with a Y44+500+730F + T491V alteration, disclosed in Ling et al, 2016, 18.7.2016, Hum Gene Ther Methods.), AAV-3 and AAV-3 variants (e.g., AAV3-ST variants comprising an engineered AAV3 capsid with a two amino acid alteration of S663V + T492V, disclosed in Vercauteren et al, 2016, Mol. Ther.24 volume (6), p.1042), AAV-3B and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-6 variants (e.g., 6 capsid Y731F/Y705F/T492V forms comprising a triple mutation, disclosed in Rosario et al, 2016, Mol The 3. 26.705), AAV6 variants, disclosed in AAV 1607.26.26. multidot., AAV-8, AAV-9, AAV-2G9, AAV-10, e.g., AAVcy10 and AAV-rh10, AAV-rh74, AAV-rh74-9 disclosed in EP18305399 (e.g., the hybrid capsid rh74-9 serotype described in the examples of EP 18305399; the rh74-9 serotype also referred to herein as "-rh 74-9", "AAVrh 74-9" or "AAV-rh 74-9"), AAV-9-rh74 disclosed in EP18305399 (e.g., the hybrid capsid 9-rh74 serotype described in the examples of EP 18305399; the-9-rh 74 "," AAV 5-rh 74 "," AAV-9-rh74 "or" rh74-AAV9 "), AAV-dr 74 serotype described in pig AAV-9, Anc 9, AAV-2G 68656, AAV-10, e.g., AAVcy10 and AAV-rh74 serotypes such as Vpo 8653, AAV-V-rh 4, and AAV serotypes described in AAV 18305399, Lysine and serine capsid mutants, and the like. In addition, other non-native engineered variants and chimeric AAVs may also be useful.

AAV viruses can be engineered using conventional molecular biology techniques such that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for modulating stability and particle lifetime, for efficient degradation, for precise delivery to the nucleus.

The desired AAV fragments for assembly into vectors include capsid proteins, including vp1, vp2, vp3 and hypervariable regions, rep proteins, including rep 78, rep 68, rep 52 and rep 40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of different vector systems and host cells.

AAV-based recombinant vectors lacking Rep proteins integrate inefficiently in the host genome and exist primarily as stable circular episomes, which can persist in target cells for years.

Instead of using AAV native serotypes, artificial AAV serotypes may also be used in the context of the present invention, including but not limited to AAV having non-naturally occurring capsid proteins. Such artificial capsids can be produced 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 portions of the same AAV serotype, non-AAV viral or non-viral sources. The artificial AAV serotype can have a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid, but is not limited thereto.

In the context of the present invention, the AAV vector comprises an AAV capsid capable of transducing a target cell of interest, i.e., a muscle cell.

According to certain embodiments, the AAV vector is AAV-1, AAV-2 variants (e.g., quadruple mutated capsid optimized AAV-2 comprising an engineered capsid with an alteration of Y44+500+730F + T491V, disclosed in Ling et al, 2016, 7/18, Hum Gene Ther Methods [ pre-imprinted electronic edition ]), AAV-3 and AAV-3 variants (e.g., AAV3-ST variants comprising an engineered AAV3 capsid with two amino acid alterations S663V + T492V, disclosed in Vercauteren et al, 2016, mol.Therr.24 volume (6), p.1042), AAV-3B and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-6 variants (e.g., AAV 6Y capsid Y731/Y705F/T6 variants comprising a triple mutation, AAV Theri 3/Y731F/Y F/T36 492V, disclosed in Therlo et al, Ros et al, 2016, page 16026), AAV-7, AAV-8, AAV-9, AAV-2G9, AAV-10 such as AAV-cy10 and AAV-rh10, AAV-rh39, AAV-rh43, AAV-rh74, AAV-rh74-9, AAV-dj, Anc80, LK03, AAV. php, AAV2i8, porcine AAV serotypes such as AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes. In particular embodiments, the AAV vector is 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 another specific embodiment, 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 one whose genome is derived from one of the AAV serotypes described above and whose capsid is derived from another serotype. For example, the genome of the pseudotyped vector may have capsids derived from AAV8, AAV9, AAVrh74, AAVrh74-9, or AAV2i8 serotypes, and its genome is derived from a different serotype. In particular embodiments, the AAV vector has a capsid of an AAV8, an AAV9, an AAVrh74, or an AAVrh74-9 serotype, particularly an AAV8 or AAV9 serotype, more particularly an AAV8 serotype.

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

In particular 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 incorporates regions or domains of VP proteins derived from at least two AAV serotypes. For example, a chimeric AAV vector may be derived from the combination of AAV8 capsid sequences with sequences of an AAV serotype different from the AAV serotype 8, such as any of those specifically mentioned above.

In another embodiment, the modified capsid may also be derived from a capsid modification inserted by error-prone PCR and/or peptide insertion (e.g., as described in Bartel et al, 2011). In addition, capsid variants may include single amino acid changes such as tyrosine mutants (e.g., as described in Zhong et al, 2008).

