Combined therapy for muscular diseases

Abstract

To provide a technique relating to the treatment of muscular diseases.SOLUTION: Provided is a GDF5 pathway-activating substance for use in a method of treating a muscular disease by gene therapy, the GDF5 pathway-activating substance being used in combination with another active ingredient suitable for treatment of the muscular disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the treatment of muscle disorders. [Background technology]

[0002] Myopathy is a disorder that affects the muscles and can cause weakness, pain, or even paralysis.

[0003] Muscular dystrophies are a group of muscle diseases that cause progressive weakening and destruction of skeletal muscles. Dystrophinopathy, in particular, is a condition caused by abnormalities in the DMD gene, which encodes a subsarcolemmal protein called dystrophin. Regarding large deletions, the most frequent genetic alteration, the severity of the phenotype primarily depends on the effect of the mutation on the protein reading frame of the dystrophin transcript. The dystrophin structure (a central rod domain composed of 24 spectrin-like repeats) tolerates large internal deletions (1), leading to the development of two major therapeutic strategies: classical gene therapy involving the transfer of functional microdystrophin cDNA into muscle, and targeted exon skipping. The present inventors previously demonstrated in International Publication No. 2016198676 that a two-step combination therapy for muscular dystrophy is more advantageous than treatment strategies known in the prior art. This combination therapy involves administering isolated AONs suitable for inducing exon skipping in dystrophin pre-mRNA and inducing muscle cells to produce mRNA transcripts encoding functional dystrophin protein. In a second step, this therapy involves administering to the same subject at least one viral vector encoding a Duchenne muscular dystrophy treatment. The viral vector (or therapeutic viral vector) is designed to restore dystrophin function in muscle cells. For example, the at least one viral vector capable of restoring dystrophin function in muscle cells is (i) an antisense oligonucleotide (also referred to as "AON-encoding virus" in the following description) that can induce exon skipping in dystrophin pre-mRNA and induce muscle cells to produce mRNA transcripts encoding functional dystrophin protein; (ii) designed to introduce into muscle cells a means for correcting the dystrophin gene in the genome of the muscle cell, for example, a genome editing means that implements one or more endonucleases specific to the dystrophin gene; or (iii) a viral vector encoding functional dystrophin protein.

[0004] It is shown herein that such strategies, and other strategies for treating not only muscular dystrophies but also other muscle diseases, would benefit from the administration of GDF5 pathway activators. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016198676 [Patent Document 2] International Publication No. 201308649 [Patent Document 3] International Publication No. 2016016449 [Patent Document 4] International Publication No. 2013 / 053928 [Patent Document 5] International Publication No. 11036640 [Patent Document 6] International Publication No. 13163628 [Patent Document 7] International Publication No. 14009567 [Patent Document 8] International Publication No. 14197748 [Patent Document 9] WO 02 / 29056 [Patent Document 10] International Publication No. 01 / 83695 [Patent Document 11] International Publication No. 08 / 088895 [Patent Document 12] International Publication No. 2011 / 113889 [Patent Document 13] International Publication No. 2006 / 021724 [Patent Document 14] International Publication No. 2017098187 [Non-patent literature]

[0006] [Non-Patent Document 1] Nakamura, K. et al. (1999) Exp. Cell Res. 250:351 [Non-patent document 2] Charbord et al., Stem Cells. 2013 September;31(9):1816-28 [Non-patent document 3] http: / / rulai.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi?process=home [Non-patent document 4] Collins and Morgan, lnt J Exp Pathol 84:165~172, 2003 [Non-Patent Document 5] Zincarelli Mol Ther. 2008 [Non-patent document 6] Schultz Mol Ther. 2008 Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, the present invention relates to a GDF5 pathway activator for use in a method for treating a muscular disease by gene therapy, the GDF5 pathway activator being used in combination with another active ingredient suitable for treating the muscular disease.

[0008] In a second aspect, the present invention relates to a kit comprising (i) a GDF5 pathway activator and (ii) an active ingredient suitable for treating a muscle disorder, the kit being useful for carrying out the therapeutic methods described herein. DETAILED DESCRIPTION OF THE INVENTION

[0009] The inventors herein show that GDF5 overexpression leads to an increase in muscle mass of both denervated and innervated muscles. This observation may be advantageously made in a method for treating a muscle disease, in which an active ingredient suitable for treating the muscle disease is administered to a subject in need thereof, and a GDF5 pathway activator is used in combination with said active ingredient to increase or stabilize muscle mass and / or muscle function.

[0010] GDF5 pathway activators In a particular embodiment, the GDF5 pathway activator is a GDF5 peptide, in particular a synthetic or recombinant GDF5, more particularly a recombinant GDF5, such as recombinant human GDF5. Unprocessed wild-type human GDF-5 (Uniprot accession number P43026) has the following sequence:

[0011] [ka]

[0012] (SEQ ID NO: 8)

[0013] SEQ ID NO:8 contains a signal peptide at amino acid positions 1-27, a propeptide at amino acid positions 28-381, and a portion at amino acid positions 382-501 that corresponds to the mature peptide, which is underlined in the sequence provided above.

[0014] Such a mature peptide therefore has the sequence shown in SEQ ID NO: 9 below: APLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO: 9).

[0015] Other recombinant human GDF5s are commercially available, such as the protein having the sequence shown in SEQ ID NO: 3 available from Thermo Fisher (catalog number RP-8663): APSATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO: 10)

[0016] In the context of the present invention, the peptide shown in SEQ ID NO: 9 or SEQ ID NO: 10 may be referred to as "reference recombinant human GDF5".

[0017] In another specific embodiment, the GDF5 pathway activator is a functional derivative of a GDF5 peptide. A functional derivative in the context of the present invention is a peptide that possesses at least one, particularly all, of the activities of the reference peptide. In the context of the present invention, a functional variant of a GDF5 peptide may have the ability to induce alkaline phosphatase production by ATDC5 mouse chondrogenic cells (Nakamura, K. et al. (1999) Exp. Cell Res. 250:351) with an ED50 of 0.01-10 μg / mL, e.g., 0.2-4 μg / mL, e.g., 0.2-1.2 μg / mL. In particular, a functional variant of a GDF5 peptide is a peptide that can treat or prevent sarcopenia in animal models or human subjects with conditions such as those provided in the experimental section of this application. GDF5 signaling can also be assessed by measuring SMAD1 / 5 / 8 phosphorylation, SMAD4 nuclear translocation, and Id-1 transcription, as provided in the experimental section below. The activity of a functional variant may be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% of the activity of the reference GDF5 peptide. In certain embodiments, the functional peptide has an activity that is greater than the activity of the reference GDF5 peptide, for example, at least 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or at least 150% of the activity of the reference GDF5 peptide. Additionally, in the present invention, functional variants of GDF5 peptides have at least 80% sequence identity with the reference GDF5 amino acid sequence, in particular at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the reference human recombinant GDF5.For example, a functional variant of a GDF5 peptide can contain 1 to 20 amino acid modifications (i.e., amino acid additions, deletions, or substitutions) compared to a reference recombinant human GDF5, such as 1 to 15 amino acid modifications, particularly 1 to 10 amino acid modifications, more particularly 1 to 6 amino acid modifications, and even more particularly 1, 2, 3, 4, 5, or 6 amino acid modifications compared to a reference recombinant human GDF5. Such functional variants of recombinant human GDF5 can be naturally occurring variants of GDF5. In certain embodiments, the functional variant is an optimized GDF5 peptide. Optimization can include various changes to the peptide, such as the amino acid modifications provided above, glycosylation, acetylation, phosphorylation, or the inclusion of at least one D-amino acid, e.g., at least two, at least three, at least four, or at least five D-amino acids. In another embodiment, the GDF5 peptide contains at least one unnatural amino acid, included by insertion, addition, or substitution with another amino acid in the GDF5 sequence. In yet another embodiment, the recombinant GDF5 may be fused to another moiety, such as another peptide moiety, which may, for example, stabilize the peptide.