In addition, the genome of an AAV vector can be a single-stranded or self-complementary double-stranded genome (McCarty et al, Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are produced by deleting the terminal dissociation site from one of the AAV terminal repeats. These modified vectors, which replicate genomes half as long as the wild-type AAV genome, have a tendency to package DNA dimers. In a preferred embodiment, the AAV vector used in the practice of the invention has a single-stranded genome and further preferably comprises an AAV8, AAV9, AAVrh74, AAVrh74-9 or AAV2i8 capsid, in particular an AAV8, AAV9, AAVrh74 or AAVrh74-9 capsid, such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. Other suitable sequences may be introduced into the nucleic acid constructs of the invention, as known in the art, to obtain a functional viral vector. Suitable sequences include AAV ITRs.

Of course, in designing the nucleic acid sequences of the invention and the expression cassettes of the invention, one skilled in the art should be aware of the size limitations of the vectors used to deliver the constructs to cells or organs. In particular, as mentioned above, where the vector is an AAV vector, the skilled person will be aware that a major limitation of an AAV vector is its carrying capacity, which may vary among different AAV serotypes, but is believed to be limited to about the size of the parental viral genome. For example, 5kb is the largest size generally considered for packaging in the AAV8 capsid (Wu Z. et al, Mol Ther.,2010,18(1): 80-86; Lai Y. et al, Mol Ther.,2010,18(1): 75-79; Wang Y. et al, Hum Gene Ther Methods,2012,23(4): 225-33). Therefore, the person skilled in the art will take care in practicing the present invention to select the components of the nucleic acid construct of the invention such that the resulting nucleic acid sequence, including the sequences encoding the AAV 5 '-and 3' -ITRs, preferably does not exceed 110%, in particular preferably does not exceed 5.5kb of the carrying capacity of the AAV vector used.

The invention also relates to an isolated cell, e.g., a muscle cell, which is transformed with a nucleic acid sequence of the invention or an expression cassette of the invention. The cells of the invention may be delivered to a subject in need thereof by injection into the tissue of interest or into the bloodstream of the subject. In a particular embodiment, the invention relates to introducing a nucleic acid molecule or expression cassette of the invention into cells of a subject to be treated and administering said cells into which the nucleic acid or expression cassette has been introduced back to said subject.

The invention also provides pharmaceutical compositions comprising the nucleic acid molecules, vectors or cells of the invention. Such compositions comprise a therapeutically effective amount of a nucleic acid sequence, vector or cell of the invention, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. or european pharmacopeia or other generally recognized pharmacopeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions may 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.

The composition 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 contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, e.w. martin. Such compositions should contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide a form suitable for administration to a subject. In a particular embodiment, the nucleic acid sequence, expression cassette, vector or cell of the invention is formulated in a composition comprising phosphate buffered saline supplemented with 0.25% human serum albumin. In another particular embodiment, the carrier of the present 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%, such as 0.001%, by weight of the total composition. The formulation may also comprise serum albumin, in particular human serum albumin, for example 0.25% human serum albumin. Other suitable formulations for storage or administration are known in the art, in particular from WO 2005/118792 or Allay et al, 2011.

In a preferred embodiment, the composition is formulated in accordance with conventional procedures as a pharmaceutical composition suitable for intravenous or intramuscular administration, preferably intravenous administration, to a human. Typically, 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 lidocaine to relieve pain at the injection site.

In one embodiment, the nucleic acid sequence, expression cassette or vector of the invention may be delivered in a vesicle, particularly a liposome. In yet another embodiment, the nucleic acid sequence, expression cassette or vector of the invention may be delivered in a controlled release system.

Method for using carrier

Thanks to the invention, it is possible to express transgenes of interest in muscle or muscle cells.

The nucleic acid molecules, expression cassettes or vectors of the invention can be used to express genes in muscle cells. Accordingly, the present invention provides a method for expressing a transgene of interest in a muscle cell, wherein the expression cassette of the present invention is introduced into the muscle 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 muscle cells.

The nucleic acid molecules, expression cassettes or vectors of the invention can also be used in gene therapy. Thus, in one aspect, the invention relates to a nucleic acid molecule, expression cassette, vector, cell or pharmaceutical composition as described above for use as a medicament. Thus, in one aspect, the invention relates to a nucleic acid molecule, expression cassette or vector disclosed herein for use in therapy, in particular gene therapy. Likewise, the cells of the invention may be used in therapy, in particular in cell therapy.

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

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

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

The neuromuscular disorder is in particular a genetic or acquired disorder, such as a genetic or acquired neuromuscular disease. Of course, the therapeutic transgene and the promoter driving expression in the tissue of therapeutic interest should be selected based on the disorder to be treated.