[0018] In another specific embodiment, the substance is a functional variant of a GDF5 peptide corresponding to a GDF5-related protein described in WO201308649, which has improved affinity for BMP receptor IB (BMPR-IB) and / or decreased affinity for BMP receptor IA (BMPR-IA). In a specific embodiment, the protein is derived from human wild-type GDF5. In a specific embodiment, the GDF5-related protein is obtained by replacing at least one amino acid residue involved in the BMPR-IB and / or BMPR-IA binding site in the amino acid sequence of the GDF5 peptide, preferably by genetic engineering techniques. In a further embodiment, at least one hydrophobic amino acid in the BMPR-IB and / or BMPR-IA binding site of the GDF5 peptide is replaced with a hydrophilic or polar amino acid residue, for example, a hydrophilic or polar amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, serine, and threonine. In an alternative embodiment, at least one hydrophilic or polar amino acid in the BMPR-IB and / or BMPR-IA binding site of the GDF5 peptide is replaced with a hydrophobic amino acid, for example, a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In another alternative embodiment, the GDF5-related protein comprises a conservative substitution of at least one amino acid in the BMPR-IB and / or BMPR-IA binding site of the GDF peptide, particularly where a hydrophobic amino acid is replaced with a smaller or larger hydrophobic amino acid, or a hydrophilic or polar amino acid is replaced with a smaller or larger hydrophilic or polar amino acid. The regions of the GDF-5-related protein involved in binding to BMPR-IA and / or BMPR-IB are well known in the art or can be readily determined using methods within the ordinary skill in the art. With reference to the full-length unprocessed amino acid sequence of wild-type human GDF5 of SEQ ID NO: 8, certain embodiments realize the substitution of one or more of the following amino acids with any different amino acid: R399, any one of F409 to W417, in particular M412, G413, W414, and / or W417; any one of E434 to M456, in particular F435, P436, L437, R438, S439, H440, P443, N445, V448, I449, L452, M453, S455, and / or M456; S475, I476, F478, Any one of K488 to M493, particularly K488, Y490, and / or D492.

[0019] In certain embodiments, with reference to the full-length unprocessed amino acid sequence of SEQ ID NO: 8, one or more of the following amino acids are replaced with the specified amino acid: - R399 is replaced with V, L, I, M, F, Y, W, E, or D; - M412 is replaced by V, L, I, F, Y, W, H, K, or R; - W414 is replaced with R, K, F, Y, H, E, or D; - W417 is replaced with R, K, F, Y, H, E, or D; - F435 is replaced by V, L, I, M, P, Y, W, H, K, or R; - P436 is replaced with V, L, I, M, F, Y, or W; - L437 is replaced with D or E, - R438 is replaced by K, D, H, N, M, E, Q, S, T, Y, or W; - S439 is replaced with K, D, E, H, R, M, T, N, Q, Y, or W; - H440 is replaced by V, I, M, F, Y, W, E, or D; - P443 is replaced by V, L, I, M, F, Y, W, A, or S; - N445 is replaced with D, Q, H, F, L, R, K, M, S, Y, or W; - V448 is replaced by F, L, I, M, P, Y, or W; - I449 is replaced by F, L, V, M, P, Y, or W; - L452 is replaced with F, I, V, M, P, Y, or W; - M456 can be replaced with F, I, L, P, Y, W, S, T, N, Q, K, or D; - S475 may be replaced with M, T, N, Q, Y, or W; - K488 can be replaced with R, M, S, T, N, Q, Y, or W, - Y490 is replaced with E, H, K, R, Q, F, T, M, S, N, Q, or W; - D492 is replaced with G, E, M, S, T, N, Q, Y, W, H, K, or R; - I476 is replaced by G, A, V, L, M, F, Y, or W, - F478 can be replaced with G, A, V, L, I, Y, or W.

[0020] In another specific embodiment, with reference to the full-length unprocessed amino acid sequence of SEQ ID NO: 8, one or more of the following amino acids are replaced with the specified amino acid: R399 can be replaced with M or E, W414 is replaced with R, W417 can be replaced with R or F, R438 is replaced with K, S439 is replaced with K or E, I449 is replaced by V.

[0021] The corresponding position in the mature peptide (eg, SEQ ID NO: 9 or SEQ ID NO: 10) can be readily derived from the above information regarding unprocessed full-length wild-type human GDF-5.

[0022] In a particular embodiment of the invention, the agent is a GDF5 peptide whose amino acid sequence consists of SEQ ID NO: 9 or SEQ ID NO: 10. In another particular embodiment, the agent is a GDF5 peptide whose amino acid sequence consists of SEQ ID NO: 9 or SEQ ID NO: 10 with an additional N-terminal methionine residue. In another embodiment, the agent is a GDF5 peptide whose amino acid sequence consists of SEQ ID NO: 9 or SEQ ID NO: 10 in which the first alanine residue has been replaced with a methionine residue.

[0023] In a further specific embodiment, the GDF5 pathway activator is an agent that induces the CaVβ1-E / GDF5 axis. Within this embodiment, variations include the use of agents that are small chemical molecules. In a non-limiting variation of this embodiment, the GDF5 pathway activator is an inhibitor of NRSF (neuron-specific transcriptional repressor; also known as REST or RE1 repressive transcription factor).

[0024] In a particular embodiment, the GDF5 pathway activator as the NRSF inhibitor is valproic acid. In a further particular embodiment, the GDF5 pathway activator is an NRSF inhibitor disclosed in Charbord et al., Stem Cells. 2013 Sep;31(9):1816-28, in particular 2-(2-hydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid allyloxy-amide (X5050), 2-thiophen-2-yl-1H-benzimidazole-5-carboxylic acid (2-ethyl-hexyl)-amide (X5917), 3-[1-(3-bromo-phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]-1-{4-[5-(mol 3-[1-(2,5-difluoro-phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]-1-{4-[5-(morpholine-4-carbonyl)-pyridin-2-yl]-2-phenyl-piperazin-1-yl}-propan-1-one (X38210), or 3-[1-(2,5-difluoro-phenyl)-3,5-dimethyl-1H-pyrazol-4-yl]-1-{4-[5-(morpholine-4-carbonyl)-pyridin-2-yl]-2-phenyl-piperazin-1-yl}-propan-1-one (X38207) molecule, more particularly the X5050 molecule disclosed therein.

[0025] In yet another embodiment, the GDF5 pathway activator is a vector comprising a nucleic acid encoding GDF5, such as human GDF5 or a functional variant thereof. In certain embodiments, the vector is a plasmid vector or a viral vector, such as an adenoviral vector or an adeno-associated viral (AAV) vector. In certain embodiments, the viral vector is suitable for transducing muscle cells and / or neuronal cells. In more specific embodiments, such a viral vector suitable for transducing muscle cells and / or neuronal cells is an AAV vector, for example, an AAV vector having an AAV2 / 2, AAV2 / 6, AAV2 / 8, AAV2 / 9, or AAV2 / 10 capsid. In further specific embodiments, the GDF5 coding sequence may be under the control of regulatory sequences, such as a promoter, an enhancer, a repressor, and a polyadenylation signal. In certain embodiments, the vector comprises an expression cassette comprising, in this order, a promoter, a GDF5 coding sequence, and a polyadenylation signal. The promoter may be ubiquitous or tissue-selective. In certain embodiments, the promoter is the native promoter of the GDF5 gene, such as the promoter of the human GDF5 gene.

[0026] In another specific embodiment, the GDF5 pathway activator is an agent that increases the activity or expression of GDF5.

[0027] In a further embodiment, the GDF5 pathway activator is a vector comprising a nucleic acid encoding CaVβ1-E, such as human CaVβ1-E. In certain embodiments, the vector is a plasmid vector or a viral vector, such as an adenovirus vector or an adeno-associated virus vector. Thus, the present invention also relates to a viral vector, such as the adenovirus or AAV vector described above, comprising a CaVβ1-E coding sequence. The CaVβ1-E coding sequence may be under the control of regulatory sequences, such as a promoter, an enhancer, a repressor, and a polyadenylation signal. In certain embodiments, the vector comprises an expression cassette comprising, in this order, a promoter, a CaVβ1-E coding sequence, and a polyadenylation signal. The promoter may be ubiquitous or tissue-selective. In certain embodiments, the promoter is the native promoter of the CaVβ1-E gene, such as the promoter of the human CaVβ1-E gene.

[0028] In certain embodiments, the GDF5 pathway activator is either (i) a vector, such as an AAV vector, comprising a nucleic acid encoding GDF5, such as human GDF5 or a functional variant thereof, or (ii) recombinant GDF5, such as recombinant human GDF5 or a functional variant thereof, as disclosed above. In yet another particular embodiment, the GDF5 pathway activator is recombinant GDF5, such as recombinant human GDF5 or a functional variant thereof.

[0029] In certain embodiments, the GDF5 pathway activator is administered separately, sequentially, or simultaneously with other active ingredients. For example, administration can be simultaneous (e.g., because the agent and ingredient are contained in the same composition, or because two different compositions, each containing the agent and active ingredient, respectively, are administered simultaneously) or separated by hours, days, weeks, or months. In another specific embodiment, the GDF5 pathway activator is a vector, such as a plasmid vector or a viral vector, particularly an AAV vector, and the agent is administered only once. In further embodiments, the GDF5 pathway activator is a recombinant protein as described above in each of the above embodiments, and the recombinant GDF5 protein can be administered only once, e.g., before, during, or after the administration of the other active ingredients, to increase muscle mass and / or function before, during, or after the other active ingredients can act. In another embodiment, the GDF5 pathway activator is a recombinant protein as described above in each of the above embodiments, and the recombinant GDF5 protein can be administered several times to maintain adequate muscle mass and / or function throughout the treatment protocol. For example, administration of GDF5 recombinant protein may be carried out at least once a year, for example at least once every six months, for example at least once a quarter, for example at least once a month, for example at least once a week, for example at least once a day.

[0030] Muscle disorders and active ingredients suitable for their treatment The present invention relates to the treatment of muscle disorders. Genetic disorders are among the muscle disorders that may benefit from the present invention. In certain embodiments, the muscle disorder is a neuromuscular disorder or a musculoskeletal disorder.

[0031] In certain embodiments, the muscle disease is a motor neuron disease such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA).

[0032] In a specific embodiment, the present invention relates to a treatment for ALS, wherein an active ingredient suitable for treating ALS is an AON targeting human SOD1 pre-mRNA, and the AON is suitable for inducing exon skipping in the pre-mRNA. Those skilled in the art will recognize which strategies are relevant for treating ALS. For example, see the disclosure of International Publication No. 2016016449. In a specific embodiment, the AON is encoded by a nucleotide sequence of interest introduced into a vector, such as a plasmid or viral vector, e.g., an AAV vector. General information useful for designing AONs is available to those skilled in the art and is provided below for AONs targeting dystrophin pre-mRNA; the same principles apply to the design of AONs targeting SOD1 pre-mRNA.

[0033] In a further particular embodiment, the present invention relates to the treatment of spinal muscular atrophy, wherein the active ingredient is a vector, such as a plasmid or viral vector, for example an AAV vector, comprising a gene encoding a motor neuron survival protein, such as the SMN1 or SMN2 gene.

[0034] In yet another embodiment, the muscle disease is a muscular atrophy, such as a muscular dystrophy. Non-limiting examples of muscular dystrophies that can be treated by the present invention include distal myopathy, glycogen storage disease type VII, limb-girdle muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, and myotonic dystrophy.

[0035] In a specific embodiment, the muscular disease to be treated is Duchenne muscular dystrophy. In a further specific embodiment, the active ingredient for treating DMD is that disclosed in International Publication No. 2016198676, in which a two-step combination therapy for muscular dystrophy is performed. In this embodiment, an isolated AON suitable for inducing exon skipping in dystrophin pre-mRNA and inducing muscle cells to produce mRNA transcripts encoding functional dystrophin protein is first administered. In the second step, the same subject is administered at least one viral vector encoding a DMD therapeutic. The viral vector (or therapeutic viral vector) is designed to restore dystrophin function in muscle cells. For example, at least one viral vector capable of restoring dystrophin function in muscle cells is: (i) encoding an antisense oligonucleotide capable of inducing exon skipping in the dystrophin pre-mRNA and inducing muscle cells to produce mRNA transcripts encoding functional dystrophin protein (also referred to as "AON-encoding viruses" in the following description); or (ii) engineered to introduce into the muscle cell a means for correcting the dystrophin gene in the genome of said muscle cell, for example, a genome editing means implementing one or more endonucleases specific for the dystrophin gene; or (iii) encodes a functional dystrophin protein It is a viral vector.

[0036] The terms "antisense oligonucleotide" and "AON" are used interchangeably and refer to a single-stranded nucleic acid sequence, e.g., a DNA or RNA sequence, that is complementary to a portion of a pre-mRNA encoding a dystrophin protein and can therefore form a heteroduplex within the target sequence by Watson-Crick base pairing. In particular, the AONs of the present invention are designed to block splice acceptor (SA) sites, exon splicing enhancers (ESEs), branch points in the dystrophin pre-mRNA, and / or splice donor (SD) sites, and / or any sequences that may regulate pre-mRNA splicing. That is, the AONs of the present invention are designed to be complementary to portions of the dystrophin pre-mRNA that contain SAs, ESEs, branch point sequences, SDs, and / or any sequences that may regulate pre-mRNA splicing (14, 15). In certain embodiments, the targeted sequence within the dystrophin pre-mRNA may include the 3' or 5' splice site or branch point of the pre-mRNA. Target sequences may be within exons or introns, or may overlap intron-exon or exon-intron junctions. Splice site target sequences may include mRNA sequences having a 5' end from 1 to about 50 base pairs downstream from the normal splice acceptor junction in the unprocessed mRNA. Preferred target sequences for splicing are any region of the pre-mRNA that includes a splice site and / or is contained entirely within the exon coding sequence and / or spans the splice acceptor and / or donor site. Of course, target sequences may include more than one of these sequences capable of modulating pre-mRNA splicing, and several such target sequences can be combined to achieve a desired effect.

[0037] Tools are available for identifying SA, ESE, SD, and branchpoint sequences in a pre-mRNA of interest. As is well known to those skilled in the art, SA is a conserved sequence located at the 3' end of an intron and terminates the intron with the almost invariant AG sequence. SD is a conserved sequence located at the 5' end of an intron and initiates the intron with the almost invariant GT sequence. In addition, the ESEfinder software tool (http: / / rulai.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi?process=home) can be used to predict ESE motifs in exon sequences intended to be skipped. AON design can then be performed according to the principles published in Aartsma-Rus et al. (16).

[0038] The AONs of the present invention are designed to complement appropriate sequences within the dystrophin pre-mRNA required for accurate splicing of the targeted exon, thereby blocking the splicing reaction that incorporates the targeted exon into the mature mRNA.

[0039] Mutated human dystrophin genes do not express any measurable dystrophin in the muscles of patients with Duchenne muscular dystrophy. To treat this condition, the antisense oligonucleotides of the present invention typically hybridize to selected regions of the pre-mRNA of the mutated human dystrophin gene, inducing exon skipping in the dystrophin mRNA, thereby causing muscle cells to produce mRNA transcripts encoding functional dystrophin protein. The exon skipping strategy converts out-of-frame mutations into in-frame mutations, resulting in partially functional dystrophin with an internal deletion. Thus, depending on the exon skipped, the rescued protein can, in the best case, improve the dystrophic phenotype toward a milder Becker-like phenotype. In certain embodiments, the resulting dystrophin protein is not necessarily a "wild-type" form of dystrophin, but rather a truncated form, but still functional or semi-functional dystrophin. By increasing the levels of functional dystrophin protein in muscle cells, these and related embodiments may be useful in the prevention and treatment of Duchenne muscular dystrophy. The combination therapies described herein offer important practical advantages over alternative methods of treating Duchenne muscular dystrophy.

[0040] "Exon skipping" generally refers to the process by which an entire exon, or a portion thereof, is removed from a given pre-mRNA, thereby excluding it from being present in the mature mRNA. Thus, the portion of the protein that would otherwise be encoded by the skipped exon is absent in the expressed form of the protein, which typically produces an altered, but still functional, form of the protein. In certain embodiments, the skipped exon is an aberrant exon from the human dystrophin gene, which may contain a mutation or other alteration in its sequence. In certain embodiments, the skipped exon is any one or more of exons 1-79 of the dystrophin gene, although any one or more of exons 23, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and / or 55 of the human dystrophin gene are preferred. Depending on the location of the DMD patient mutation, one or several exons are selected for skipping to restore the dystrophin coding frame and generate a partially functional dystrophin with an internal deletion. "Dystrophin" is a rod-shaped cytoplasmic protein that is an integral part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix via the cell membrane. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin-binding domain approximately between amino acids 14 and 240 and a central rod domain approximately between amino acids 253 and 3040. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids that share homology with alpha-actinin and spectrin. Repeats are typically separated by four proline-rich non-repeat segments, also known as hinge regions. Repeats 15 and 16 are separated by an 18-amino acid stretch that is thought to provide the primary site for proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10 to 25%. One repeat contains three alpha-helices 1, 2, and 3.Alpha-helices 1 and 3 are each formed by seven helical turns that interact as a coiled coil, presumably via a hydrophobic interface. Alpha-helix 2 has a more complex structure, formed by four- and three-helical turn segments separated by glycine or proline residues. Each repeat is encoded by two exons, typically separated by an intron between amino acid positions encoding amino acids 47 and 48 in the first part of alpha-helix 2. Other introns are found at various positions in the repeat-encoding region, typically dispersed within helix 3. Dystrophin also contains a cysteine-rich domain approximately between amino acids 3080 and 3360, including a cysteine-rich segment (i.e., 15 cysteines out of 280 amino acids) that shows homology to the C-terminal domain of slime mold (Dictyostelium discoideum) alpha-actinin. The carboxy-terminal domain is located approximately between amino acids 3361 and 3685.