The term "neuromuscular disorder" encompasses diseases and conditions that impair muscle function either directly (voluntary myopathy) or indirectly (neuro or neuromuscular junction disease). Illustrative neuromuscular disorders include, but are not limited to, muscular dystrophy (e.g., myotonic dystrophy (Steinert'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 disease (e.g., Amyotrophic Lateral Sclerosis (ALS), spinal muscular dystrophy (infantile progressive spinal muscular dystrophy (type 1, Werdnig-Hoffmann disease), intermediate spinal muscular dystrophy (type 2), juvenile spinal muscular dystrophy (type 3, Kugelberg-Welander disease), adult spinal muscular dystrophy (type 4)), spinal muscular dystrophy (kennedy's disease)), inflammatory myopathy (e.g., polymyositis dermatomyositis, multiple sclerosis, multiple, or multiple, or multiple, or multiple, or multiple, or multiple, or multiple, or multiple, or multiple, multiple, Inclusion body myositis), neuromuscular junction diseases (e.g. myasthenia gravis, Lambert-Eaton syndrome, myasthenia congenital syndrome), peripheral neurological diseases (e.g. Charcot-Marie-Tooth disease, Friedel's ataxia, Dejerine-Sottas disease), metabolic muscle diseases (e.g. phosphorylase deficiency (Mackade's disease), acid maltase deficiency (Pompe disease), phosphofructokinase deficiency (Tarui disease), debranching enzyme deficiency (Cori disease or Forbes disease), mitochondrial myopathy, carnitine deficiency, carnitine palmitoyl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, adenylate deaminase deficiency), endocrine abnormality-induced myopathies (e.g. hyperthyroid myopathy, hypothyroidism) and other myopathies (e.g. myotonia congenital myopathy, myotonia), Myotonia congenita, central axonopathy, linear myopathy, myotubular myopathy, periodic paralysis). In this embodiment, the nucleic acid sequence of the invention comprises liver-selective, muscle-selective and/or neuron-selective transcriptional regulatory elements, such as liver-selective and muscle-selective transcriptional regulatory elements, liver-selective and neuron-selective transcriptional regulatory elements and liver-selective, muscle-selective and neuron-selective transcriptional regulatory elements.

In a particular embodiment, the disorder is glycogen storage disease. The expression "glycogen storage disease" denotes a group of inherited metabolic disorders involving enzymes responsible for the synthesis and degradation of glycogen. In a more specific embodiment, the glycogen storage disease may be GSDI (gouge's disease), GSDII (pompe's disease), GSDIII (cori's disease), gsdivv, GSDV, GSDVI, GSDVII, GSDVIII, or a lethal congenital glycogen storage disease of the heart. More specifically, the glycogen storage disease is selected from the group consisting of GSDI, GSDII and GSDIII, even more specifically from 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 to treat GAA-deficient disorders or other disorders accompanied by glycogen accumulation, such as GSDI (behcet's disease), GSDII (pompe's disease), GSDIII (cori disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and lethal congenital glycogen storage diseases of the heart, more particularly GSDI, GSDII or GSDIII, even more particularly GSDII and GSDIII. In another specific embodiment, the disorder is pompe disease and the therapeutic transgene is a gene encoding acid alpha-Glucosidase (GAA) or a variant thereof. Variants of such GAA are specifically disclosed in applications PCT/2017/072942, PCT/EP2017/072945 and PCT/EP2017/072944, which are incorporated herein by reference in their entirety. In this embodiment, the sequences of the invention comprise liver-selective, muscle-selective and/or neuron-selective transcriptional regulatory elements, such as liver-selective and muscle-selective transcriptional regulatory elements, liver-selective and neuron-selective transcriptional regulatory elements, muscle-selective and neuron-selective transcriptional regulatory elements, and liver-selective, muscle-selective and neuron-selective transcriptional regulatory elements. In a particular embodiment, the disorder is Infancy Onset Pompe Disease (IOPD) or late pompe disease (LOPD). Preferably, the disorder is IOPD.

The transgenes of interest useful in the treatment of these and other disorders by gene therapy are known to those skilled in the art. For example, the therapeutic transgene is: the lysosomal enzyme α -L-iduronidase [ IDUA (α -L-iduronidase) ], acid α -glucosidase for pompe disease (GAA), Glycogen Debranching Enzyme (GDE) or shortened form of GDE (also known as truncated form of GDE or mini-GDE) for cori disease (GSDIII), G6P for GSDI, α -myosin (SGCA) for LGMD2D, dystrophin for DMD or shortened form thereof, and SMN1 for SMA. The transgene of interest may also be a transgene that provides other therapeutic properties than providing RNA that loses protein or inhibits expression of a given protein. For example, a transgene of interest can include, but is not limited to, a transgene that can increase muscle strength.

Specific examples of therapeutic transgenes of interest that can be operably linked to the hybrid promoters of the invention for use in particular diseases are provided below.

In a particular embodiment, the disease is coriolis and the transgene of interest encodes GDE or a shortened form of GDE. Foreshortened forms of GDEs suitable for use in the present invention may include, but are not limited to, those described in EP 18306088. Alternatively, the invention uses a dual AAV vector system for expressing GDEs, such as the dual AAV vector system disclosed in WO 2018162748. In this embodiment, the vector of the invention may correspond to a first AAV vector of the dual AAV vector system comprising between the 5 'and 3' AAV ITRs a first nucleic acid sequence encoding the N-terminal portion of the GDE under the control of the nucleic acid molecule of the invention.

In another specific embodiment, the disease is pompe disease and the transgene of interest encodes acid alpha-Glucosidase (GAA) or modified GAA. Modified GDEs suitable for use in the present invention include, but are not limited to, those disclosed in WO2018046772, WO2018046774, and WO 2018046775.