[0041] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the muscle cell membrane. DAPC contains dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal inherited muscular dystrophies. In the absence of dystrophin, the DAPC becomes unstable, leading to a decrease in the levels of its constituent proteins, which in turn causes progressive fiber damage and membrane leakage. In various forms of muscular dystrophies, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin or no dystrophin at all, primarily due to mutations in the gene sequence. Predominant expression of defective dystrophin protein or the complete absence of dystrophin or dystrophin-like proteins leads to the rapid progression of muscle degeneration, as shown above. In this regard, a "defective" dystrophin protein can be characterized by the form of dystrophin produced in certain subjects with DMD or BMD known in the art, or by the absence of detectable dystrophin.

[0042] As used herein, terms such as "function" and "functionality" refer to biological, enzymatic, or therapeutic functions. A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to inhibit the progressive degradation of muscle tissue, typically compared to an altered or "defective" form of dystrophin present in a particular subject with DMD or BMD, otherwise characteristic of muscular dystrophy. In certain embodiments, a functional dystrophin protein can have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the in vitro or in vivo biological activity of wild-type dystrophin (including all integers therebetween), as measured according to routine techniques in the art. As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (17). Animal models are also valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activities. The two most widely used animal models for DMD research are the dystrophin-negative mdx mouse and the Golden Retriever Muscular Dystrophy (GRMD) dog (see, e.g., Collins and Morgan, lnt J Exp Pathol 84:165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins, including truncated forms of dystrophin, such as those produced by some of the exon-skipping antisense compounds of the present invention.

[0043] The isolated AONs of the present invention can be of any suitable type. Exemplary types of AONs include oligodeoxyribonucleotides, oligoribonucleotides, morpholinos (e.g., phosphorodiamidate morpholinos (PMOs) or peptide-phosphorodiamidate morpholinos (PPMOs)), 2'-O-methyl-phosphorothioates (2'OMePS), 2'-O-2-methoxyethyl-antisense oligonucleotides, tricyclo-DNA-antisense oligonucleotides, tricyclo-phosphorothioate DNA oligonucleotides, LNAs, small nuclear RNA-modified AONs, such as U7-, U1-, or U6-modified AONs (or other UsnRNPs), or conjugated products thereof, such as peptide-conjugated or nanoparticle-conjugated AONs.

[0044] In particular, for in vivo use, AONs can be stabilized, for example, via phosphate backbone modifications. For example, stabilized AONs of the present invention can have modified backbones, such as phosphorothioate linkages. Other possible stabilizing modifications include phosphodiester modifications, combinations of phosphodiester and phosphorothioate modifications, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. Chemically stabilized modified forms of AONs also include "morpholinos" (phosphorodiamidate morpholino oligomers, PMOs), 2'-O-methyl oligomers, tricyclo-DNA, tricyclo-DNA-phosphorothioate AON molecules (WO 2013 / 053928), or U small nuclear (sn)RNA. The latter forms of AONs that can be used to this effect can bind to molecules of small nuclear RNAs such as U1, U6, or U7 (or other UsnRNPs).

[0045] In certain embodiments, the isolated AON used in the present invention is a 2'OMePS oligonucleotide or a PPMO oligonucleotide. Preferably, the AON is a PPMO oligonucleotide.

[0046] AONs used in the practice of the present invention are generally about 10 to about 40 nucleotides in length, and can be, for example, about 10, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40 nucleotides in length, or more, depending on the targeting sequence within the dystrophin pre-mRNA and the AON chemistry.

[0047] Exemplary AONs for practicing the present invention result in exon 51 skipping or exon 53 skipping of human dystrophin pre-mRNA. In more specific embodiments, AONs for practicing the present invention result in exon 51 skipping of human dystrophin pre-mRNA. In another specific embodiment, an AON for practicing the present invention can be CATTCAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTGTAC (SEQ ID NO: 1) and can result in exon 53 skipping of human dystrophin pre-mRNA.

[0048] Of course, any AON having the properties described above can be used in the practice of the present invention.

[0049] In one embodiment, the isolated AON of the invention has the sequence set forth in SEQ ID NO: 1 and is a PPMO oligonucleotide.

[0050] For stable and efficient in vivo delivery, the isolated AONs used in the practice of the present invention may also be fused to or co-administered with any cell-penetrating peptide and signal peptide that mediates protein secretion. The cell-penetrating peptide may be the RVG peptide (18), PiP (19), e.g., Pip6a-PMO (20), P28 (21), or a protein transduction domain such as TAT ​​(22) or VP22 (23). In certain embodiments, the isolated AON is a PPMO oligonucleotide, i.e., a PMO oligonucleotide fused to a peptide moiety, more particularly, Pip6a-PMO, and even more particularly, Pip6a-PMO. In certain embodiments, the PPMO oligonucleotide moiety comprises or consists of the sequence set forth in SEQ ID NO:1. In further specific embodiments, the isolated AON is a Pip6a-PMO, the oligonucleotide moiety comprises or consists of the sequence set forth in SEQ ID NO:1.

[0051] Additionally, the isolated AONs used in the practice of the present invention can be administered in compositions further comprising a pharmaceutically acceptable carrier and an agent that improves the efficiency of oligonucleotide delivery, including, but not limited to, F127 (24).

[0052] In certain embodiments of the invention, the isolated AON of the invention is capable of inducing functional dystrophin expression that is at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably 50%, more preferably at least 51%, 52%, 53%, 54%, 55%, or at least 56% compared to the normal expression level of wild-type dystrophin. Of course, higher functional dystrophin expression is also preferred, for example, expression that is at least 60%, 70%, 80%, or even at least 90% compared to the normal expression level of wild-type dystrophin.

[0053] In a second step of the method invention, a viral vector encoding a therapeutic is also administered to the same patient in need of treatment. In the context of the present invention, the therapeutic is capable of restoring dystrophin function in muscle cells in need thereof.

[0054] In certain embodiments, the therapeutic-encoding viral vector is an AON-encoding virus, where the AON encoded by this virus is as defined above and is capable of inducing exon skipping in the dystrophin pre-mRNA and inducing muscle cells to produce mRNA transcripts encoding functional dystrophin protein.

[0055] In another specific embodiment, one or several viral vectors encode a means for correcting the dystrophin gene in the genome of muscle cells. In this embodiment, a viral vector capable of restoring dystrophin function in muscle cells is designed to correct the mutant dystrophin gene in a subject by introducing a genome editing system into the genome of the cell. For example, a site-specific nuclease may be encoded by the viral vector, thereby restoring the expression of a fully functional or partially functional dystrophin protein using a repair template or donor DNA that can replace the entire dystrophin gene or the region containing the mutation. The site-specific nuclease may be used to introduce a site-specific double-strand break into the targeted genomic locus. The site-specific double-strand break occurs when the site-specific nuclease binds to the target DNA sequence, thereby allowing the target DNA to be cut. This DNA break can stimulate natural DNA repair mechanisms to trigger one of two possible repair pathways: homology-directed repair (HDR) or non-homologous end joining (NHEJ). This embodiment may include introducing into muscle cells a genome editing tool that implements one or more endonucleases specific to the dystrophin gene (e.g., one or more meganucleases, TALENs, ZFNs, or CRISPR / Cas9 endonucleases) and one or more repair matrices. Such systems are described, for example, in WO 11036640, WO 13163628, WO 14009567, and WO 14197748, and are known to those skilled in the art.

[0056] In another embodiment, the therapeutic encoded by the viral vector is functional dystrophin. As discussed above, a "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to inhibit the progressive degradation of muscle tissue, typically compared to an altered or "defective" form of dystrophin protein present in a particular subject with DMD or BMD, otherwise characteristic of muscular dystrophy. In certain embodiments, a functional dystrophin protein can have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the in vitro or in vivo biological activity of wild-type dystrophin (including all integers therebetween), as measured according to routine techniques in the art. A functional dystrophin protein can be a truncated dystrophin protein, such as a mini- or micro-dystrophin. Such mini- and micro-dystrophins are known in the art, e.g., WO 02 / 29056, WO 01 / 83695, WO 08 / 088895, and Foster et al., 2008 (37). In certain embodiments, the micro-dystrophin is a ΔAB / R3-R18 / ΔCT or ΔR4-R23 / ΔCT micro-dystrophin (more particularly, a ΔR4-R23 / ΔCT micro-dystrophin), such as the ΔAB / R3-R18 / ΔCT or ΔR4-R23 / ΔCT micro-dystrophin described in Foster et al., 2008, more particularly, a ΔR4-R23 / ΔCT micro-dystrophin. In further particular embodiments, the mini- or micro-dystrophin-encoding gene is codon-optimized. In a further particular embodiment, the mini- or micro-dystrophin-encoding gene is codon-optimized and encodes the ΔAB / R3-R18 / ΔCT or ΔR4-R23 / ΔCT micro-dystrophin, particularly the ΔR4-R23 / ΔCT micro-dystrophin described in Foster et al., 2008. The corresponding coding sequences are set forth in SEQ ID NOs: 6 and 7, respectively.

[0057] Adeno-associated viral vector (AAV)-mediated delivery of microdystrophin to dystrophin-deficient mice with DMD has shown remarkable efficiency ( 25 , 26 , 27 ), leading to the initiation of early clinical trials ( 28 ).

[0058] Viral vectors include, but are not limited to, adenoviruses; parvoviruses such as adeno-associated viruses; and non-integrating viral vectors (or vectors that integrate into the genome of target cells with low efficiency), such as episomal vectors, including SV40-type viruses. Other vectors that are not named but are known in the art can be easily used. Among vectors that have been validated for clinical applications and can be used to deliver antisense sequences, AAV shows relatively high potential for exon skipping strategies.

[0059] In a preferred embodiment, the viral vector is a parvovirus, particularly an AAV vector. The parvovirus, adeno-associated virus (AAV), is a naturally replication-defective virus that can integrate into the genome of infected cells and establish latent infection. This latter feature is thought to be unique among mammalian viruses because integration occurs at a specific site in the human genome, designated AAVS1, located on chromosome 19 (19q13.3-qter). AAV-based recombinant vectors lack Rep proteins, integrate with low efficiency, and exist primarily as stable, circular episomes that can persist in target cells for months, or even years. Therefore, AAV has attracted considerable interest as a potential vector for human gene therapy. Its lack of association with any human disease and the wide range of cell lines derived from various tissues that it can infect are favorable characteristics of this virus. Indeed, 12 AAV serotypes (AAV1-12) and up to 120 variants, each with distinct tissue tropism, are known (29, 30). Thus, the present invention relates to an AAV vector encoding the AON described above, which is configured to target dystrophin pre-mRNA, induce exon skipping in the human pre-mRNA, and induce the production of functional dystrophin protein in muscle cells. In a specific embodiment, the AAV genome is derived from AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 serotype. In a preferred embodiment, the AAV capsid is derived from AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 serotype or an AAV variant. In a further specific embodiment, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are derived from AAVs of different serotypes. For example, a pseudotyped AAV vector can be a vector whose genome is derived from AAV2 serotype and whose capsid is derived from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Additionally, the genome of an AAV vector can be a single-stranded or self-complementary double-stranded genome (31).Self-complementary double-stranded AAV vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genomes are half the length of the wild-type AAV genome, tend to package DNA dimers.

[0060] Preferably, the AAV vector implemented in the practice of the present invention is a vector that targets muscle cells. In particular, AAV1, 6, 8, and 9 exhibit high tropism for striated muscle (Zincarelli Mol Ther. 2008, Schultz Mol Ther. 2008), and are particularly preferred. In a preferred embodiment, the AAV vector has an AAV1, 6, 8, or 9 capsid, and this vector is optionally a pseudotyped vector.

[0061] In certain embodiments, the AON encoded by the AON-encoding vector described above is linked to a small nuclear RNA molecule, such as U1, U2, U6, U7, or any other small nuclear RNA (snRNA), or a chimeric small nuclear RNA (SNRNA) (32, 33). Information regarding U7 modifications can be found, inter alia, in Goyenvalle et al. (34), WO 2011 / 113889, and WO 2006 / 021724. In certain embodiments, the U7 cassette described by D. Schumperli is used (35). It contains the native U7 promoter (positions -267 to +1), the U7smOpt snRNA, and downstream sequences up to position 116. The 18-nt native sequence complementary to the histone pre-mRNA in U7 snRNA is replaced with one or two (either the same sequence used twice or two different sequences) or more repeats of a selected AON sequence, using, for example, PCR-mediated mutagenesis, as previously described ( 34 ).

[0062] In certain embodiments, the U7-modified AON comprises the sequence set forth in SEQ ID NO: 2: CATTCAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTGTAC (SEQ ID NO: 2) and confers exon 53 skipping in the dystrophin pre-mRNA. More particularly, the U7-modified AON that confers exon 53 skipping in the dystrophin pre-mRNA is as disclosed in WO2017098187.

[0063] In certain embodiments, small nuclear RNA-modified AONs, particularly U7-modified AONs, are carried on AAV vectors.

[0064] Typically, viral vectors, particularly functional dystrophin-encoding vectors or AON-encoding vectors, may also contain regulatory sequences, such as promoters, enhancers, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTDs), and the like, that enable expression of the encoded functional dystrophin or AON. In this regard, the vector most preferably contains a promoter region operably linked to the coding sequence to induce or improve expression of the AON. Such promoters may be ubiquitous promoters, tissue-specific promoters, strong promoters, weak promoters, regulated promoters, chimeric promoters, and the like, enabling efficient and suitable production of AONs. The promoter may be a cellular promoter, a viral promoter, a fungal promoter, a plant promoter, or a synthetic promoter. The most preferred promoters for use in the present invention are those that are functional in muscle cells. Non-limiting examples of muscle-specific promoters include the desmin promoter, the C5-12 synthetic promoter, and the muscle creatine kinase (MCK) promoter. For embodiments involving expression of AONs, the promoter can be selected from small nuclear RNA promoters, such as U1, U2, U6, U7, or other small nuclear RNA promoters, or chimeric small nuclear RNA promoters. Other representative promoters include RNA polymerase III-dependent promoters, such as the H1 promoter, or RNA polymerase II-dependent promoters. Examples of regulated promoters include, but are not limited to, promoters containing Tet on / off elements, rapamycin-inducible promoters, and metallothionein promoters. Examples of ubiquitous promoters include viral promoters, particularly CMV promoters, RSV promoters, SV40 promoters, hybrid CBA (chicken beta actin / CMV) promoters, and cellular promoters, such as PGK (phosphoglycerate kinase) or EF1 alpha (elongation factor 1 alpha) promoters.

[0065] A practitioner can use a composition comprising the isolated AON or viral vector described above in a pharmaceutically acceptable carrier. In addition to the AON or virus, the pharmaceutical composition of the present invention can also contain a pharmaceutically or physiologically acceptable carrier, such as saline, sodium phosphate, etc. The composition will generally be in liquid form, although this need not always be the case. Suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl and propyl hydroxybenzoates, mineral oil, etc. The formulation can also include lubricants, wetting agents, emulsifiers, preservatives, buffers, etc. Notably, the present invention involves the administration of isolated AONs or AON-encoding viruses and thus is somewhat similar to gene therapy. Those skilled in the art will recognize that nucleic acids are often delivered in association with lipids (e.g., cationic or neutral lipids, or mixtures thereof), often in the form of liposomes or other suitable micro- or nanostructured materials (e.g., micelles, lipid complexes, dendrimers, emulsions, cubic phases, etc.).

[0066] The compositions of the present invention are generally administered via the enteral or parenteral route, for example, intravenously (iv), intraarterially, subcutaneously, intramuscularly (im), intracerebrally, intracerebroventricularly (icv), intrathecally (it), intraperitoneally (ip), although other types of administration are not excluded.

[0067] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Delivery can be local (i.e., in situ, i.e., directly to tissues such as muscle tissue) or systemic, but typically delivery will be local to affected muscle tissues, such as skeletal muscle, smooth muscle, cardiac muscle, etc. Depending on the form of AON or viral vector administered and the tissue or cell type targeted, techniques such as electroporation, sonoporation, or "gene gun" (delivery of nucleic acid-coated gold particles) can be used.

[0068] Those skilled in the art will recognize that the amount of isolated AON or viral vector administered will be sufficient to induce remission of undesirable muscular dystrophy symptoms. Such amounts may vary depending on factors such as the patient's sex, age, weight, and general health, among others, and can be determined on an individual basis. Amounts may also vary according to other components of the treatment protocol (e.g., administration of other medications). Generally, suitable doses range from about 1 mg / kg to about 100 mg / kg, more usually from about 2 mg / kg / day to about 10 mg / kg. In the case of viral-based delivery of AONs, suitable doses will depend on various factors, such as the virus used and the route of delivery (intramuscular, intravenous, intraarterial, or other), but will typically range from 10e9 to 10e15 viral particles / kg. Those skilled in the art will recognize that such parameters will usually become apparent during clinical trials. Furthermore, those skilled in the art will recognize that, while disease symptoms may be completely alleviated by the treatments described herein, this need not be the case. Even partial or intermittent relief of symptoms may be of significant benefit to the recipient. Additionally, patient treatment can be a single event, or the patient can receive multiple doses of AONs and / or viral vectors, with doses spaced days, weeks, months, or even years apart, depending on the results obtained.

[0069] Combination therapy using GDF5 pathway activators As noted above, the present invention relates to GDF5 pathway activators for use in combination with another active ingredient in methods for the treatment of muscular disorders by gene therapy.

[0070] The present invention therefore defines a combination comprising a GDF5 pathway activator and another active ingredient suitable for the treatment of a muscle disorder.

[0071] In certain embodiments, the combination comprises a vector, such as a viral vector, particularly an AAV vector, encoding GDF5, eg, one of the GDF5 proteins disclosed above.