In another specific embodiment, the disorder is selected from duchenne muscular dystrophy, myotubular myopathy, spinal muscular dystrophy, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2A, limb-girdle muscular dystrophy type 2B, limb-girdle muscular dystrophy type 2C or limb-girdle muscular dystrophy type 2D and type 1 myotonic dystrophy.

Methods of administration of the vectors of the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, local administration as described in WO2015158924, and oral routes. In particular embodiments, the administration is by intravenous or intramuscular route. The vectors of the invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Administration may be systemic or topical.

In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the present invention topically to an area in need of treatment, such as the liver or muscle. This may be achieved, for example, using an implant that is a porous, non-porous or gel-like material, including membranes such as silicone rubber membranes or fibers.

The amount of a vector of the invention effective in the treatment of a disorder to be treated can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be used to help predict optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease and should be decided according to the judgment of the practitioner and each patient's circumstances. The dosage of the vectors of the invention to be administered to a subject in need thereof will vary depending upon several factors, including but not limited to the route of administration, the particular disease being treated, the subjectAge or expression level necessary to obtain a therapeutic effect. The required dosage can be readily determined by one skilled in the art based on these and other factors, based on the knowledge in the art. Where treatment involves administering an AAV vector to a subject, a typical dose of vector is at least 1X 10 per kilogram body weight⁸Copies of the vector genome (vg/kg), e.g., at least 1X 10⁹vg/kg, at least 1X 10¹⁰vg/kg, at least 1X 10¹¹vg/kg, at least 1X 10¹²vg/kg, at least 1X 10¹³vg/kg, at least 1X 10¹⁴vg/kg or at least 1X 10¹⁵vg/kg。

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

Examples

Materials and methods

In vivo studies

All mouse studies were performed according to French and European legislation on animal care and experiments (2010/63/EU) and were approved by the local institutional ethics committee (protocol No. 2016-002B). AAV vectors were administered intravenously via tail vein to 6-8 week old male C57Bl6/J mice. PBS injected littermates were used as controls. Mice were perfused with PBS immediately after sacrifice to avoid blood contamination in the tissues. After sampling, the tissues were homogenized in DNase/RNase-free water using a Fastprep tube (4 m/s; 60 seconds).

mSeAP Activity

mSeAP activity in tissues was measured using the Phospha-LightTMSEAP reporter assay System (ThermoFisher) following the manufacturer's instructions.

Plasmid construction

Enhancer/promoter (EP) sequences are purchased from commercial sources. The mSeAP cDNA was ligated to each EP sequence using the XhoI and Mlu1 restriction enzymes. The resulting transgene expression cassette was cloned between two ITRs derived from AAV2 using XbaI restriction sites flanking the sequence.

The promoter/enhancer combinations used in the experimental section are described in table 1 below.

TABLE 1 description of promoter/enhancer combinations used in the figures

Names in the figures	SEQ ID NO	Enhancer	Promoters
				spC5-12	2	-	spC5-12¹
MCK-spC5-12	35	MCK²	spC5-12¹
				H1-MCK-spC5-12	10	H1³,MCK²	spC5-12¹
H3-MCK-spC5-12	12	H3⁴,MCK²	spC5-12¹
				H3-spC5-12	11	H3⁴	spC5-12¹
CK6	6	-	CK6⁵
				H3-CK6	36	H3⁴	CK6⁵
CK8	7	-	CK8⁶
				H3-CK8	37	H3⁴	CK8⁶
H3-ACTA1	38	H3⁴	ACTA1⁷
				F-spC5-12	39	F⁸	spC5-12¹

¹The spC5-12 promoter (SEQ ID NO: 2);²the MCK enhancer (SEQ ID NO: 5);³SEQ ID NO: 1, a copy;⁴SEQ ID NO: 1, three copies;⁵the CK6 promoter (SEQ ID NO: 6);⁶the CK8 promoter (SEQ ID NO: 7);⁷the ACTA1 promoter (SEQ ID NO: 8);⁸the fibrinogen alpha chain enhancer (SEQ ID NO: 30).

Results

We evaluated tissue-specific expression driven by four combinations of enhancers and promoters reported in figure 1. These promoters consist of a combination of the muscle-specific promoter (spC5-12) and the muscle enhancer MCK (MCK-spC5-12, Table 1). This promoter and enhancer combination is referred to in the literature as E-Syn (Wang B. Gene therapy 2008). Novel hybrid promoters were obtained by fusing one (H1) or three repeats (H3) of the liver enhancer HS-CRM8 at position 1 of the MCK-spC5-12 promoter/enhancer combination (H1-MCK-spC5-12 and H3-MCK-spC5-12, respectively, Table 1). To verify the tissue specificity of these constructs, we used a mouse secreted alkaline phosphatase (mSeAP) reporter. The transgene expression cassette with this reporter gene and the four promoter/enhancer combinations was pseudotyped in AAV9 vector generated by triple transfection and cesium chloride gradient purification.

The mSeAP-AAV9 vector was administered at 2X 10 per mouse¹¹Doses of duplicate vector genomes were injected intravenously into 2-month-old C57Bl6/J male mice. In parallel, mice were injected with Phosphate Buffered Saline (PBS) as a pairAnd (6) irradiating. Animals were sacrificed 1 month after vehicle injection. Mice were perfused with PBS to avoid blood contamination in the tissues. Biochemical analysis was performed on muscle and non-muscle tissue to quantify mSeAP enzyme activity. In muscle, the spC5-12 promoter or the MCK-spC5-12 enhancer/promoter did not elicit significant and detectable mEAP activity compared to PBS injected mice (FIG. 2). Interestingly, AAV9 expressing mSAPs under the transcriptional control of the H1-MCK-spC5-12 and H3-MCK-spC5-12 hybrid promoters allowed a 5 to 10-fold dramatic increase in mSAPs activity in different skeletal muscles (FIG. 2). In the diaphragm and posterior tibialis, we observed a higher increase in mSEPA activity, 150 and 20 fold for the H3-MCK-spC5-12 enhancer/promoter, respectively. In the liver, we did not observe any significant increase in mEAP expression (FIG. 3). In the kidney and brain, a 2 to 3 fold significant increase in mEAP expression was observed in mice receiving the vector with H1-MCK-spC5-12 and H3-MCK-spC5-12 (FIG. 3).

Given that mSEAP expression in muscle is much higher than in other tissues, these data demonstrate that fusing one or three copies of HS-CRM8 5' to the synthetic muscle promoter (MCK-spC5-12) specifically increases transgene expression in muscle, thus providing a new tool for gene therapy of neuromuscular disorders.

Then, we reduce the size of the promoter by removing the MCK enhancer. We generated AAV9 vectors that express mSEAP under the transcriptional control of the following promoters: (i) the spC5-12 promoter, (ii) the spC5-12 promoter fused directly to three copies of HS-CRM8 (H3-spC5-12), or (iii) the H3-MCK-spC5-12 hybrid promoter. The mSeAP-AAV9 vector was administered at 4X 10 per mouse¹¹Doses of duplicate vector genomes were injected into C57Bl/6 male mice. In parallel, mice were injected with Phosphate Buffered Saline (PBS) as a control. Animals were sacrificed 1 month after vehicle injection. Mice were perfused with PBS to avoid blood contamination in the tissues. Muscle tissue was analyzed to quantify the mSeAP enzyme activity. Importantly, in muscle, unlike the spC5-12 promoter, fusion of the spC5-12 promoter to H3 or H3-MCK resulted in significant and detectable mEAP activity compared to PBS injected mice (FIG. 4). These data indicate the transfer intoThe increase in muscle expression of the gene is independent of the presence of the MCK enhancer. The transgene expression cassette constructs described below do not include this enhancer.

To demonstrate the robustness of the effect observed when fusing the H3 enhancer to the muscle promoter, we generated two new promoter/enhancer combinations comprising the CK6 and CK8 promoters commonly used in vivo gene therapy. We prepared AAV9 vectors expressing mSEAP under the transcriptional control of CK6 or CK8 promoters or under the control of CK6 or CK8(H3-CK 6 and H3-CK8, respectively) fused directly to three copies of HS-CRM 8. The mSeAP-AAV9 vector was administered at 5X 10 per mouse¹¹Doses of duplicate vector genomes were injected into C57Bl/6 male mice. Animals were sacrificed 15 days after vehicle injection. Mice were perfused with PBS to avoid blood contamination in the tissues. Muscle tissue was analyzed to quantify the mSeAP enzyme activity. Importantly, in muscle, fusion of H3 with both muscle specific promoters CK6 and CK8 resulted in significant and detectable mSEAP activity compared to the parental CK6 and CK8 promoters, respectively (fig. 5). Similar findings have also been reported for different promoters, ACTA1, when fused to three copies of HS-CRM8 (H3-ACTA1), resulted in expression levels of mSEPA transgene in muscle that were similar to those measured using the H3-spC5-12 hybrid promoter (FIG. 6). Notably, the ACTA1 promoter showed comparable potency in muscle as the spC5-12 promoter in different experimental settings (data not shown). These data indicate that H3 induces an increase in promoter potency regardless of the muscle-selective promoter.

Finally, to confirm that other liver selective enhancers have similar effects, we tested regulatory sequences controlling fibrinogen alpha chain transcription fused to the spC5-12 promoter (described as HS-CRM11 in Chuah et al, molecular Therapy,2014, Vol.22, p.9, p.1605) (F-spC 5-12). The F-spC5-12 hybrid promoter resulted in a significant increase in mSEPA transgene expression compared to the spC5-12 promoter (FIG. 7), thus indicating that similar to H3, other liver-specific enhancers also increase transgene expression in muscle driven by muscle-specific promoters.