[0072] In yet another embodiment, the combination comprises recombinant GDF5, in particular recombinant human GDF5, such as one of the GDF5 proteins disclosed above.

[0073] In another embodiment, the combination comprises an active ingredient suitable for treating a muscle disease, e.g. - AONs suitable for exon skipping in SOD1 pre-mRNA for the treatment of ALS; - a vector, such as a viral vector, in particular an AAV vector, comprising a gene encoding an SMN protein, such as the SMN1 or SMN2 gene, more particularly the SMN1 gene; - a vector, such as a viral vector, in particular an AAV vector, comprising a gene encoding a functional dystrophin as described above for the treatment of DMD, or - (i) an AON suitable for exon skipping in dystrophin pre-mRNA as described above or a vector encoding the same, and (ii) a vector such as a viral vector, in particular an AAV vector, comprising a gene encoding functional dystrophin as described above for the treatment of DMD. Includes:

[0074] In further specific embodiments, the components of the combination are for sequential, separate, or simultaneous use. For example, the GDF5 pathway activator may be administered before, during, or after administration of the gene therapy vector or AON.

[0075] For example, a GDF5 pathway activator can be administered prior to administration of an AON, such as an AON for use in treating ALS or DMD.

[0076] For example, a GDF5 pathway activator can be administered during administration of an AON, such as an AON for use in treating ALS or DMD. In this embodiment, the GDF5 pathway activator and the AON can be in the same composition or in different compositions.

[0077] In another example, a GDF5 pathway activator can be administered following administration of an AON, such as an AON for use in treating ALS or DMD.

[0078] In another embodiment, a GDF5 pathway activator may be administered prior to administration of a gene therapy vector for use in treating a muscle disease, such as the gene therapy vectors described above for the treatment of SMA or DMD.

[0079] In certain embodiments, the GDF5 pathway activator can be administered during the administration of a gene therapy vector, e.g., a gene therapy vector for use in treating a muscle disease, e.g., a gene therapy vector for the treatment of SMA or DMD, as described above. In this embodiment, the GDF5 pathway activator and the gene therapy vector can be in the same composition or in different compositions.

[0080] In another specific embodiment, the GDF5 pathway activator may be administered following administration of a gene therapy vector for use in treating a muscle disease, such as the gene therapy vectors described above for the treatment of SMA or DMD.

[0081] Details regarding the treatment of DMD according to the two-step method disclosed above are provided below, however, one skilled in the art will readily adapt the treatment strategy to other active ingredients and diseases due to the information provided herein and common general knowledge in the field of gene therapy.

[0082] Treatment for Duchenne muscular dystrophy is - first, an isolated antisense oligonucleotide that is complementary to a portion of the dystrophin pre-mRNA and is capable of inducing exon skipping during the processing of this pre-mRNA into mRNA; and - second, a viral vector encoding a Duchenne muscular dystrophy therapeutic, for example, (i) encoding an antisense oligonucleotide capable of inducing exon skipping in dystrophin pre-mRNA, (ii) encoding a dystrophin gene editing means, or (iii) encoding functional dystrophin. This may involve two-step administration of

[0083] In certain embodiments, the GDF5 pathway activator may be administered prior to the first step.

[0084] In a further particular embodiment, a GDF5 pathway activator may be administered during the first step.

[0085] In another embodiment, the GDF5 pathway activator may be administered between the first and second steps.

[0086] In another embodiment, the GDF5 pathway activator may be administered after the second step.

[0087] In a further particular embodiment, a GDF5 pathway activator may be administered during the second step.

[0088] In certain embodiments, the therapeutic is a U7-modified AON, particularly a U7-modified AON comprising the sequence set forth in SEQ ID NO:2 that targets exon 53 of the dystrophin gene.

[0089] As described above, the first administration of isolated AONs is likely to induce sufficient functional dystrophin expression in muscle cells to ensure membrane integrity, thus limiting the loss of viral vectors, particularly AON-encoding viruses (e.g., AAV), dystrophin-correcting viruses (e.g., AAV), or functional dystrophin-encoding viruses (e.g., AAV), due to repeated cycles of necrosis-regeneration of otherwise dystrophic muscle fibers.

[0090] The period between the injection of the isolated AON and the injection of the viral vector can vary depending on many factors, such as the stage of the disease, the age or condition of the patient, and the dosage of the therapy. In either event, the time between the first and second steps of the method is sufficient to realize the long-lasting benefits of viral vector treatment. The method allows for the maintenance of a high viral therapeutic genome content and improved transgene expression in dystrophic muscle. The result of these early events is a stronger therapeutic benefit of AAV-based therapy that lasts longer than non-combined therapy. Thus, the time between the first and second steps of the method of the invention may be from 1 to 40 days, for example at least 1 day or more than 2 days, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days; or at least 1 week or more than 2 weeks, for example at least 1, 2, 3, 4, or 5 weeks. In particular embodiments, the period between both administrations is 2 weeks (i.e., about 12-16 days, e.g., about 12, 13, 14, 15, or 16 days), about 3 weeks (i.e., about 19-23 days, e.g., about 19, 20, 21, 22, or 23 days), or about 4 weeks (i.e., about 26-30 days, e.g., about 26, 27, 28, 29, or 30 days). More particularly, this period is comprised between 14 and 28 days, more particularly 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In another embodiment, the period between both administrations is about 14 (i.e., 13, 14, or 15 days, more particularly 14 days), 21 (i.e., 20, 21, or 22 days, more particularly 21 days), or 28 days (i.e., 27, 28, or 29 days, more particularly 28 days).

[0091] Further aspects and advantages of the present invention will be disclosed in the following experimental section, which is intended to be merely illustrative and not limiting the scope of this application. [Brief explanation of the drawings]

[0092] [Figure 1] This figure shows that the viral genome is efficiently maintained in Pip6a-PMO-rescued mdx muscle. (a) TAs from mdx and wt mice were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before injection of 1E+11vg of non-therapeutic AAV1-U7scr vector (day 0, d0). Control mdx and wt TAs were injected with the AAV1-U7scr vector alone. Four TAs were injected per group. Mice were sacrificed three weeks later (3w). (b) Dystrophin rescue monitored by immunostaining with NCL-DYS2 monoclonal antibody on transverse sections of TA muscle. One representative immunostained section per condition is shown. (c) Dystrophin restoration assessed by Western blotting with NCL-DYS1 monoclonal antibody on total protein extracts from PPMO-treated muscles (upper panel) (lower panel: α-actinin). Dystrophin restoration was quantified using ImageJ software and is expressed as a percentage of dystrophin expression in wt muscle. (d) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as the number of AAV genomes relative to the value obtained for non-PPMO-treated mdx muscle. Data represent the mean ± SEM of four muscles per group. ns: not significant; ***p<0.001, Student's t-test. One of two representative experiments is shown. [Figure 2]Figure 1 shows that Pip6a-PMO pretreatment allows significant dystrophin rescue at low AAV-U7ex23 doses after 6 months. (a) Mdx TAs were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before injection of 1E+10vg of therapeutic AAV1-U7ex23 vector (day 0, d0). Control mdx TAs were injected with PPMO or AAV1-U7ex23 vector alone. Four TAs were injected per group. Mice were sacrificed after 6 months (6m). (b) Levels of exon 23 skipping estimated by nested RT-PCR. The 901-bp PCR product corresponds to the full-length dystrophin transcript, whereas the 688-bp product corresponds to a transcript lacking exon 23. (c) Quantification of exon 23 skipping performed by relative TaqMan qPCR and expressed as a percentage of total dystrophin transcript. (d) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as the number of AAV genomes relative to the value obtained for non-PPMO-treated mdx muscles. Data presented in (c) and (d) represent the mean ± SEM of four TAs per group. *p<0.05, ***p<0.001, Student's t-test. (e) Dystrophin restoration (upper panel) assessed by Western blotting using the NCL-DYS1 monoclonal antibody on total protein extracts from treated muscles (lower panel: α-actinin). Dystrophin restoration was quantified using ImageJ software and is expressed as a percentage of dystrophin expression in wt muscle. [Figure 3]Figure 1 shows the effect of Pip6a-PMO pretreatment on AAV1-mediated microdystrophin gene therapy. (a) Mdx TAs were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before injection (day 0, d0) of 1E+10 vg of AAV1-MD1 microdystrophin expression vector. Control mdx TAs were injected with PPMO or AAV1-MD1 vector alone. Five TAs were injected per group. Mice were sacrificed four weeks later (4w). (b) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as AAV genome number relative to the value obtained for non-PPMO-treated mdx muscles. Data represent the mean ± SEM of five muscles per group. *p<0.05, Student's t-test. (c) PPMO-induced dystrophin (DYS, 427 kDa) and microdystrophin (μDYS, 132 kDa) expression (upper panel) (lower panel: α-actinin) assessed by Western blotting using MANEX1011B monoclonal antibody on total protein extracts from treated muscles. [Figure 4]Figure 1 shows GDF5 overexpression in muscle from young mice. A-C: RT-qPCR of (A) Gdf5, (B) Cacnb1-E (Ex2-3), and (C) Cacnb1-D in 15-day innervated (Inn) or denervated (Den) adult TA treated with Scra or GDF5. A: Minimum value of middle ellipses = 150, minimum value of upper ellipses = 3000. D, E: Immunofluorescence images of TA Inn (top) or Den (bottom) treated with (D) Scra or (E) GDF5, stained with GDF5 (magenta) and CaVβ1E (yellow). Bar: 10 μm. F, G: RT-qPCR of (F) Id1 and (G) Id2 in TA Inn or Den treated with Scra or GDF5. H, I: Hematoxylin and eosin (H / E) staining of TA Inn (top) or Den (bottom) treated with (H) Scra or (I) GDF5. Bar 100 μm. J, K: Sirius red (SR) staining of TA Inn (top) or Den (bottom) treated with (J) Scra or (K) GDF5. Bar 100 μm. L: Muscle / body weight ratio of Inn or Den adult TA treated with Scra or GDF5. A: Mean ± sem (n = 6) *P < 0.05, ***P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001; (Benjamini, Krieger, and Yekutieri two-step linear ascending procedure, Q = 1%. Each row was analyzed individually without assuming a consistent SD). B, C, F–G, L: Mean ± sem (n = 6) *P < 0.05, ***P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001 (ordinary one-way ANOVA with Sidak test). [Example]