Sequence listing

<110> Genesson pine Corp (GENETION), etc

<120> hybrid promoter for muscle expression

<130> B2985PC00

<160> 39

<170> PatentIn 3.3 edition

<210> 1

<211> 72

<212> DNA

<213> Artificial

<220>

<223> HS-CRM8 (x1)

<400> 1

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60

ggctaagtcc ac 72

<210> 2

<211> 315

<212> DNA

<213> Artificial

<220>

<223> spC5.12 (1)

<400> 2

caccgcggtg gcggccgtcc gccctcggca ccatcctcac gacacccaaa tatggcgacg 60

ggtgaggaat ggtggggagt tatttttaga gcggtgagga aggtgggcag gcagcaggtg 120

ttggcgctct aaaaataact cccgggagtt atttttagag cggaggaatg gtggacaccc 180

aaatatggcg accggttcct caaccggtcg ccatatttgg gtgtccgccc tcggccgggg 240

ccgcattcct gggggccggg cggtgctccc gcccgcctcg ataaaaggct ccggggccgg 300

cggcggccca cgagc 315

<210> 3

<211> 400

<212> DNA

<213> Artificial

<220>

<223> spC5.12 (2)

<400> 3

ccgagctcca ccgcggtggc ggccgtccgc cctcggcacc atcctcacga cacccaaata 60

tggcgacggg tgaggaatgg tggggagtta tttttagagc ggtgaggaag gtgggcaggc 120

agcaggtgtt ggcgctctaa aaataactcc cgggagttat ttttagagcg gaggaatggt 180

ggacacccaa atatggccca aatatggcga cggttcctca cccgtcgcca tatttgggtg 240

tccgccctcg gccggggccg cattcctggg ggccgggcgg tgctcccgcc cgcctcgata 300

aaaggctccg gggccggcgg cggcccacga gctacccgga ggagcgggag gcgccaagct 360

ctagaactag tggatccccc gggctgcagg aattcgatat 400

<210> 4

<211> 358

<212> DNA

<213> Artificial

<220>

<223> spC5.12 (3)

<400> 4

caccgcggtg gcggccgtcc gccctcggca ccatcctcac gacacccaaa tatggcgacg 60

ggtgaggaat ggtggggagt tatttttaga gcggtgagga aggtgggcag gcagcaggtg 120

ttggcgctct aaaaataact cccgggagtt atttttagag cggaggaatg gtggacaccc 180

aaatatggcg acggttcctc acccgtcgcc atatttgggt gtccgccctc ggccggggcc 240

gcattcctgg gggccgggcg gtgctcccgc ccgcctcgat aaaaggctcc ggggccggcg 300

gcggcccacg agctacccgg aggagcggga ggcgccaagc tctagaacta gtggatct 358

<210> 5

<211> 208

<212> DNA

<213> Artificial

<220>

<223> MCK enhancer

<400> 5

cactacgggt ctaggctgcc catgtaagga ggcaaggcct ggggacaccc gagatgcctg 60

gttataatta accccaacac ctgctgcccc ccccccccca acacctgctg cctgagcctg 120

agcggttacc ccaccccggt gcctgggtct taggctctgt acaccatgga ggagaagctc 180

gctctaaaaa taaccctgtc cctggtgg 208

<210> 6

<211> 575

<212> DNA

<213> Artificial

<220>

<223> CK6 promoter

<400> 6

ctacgggtct aggctgccca tgtaaggagg caaggcctgg ggacacccga gatgcctggt 60

tataattaac cccaacacct gctgcccccc cccccccaac acctgctgcc tgagcctgag 120

cggttacccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 180

tctaaaaata accctgtccc tggtgggccc aatcaaggct gtgggggact gagggcaggc 240

tgtaacaggc ttgggggcca gggcttatac gtgcctggga ctcccaaagt attactgttc 300

catgttcccg gcgaagggcc agctgtcccc cgccagctag actcagcact tagtttagga 360

accagtgagc aagtcagccc ttggggcagc ccatacaagg ccatggggct gggcaagctg 420

cacgcctggg tccggggtgg gcacggtgcc cgggcaacga gctgaaagct catctgctct 480

caggggcccc tccctgggga cagcccctcc tggctagtca caccctgtag gctcctctat 540

ataacccagg ggcacagggg ctgcccccgg gtcac 575

<210> 7

<211> 454

<212> DNA

<213> Artificial

<220>

<223> CK8 promoter

<400> 7

ctacaaacgc tagcatgctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc 60

tggttataat taacccagac atgtggctgc cccccccccc ccaacacctg ctgcctctaa 120

aaataaccct gcatgccatg ttcccggcga agggccagct gtcccccgcc agctagactc 180

agcacttagt ttaggaacca gtgagcaagt cagcccttgg ggcagcccat acaaggccat 240

ggggctgggc aagctgcacg cctgggtccg gggtgggcac ggtgcccggg caacgagctg 300

aaagctcatc tgctctcagg ggcccctccc tggggacagc ccctcctggc tagtcacacc 360

ctgtaggctc ctctatataa cccaggggca caggggctgc cctcattcta ccaccacctc 420

cacagcacag acagacactc aggagccagc cagc 454

<210> 8

<211> 324

<212> DNA

<213> Artificial

<220>

<223> Acta1 promoter

<400> 8

aaaggcatag ccccatatat cagtgatata aatagaacct gcagcaggct ctggtaaatg 60

atgactacaa ggtggactgg gaggcagccc ggccttggca ggcatcgacc gggccaaccc 120

gctccttctt tggtcaacgc aggggacccg ggcgggggcc caggccgcga accggccgag 180

ggagggggct ctagtgccca acacccaaat atggctcgag aagggcagcg acattcctgc 240

ggggtggcgc ggagggaatg cccgcgggct atataaaacc tgagcagagg gacaagcggc 300

caccgcagcg gacagcgcca agtg 324

<210> 9

<211> 395

<212> DNA

<213> Artificial

<220>

<223> HS-CRM8 x 1 - spC5.12

<400> 9

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60

ggctaagtcc acaagcttca caccgcggtg gcggccgtcc gccctcggca ccatcctcac 120

gacacccaaa tatggcgacg ggtgaggaat ggtggggagt tatttttaga gcggtgagga 180

aggtgggcag gcagcaggtg ttggcgctct aaaaataact cccgggagtt atttttagag 240

cggaggaatg gtggacaccc aaatatggcg accggttcct caaccggtcg ccatatttgg 300

gtgtccgccc tcggccgggg ccgcattcct gggggccggg cggtgctccc gcccgcctcg 360

ataaaaggct ccggggccgg cggcggccca cgagc 395

<210> 10

<211> 601

<212> DNA

<213> Artificial

<220>

<223> HS-CRM8x 1-MCK enhancer-spC5.12

<400> 10

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60

ggctaagtcc acaagcttca ctacgggtct aggctgccca tgtaaggagg caaggcctgg 120

ggacacccga gatgcctggt tataattaac cccaacacct gctgcccccc cccccccaac 180

acctgctgcc tgagcctgag cggttacccc accccggtgc ctgggtctta ggctctgtac 240

accatggagg agaagctcgc tctaaaaata accctgtccc tggtggcacc gcggtggcgg 300

ccgtccgccc tcggcaccat cctcacgaca cccaaatatg gcgacgggtg aggaatggtg 360

gggagttatt tttagagcgg tgaggaaggt gggcaggcag caggtgttgg cgctctaaaa 420

ataactcccg ggagttattt ttagagcgga ggaatggtgg acacccaaat atggcgaccg 480

gttcctcaac cggtcgccat atttgggtgt ccgccctcgg ccggggccgc attcctgggg 540

gccgggcggt gctcccgccc gcctcgataa aaggctccgg ggccggcggc ggcccacgag 600

c 601

<210> 11

<211> 545

<212> DNA

<213> Artificial

<220>

<223> HS-CRM8x3 - spC5.12

<400> 11

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60

ggctaagtcc acaagcttgg gggaggctgc tggtgaatat taaccaaggt caccccagtt 120

atcggaggag caaacagggg ctaagtccac gggggaggct gctggtgaat attaaccaag 180

gtcaccccag ttatcggagg agcaaacagg ggctaagtcc acaagcttca caccgcggtg 240

gcggccgtcc gccctcggca ccatcctcac gacacccaaa tatggcgacg ggtgaggaat 300

ggtggggagt tatttttaga gcggtgagga aggtgggcag gcagcaggtg ttggcgctct 360

aaaaataact cccgggagtt atttttagag cggaggaatg gtggacaccc aaatatggcg 420

accggttcct caaccggtcg ccatatttgg gtgtccgccc tcggccgggg ccgcattcct 480

gggggccggg cggtgctccc gcccgcctcg ataaaaggct ccggggccgg cggcggccca 540

cgagc 545

<210> 12

<211> 751