[0093] Materials and Methods Viral vector production and animal experiments A three-plasmid transfection protocol was used to generate the single-stranded AAV1-U7ex23 (7), AAV1-U7scr (13), and AAV1-MD1 (37) vectors. pAAV(U7smOPT-SD23 / BP22), pAAV(U7smOPT-scr), and codon-optimized pΔR4-R23 / ΔCT(MD1) plasmids were used. AAV-GDF5 was generated by direct cloning of the Gdf5 ORF (NM_008109.2) flanked by EcoRI and NheI sites (GeneArt string, ThermoFisher) into the pSMD2 AAV2 vector backbone under the CMV promoter. pSUPER retro puro Scr ShRNA (SCRA) was a gift from John Gurdon (Addgene plasmid #30520). A BamHI site was inserted by PCR, and the H1-SCRA cassette was cloned into pSMD2-sh via the BamHI and SalI sites. The final virus preparation was stored in PBS at -80°C. Vector titers were determined by real-time PCR and expressed as vector genomes per milliliter (vg / ml). Three-month-old mdx mice were injected into the tibialis anterior (TA) muscle with 1 nmole of Pip6a-PMO oligonucleotide (GGCCAAACCTCGGCTTACCTGAAAT, SEQ ID NO: 11) (20). In addition, 50 μl of AAV1-U7scr, AAV1-U7ex23, or AAV1-MD1 containing 1E+10 or 1E+11 vg was injected into C57BL / 6 (wt) or mdx TA. AAV-GDF5 was injected at 5E+10 into 8-week-old C57BL / 6 TA. As a control, 8-week-old C57 / BL6 mice were injected with the SCRA AAV vector using the same procedure. Mice were sacrificed 10 or 12 weeks after injection. These animal experiments were performed at the Myology Research Center, Paris, France, in accordance with guidelines and protocols approved by the ethical review committee. A minimum of four mice per group were injected per experiment. At the time of sacrifice, muscles were collected, flash-frozen in liquid nitrogen-cooled isopentane, and stored at -80°C.

[0094] Denervation experiments Ten weeks after AAV injection into mice, the sciatic nerve was denervated (a 5 mm section of the sciatic nerve was removed) under general anesthesia (isoflurane, 3% induction, 2% maintenance) with buprenorphine (vetergesic 1 mg / kg, subcutaneous). Mice were sacrificed 1, 3, 7, or 15 days after denervation, and the TA was dissected, weighed, and then frozen in pre-cooled isopentane in liquid nitrogen and stored at -80°C until histological or molecular analysis.

[0095] Viral genome quantification Genomic DNA was extracted from mouse muscle using a Puregene Blood kit (Qiagen). The copy number of AAV genomes and genomic DNA was measured for 100 ng of genomic DNA by absolute quantitative real-time PCR using Taqman® Universal Master Mix (Applied Biosystems) on a StepOnePlus™ (Applied Biosystems). The viral genome sequence was specifically amplified using primers (forward: CTCCATCACTAGGGGTTCCTTG (SEQ ID NO: 3) and reverse: GTAGATAAGTAGCATGGC (SEQ ID NO: 4)) and a probe (TAGTTAATGATTAACCC (SEQ ID NO: 5)). A 10-fold serial dilution of pAAV plasmid (10 7 ~10 1 All genomic DNA samples were analyzed in duplicate.

[0096] RT-PCR analysis Total RNA was isolated from mouse muscle using NucleoSpin® RNA II (Macherey-Nagel), and reverse transcription (RT) was performed on 200 ng of RNA using Superscript™ II and random primers (Life Technologies). Unskipped and skipped dystrophin transcripts were detected by nested PCR and quantified as described (9).

[0097] Gene expression analysis by RT-qPCR Total RNA was prepared from TA frozen sections using TRizol (Life Technologies) according to the manufacturer's instructions. Complementary DNA was generated using Superscript II reverse transcriptase (Life Technologies), amplified using PCR Master Mix (M7505, Promega) for RT-PCR, or analyzed by real-time qPCR. Real-time qPCR was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) using Power SyberGreen PCR MasterMix (Applied Biosystems). All data were analyzed using the ΔΔCT method and normalized to PO (mouse acidic ribosomal phosphoprotein) mRNA expression levels. The reference sample used to calculate mRNA fold changes is indicated in each panel. The primers used are listed.

[0098] [Table 1]

[0099] Western blot analysis Protein extracts were obtained from pooled muscle sections treated with 125 mM sucrose, 5 mM Tris-HCl pH 6.4, 6% XT Tricine running buffer (Bio-Rad), 10% SDS, 10% glycerol, and 5% β-mercaptoethanol. Samples were purified using the Pierce Compat-Able™ Protein Assay Preparation Reagent Set (Thermo Scientific), and total protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were denatured at 95°C for 5 minutes, and 100 μg of protein was loaded onto Criterion XT Tris Acetate Precast Gels 3-8% (Bio-Rad). The membranes were probed with primary monoclonal antibodies directed against dystrophin (NCL-DYS1, 1:50, Leica Biosystems; MANEX1011B, 1:50, a kind gift from The Muscular Dystrophy Association Monoclonal Antibody Resource (38)) and α-actinin (1:1000, Sigma-Aldrich), followed by incubation with sheep anti-mouse secondary antibody (horseradish peroxidase conjugate; 1:15000) and Pierce ECL Western blotting substrate (Thermo Scientific).

[0100] Immunohistochemistry and histology Twelve-micrometer TA sections were cut and examined for dystrophin expression using NCL-DYS2 monoclonal antibody (Leica Biosystems). A rabbit polyclonal antibody against the Cavβ1 C-terminus (AP16144b) was purchased from AbGent, and a mouse monoclonal antibody against GDF5 (SC-373744) was obtained from Santa Cruz Biotechnologies. Fluorescent secondary antibodies, goat anti-rabbit and goat anti-mouse, were purchased from Life Technologies.

[0101] For H&E staining, sections were fixed in 4% PFA for 10 minutes, washed in PBS, and then stained with hematoxylin for 5 minutes and eosin for 30 seconds. Muscle sections were then dried in increasing concentrations of ethanol / water solutions, fixed in 100% xylene, and mounted in Vectamount (Vector Laboratories). Sirius red staining was performed to analyze total collagen I and III content. Muscle frozen sections were fixed in 4% PFA for 10 minutes, washed in water, and dried in 100% ethanol for 5 minutes. The sections were then stained in picrosirius red (0.3%) solution for 1 hour in the dark. After washing in oxygenated water (5 min in 0.5% v / v acetic acid), sections were fixed in 100% ethanol (three 5-min washes), finally dehydrated in 100% xylene, mounted in Vectamount, and visualized using a Nikon AZ100 zoom macroscope. Confocal images were taken using a Leica SPE or Nikon Ti2 microscope equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled to a Prime 95 sCMOS camera (Photometrics).