<212> DNA

<213> Artificial

<220>

<223> HS-CRM8x3-MCK enhancer-spC5.12

<400> 12

gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60

ggctaagtcc acaagcttgg gggaggctgc tggtgaatat taaccaaggt caccccagtt 120

atcggaggag caaacagggg ctaagtccac gggggaggct gctggtgaat attaaccaag 180

gtcaccccag ttatcggagg agcaaacagg ggctaagtcc acaagcttca ctacgggtct 240

aggctgccca tgtaaggagg caaggcctgg ggacacccga gatgcctggt tataattaac 300

cccaacacct gctgcccccc cccccccaac acctgctgcc tgagcctgag cggttacccc 360

accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc tctaaaaata 420

accctgtccc tggtggcacc gcggtggcgg ccgtccgccc tcggcaccat cctcacgaca 480

cccaaatatg gcgacgggtg aggaatggtg gggagttatt tttagagcgg tgaggaaggt 540

gggcaggcag caggtgttgg cgctctaaaa ataactcccg ggagttattt ttagagcgga 600

ggaatggtgg acacccaaat atggcgaccg gttcctcaac cggtcgccat atttgggtgt 660

ccgccctcgg ccggggccgc attcctgggg gccgggcggt gctcccgccc gcctcgataa 720

aaggctccgg ggccggcggc ggcccacgag c 751

<210> 13

<211> 91

<212> DNA

<213> Artificial

<220>

<223> SV40 intron

<400> 13

ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt 60

ttgtgtattt tagattccaa cctatggaac t 91

<210> 14

<211> 366

<212> DNA

<213> Artificial

<220>

<223> MCK promoter

<400> 14

caatcaaggc tgtgggggac tgagggcagg ctgtaacagg cttgggggcc agggcttata 60

cgtgcctggg actcccaaag tattactgtt ccatgttccc ggcgaagggc cagctgtccc 120

ccgccagcta gactcagcac ttagtttagg aaccagtgag caagtcagcc cttggggcag 180

cccatacaag gccatggggc tgggcaagct gcacgcctgg gtccggggtg ggcacggtgc 240

ccgggcaacg agctgaaagc tcatctgctc tcaggggccc ctccctgggg acagcccctc 300

ctggctagtc acaccctgta ggctcctcta tataacccag gggcacaggg gctgcccccg 360

ggtcac 366

<210> 15

<211> 1060

<212> DNA

<213> Artificial

<220>

<223> Myoglobin promoter

<400> 15

taccccctgc cccccacagc tcctctcctg tgccttgttt cccagccatg cgttctcctc 60

tataaatacc cgctctggta tttggggttg gcagctgttg ctgccaggga gatggttggg 120

ttgacatgcg gctcctgaca aaacacaaac ccctggtgtg tgtgggcgtg ggtggtgtga 180

gtagggggat gaatcaggga gggggcgggg gacccagggg gcaggagcca cacaaagtct 240

gtgcgggggt gggagcgcac atagcaattg gaaactgaaa gcttatcaga ccctttctgg 300

aaatcagccc actgtttata aacttgaggc cccaccctcg acagtaccgg ggaggaagag 360

ggcctgcact agtccagagg gaaactgagg ctcagggcca gctcgcccat agacatacat 420

ggcaggcagg ctttggccag gatccctccg cctgccaggc gtctccctgc cctcccttcc 480

tgcctagaga cccccaccct caagcctggc tggtctttgc ctgagaccca aacctcttcg 540

acttcaagag aatatttagg aacaaggtgg tttagggcct ttcctgggaa caggccttga 600

ccctttaaga aatgacccaa agtctctcct tgaccaaaaa ggggaccctc aaactaaagg 660

gaagcctctc ttctgctgtc tcccctgacc ccactccccc ccaccccagg acgaggagat 720

aaccagggct gaaagaggcc cgcctggggg ctgcagacat gcttgctgcc tgccctggcg 780

aaggattggt aggcttgccc gtcacaggac ccccgctggc tgactcaggg gcgcaggcct 840

cttgcggggg agctggcctc cccgccccca cggccacggg ccgccctttc ctggcaggac 900

agcgggatct tgcagctgtc aggggagggg aggcgggggc tgatgtcagg agggatacaa 960

atagtgccga cggctggggg ccctgtctcc cctcgccgca tccactctcc ggccggccgc 1020

ctgcccgccg cctcctccgt gcgcccgcca gcctcgcccg 1060

<210> 16

<211> 195

<212> DNA

<213> Artificial

<220>

<223> Unc45b promoter

<400> 16

tttctcattc tagaaagtac cagtcaactc tccaccagcc cagctgttgg cagacgcaca 60

cctccatccc cctgccctca gacatttgcc actgatttct cagctgtcat cccctctcca 120

taaatagacc ctatcagaga aagtccattg cactaatata aggggtgacc acatttctac 180

aaaaccacaa ttaat 195

<210> 17

<211> 101

<212> DNA

<213> Artificial

<220>

<223> linker

<400> 17

cgatgggcaa ctcatgcaat tattgtgagc aatacacacg cgcttccagc ggagtataaa 60

tgcctaaagt aataaaaccg agcaatccat ttacgaatgt t 101

<210> 18

<211> 300

<212> DNA

<213> Artificial

<220>

<223> linker

<400> 18

cgatgggcaa ctcatgcaat tattgtgagc aatacacacg cgcttccagc ggagtataaa 60

tgcctaaagt aataaaaccg agcaatccat ttacgaatgt ttgctgggtt tctgttttaa 120

caacattttc tgcgccgcca caaattttgg ctgcatcgac agttttcttc tgcccaattc 180

cagaaacgaa gaaatgatgg gtgatggttt cctttggtgc tactgctgcc ggtttgtttt 240

gaacagtaaa cgtctgttga gcacatcctg taataagcag ggccagcgca gtagcgagta 300

<210> 19

<211> 500

<212> DNA

<213> Artificial

<220>

<223> linker

<400> 19

cgatgggcaa ctcatgcaat tattgtgagc aatacacacg cgcttccagc ggagtataaa 60

tgcctaaagt aataaaaccg agcaatccat ttacgaatgt ttgctgggtt tctgttttaa 120

caacattttc tgcgccgcca caaattttgg ctgcatcgac agttttcttc tgcccaattc 180

cagaaacgaa gaaatgatgg gtgatggttt cctttggtgc tactgctgcc ggtttgtttt 240

gaacagtaaa cgtctgttga gcacatcctg taataagcag ggccagcgca gtagcgagta 300

gcattttttt catggtgtta ttcccgatgc tttttgaagt tcgcagaatc gtatgtgtag 360

aaaattaaac aaaccctaaa caatgagttg aaatttcata ttgttaatat ttattaatgt 420

atgtcaggtg cgatgaatcg tcattgtatt cccggattaa ctatgtccac agccctgacg 480

gggaacttct ctgcgggagt 500

<210> 20

<211> 1000

<212> DNA

<213> Artificial

<220>

<223> linker

<400> 20