[0102] result Effect of dystrophin restoration by AON pretreatment on untreated viral genome maintenance To induce transient dystrophin expression in the sarcolemma of Mdx muscle fibers, Mdx tibialis anterior (TA) muscles were injected with 11 μg of Pip6a-PMO, a peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotide that is particularly efficient in mdx exon skipping (20). A non-therapeutic AAV-U7scr vector (carrying a nonspecific scrambled sequence) was injected into the same muscles at a high dose (1E+11 viral genomes) 2 weeks later, when dystrophin rescue was optimal (Figure 1a). We previously showed that these U7scr vectors, which are unable to induce exon skipping and thereby rescue dystrophin expression, were dramatically lost from dystrophin-deficient mdx muscles within 3 weeks (13).

[0103] Three weeks after AAV1-U7scr injection following AON pretreatment to induce exon skipping, immunofluorescence staining revealed robust dystrophin restoration and precise localization of dystrophin at the sarcolemma of mdx-injected muscles (Figure 1b). Dystrophin levels in mdx muscles were quantified by Western blotting, which showed that AON pretreatment resulted in 56–98% para-dystrophin restoration compared to normal levels (Figure 1c). As expected (13), the viral genome content analyzed by quantitative PCR (qPCR) was six-fold higher in wild-type (wt) muscles than in non-AON-treated mdx muscles. Interestingly, the viral genome content in the AON-treated mdx group analyzed by qPCR was comparable to that observed in wt muscles (Figure 1d). Thus, the marked dystrophin expression induced by PPMO pretreatment at the time of AAV1-U7scr injection prevents rapid loss of the AAV1-U7scr genome in mdx muscle comparable to that observed in wt muscle.

[0104] Effect of Pip6a-PMO pretreatment on dystrophin rescue with low-dose therapeutic AAV-U7ex23 An AAV1 vector encoding U7ex23 (AAV1-U7ex23) enables efficient exon 23 skipping and thus quasi-dystrophin rescue in mdx muscles. To evaluate the benefit of AON pretreatment on AAV1-U7ex23-mediated quasi-dystrophin rescue, we injected 11 μg of Pip6a-PMO antisense oligonucleotide into mdx TAs, followed 2 weeks later by injection of the AAV1-U7ex23 vector (Figure 2a). We chose a low vector dose (1E+10 viral genomes) because this dose allows for weak quasi-dystrophin rescue (less than 5% of normal levels) (13).

[0105] The benefit of AAV1-U7ex23 injection was analyzed 6 months later, when the dystrophin rescue induced by a single PPMO injection had largely disappeared. The level of exon 23 skipping in mdx TAs treated with AAV1-U7ex23 or PPMO alone, analyzed by nested RT-PCR (Fig. 2b) and quantified by qPCR (Fig. 2c), was low as expected: 9 and 6% skipped transcripts, respectively, resulting in the synthesis of rescued dystrophin at approximately 2% of normal levels (Fig. 2e). In contrast, TAs treated sequentially with PPMO and AAV1-U7ex23 showed 54% skipped transcripts (Fig. 2c) and 20% normal levels of dystrophin (Fig. 2e). The AAV genome copy number, quantified by absolute qPCR, was eightfold higher in dual PPMO / AAV1-U7ex23-treated muscles than in muscles injected with AAV1-U7ex23 alone (Fig. 2d). These data demonstrate that PPMO pretreatment allowed better maintenance of the therapeutic U7ex23 genome in mdx muscles 6 months after AAV-U7 injection and, notably, resulted in a 10-fold improvement in rescued dystrophin levels.

[0106] Pip6a-PMO pretreatment significantly improves the efficacy of AAV1-mediated microdystrophin gene therapy To evaluate the efficacy of AON pretreatment for AAV-microdystrophin gene therapy, Pip6a-PMO AONs were injected into mdx TAs, followed 2 weeks later by injection of an AAV1-MD1 vector (1E+10 vg) expressing murine microdystrophin (MD1) (37) (Figure 3a). After 4 weeks, robust dystrophin restoration was observed in PPMO-treated mdx TAs induced by PPMO pretreatment (Figure 3c). AAV genome copy number and microdystrophin expression were threefold greater in PPMO / AAV1-MD1-treated muscles than in muscles treated with AAV1-MD1 alone (Figures 3b and 3c), demonstrating the benefit of PPMO pretreatment for AAV-microdystrophin gene therapy. This experiment establishes proof-of-concept that AON pretreatment can enhance all AAV-based gene therapies for DMD.

[0107] Effect of GDF5 overexpression In young TAs, we induced Cacnb1-E transcription in innervated TAs (Figures 4B, 4D, and 4E), but overexpressed GDF5 relative to scrambled (Figures 4A, 4D, and 4E) without affecting Cacnb1-D expression (Figure 4C). Nevertheless, GDF5 overexpression and its activation signaling, confirmed by Id-1 and Id-2 transcription (Figures 4F and 4G), increased innervated muscle mass in most cases (Figures 4H–L).

[0108] Consideration Due to its episomal nature, the AAV genome is rapidly lost from dystrophic muscle during AAV-U7-mediated exon skipping therapy. The vulnerability of dystrophic muscle fibers undergoing cycles of necrosis and regeneration is characterized by abnormally leaky membranes and increased exosome and microparticle excretion (36). Here, we demonstrate that significant (>60%) paradystrophin rescue after PPMO pretreatment at the time of AAV-U7 injection allows efficient maintenance of the viral genome in mdx muscles 3 weeks later. Furthermore, this initial maintenance of the viral genome enhances AAV-U7-mediated paradystrophin restoration by approximately 6-fold at the RNA level and approximately 10-fold at the protein level 6 months later.

[0109] PPMO pretreatment resulted in substantial dystrophin expression at the time of AAV-U7 injection. This likely reduces the membrane abnormalities that cause AAV genome loss before AAV-U7-induced dystrophin expression occurs, similar to that observed in normal control muscle. Once established, AAV-U7-mediated high dystrophin expression is maintained because it prevents transgene loss by itself. Thus, by allowing the maintenance of high viral genome content during the critical period between AAV injection and AAV-mediated transgene expression in treated dystrophic muscle, PPMO-mediated dystrophin restoration ensures the long-term, sustained benefits of AAV-U7 treatment.

[0110] This pretreatment could be induced by any AON (i.e., using different skippable mutations, different target sequences, and different AON chemistries such as tricyclo-DNA (36)) that allows for substantial quasi-dystrophin rescue using the principles demonstrated here with PPMO chemistry.

[0111] This AON pretreatment is applicable to all therapeutic approaches for Duchenne myopathy that use AAV vectors, particularly AAV-U7-mediated exon skipping, and classical gene therapy involving transfer of functional microdystrophin cDNA into muscle, as demonstrated by the data presented herein.

[0112] In addition, it has been shown herein that muscle mass increases when GDF5 is overexpressed, meaning that the treated muscles may be further protected during the application of the above-mentioned therapeutic strategies due to the administration of either a vector expressing GDF5 or recombinant GDF5 protein.

[0113] On the eve of clinical trials using AAV-based therapy for DMD patients, this study highlights the powerful impact of a combination approach to improve the benefits of AAV-based therapy, allowing the use of lower, and therefore safer, vector doses for greater levels of dystrophin expression over the long term.

[0114] [References] JPEG2025176070000003.jpg180170 JPEG2025176070000004.jpg216170 JPEG2025176070000005.jpg219170 JPEG2025176070000006.jpg110170

Claims

1. A combination comprising a GDF5 pathway activator and at least one other active ingredient suitable for use in the treatment of a muscular disease, for use in the treatment of said muscular disease.

2. GDF5 pathway activators a vector comprising a gene encoding GDF5, such as human GDF5; or Recombinant GDF5, such as recombinant human GDF5 2. The combination of claim 1, wherein

3. 3. The combination according to claim 1 or 2, for use in the treatment of Duchenne muscular dystrophy, wherein at least one other active ingredient is a combination of (i) an antisense oligonucleotide (AON) capable of inducing exon skipping in dystrophin pre-mRNA and (ii) a viral vector, such as an AAV vector, encoding a therapeutic agent for Duchenne muscular dystrophy, and wherein component (i) is administered before component (ii).

4. 4. The combination of claim 3, wherein the viral vector of component (ii) (a) encodes an antisense oligonucleotide capable of inducing exon skipping in dystrophin pre-mRNA, (b) encodes a dystrophin gene editing means, or (c) encodes a functional dystrophin protein.

5. 5. The combination of claim 3 or 4, wherein the AON is a peptide-phosphorodiamidate morpholino oligomer, in particular a phosphorodiamidate morpholino oligomer such as a Pip6a-PMO oligomer.

6. 6. A combination according to any one of claims 3 to 5, wherein the viral vector of component (ii) encodes a U7-AON.

7. 6. The combination according to any one of claims 3 to 5, wherein the viral vector of component (ii) encodes a functional truncated dystrophin, such as a mini- or micro-dystrophin.

8. GDF5 pathway activators - before administering component (i), - during administration of component (i), - between the administration of component (i) and the administration of component (ii), - during administration of component (ii), or - after administration of component (ii) 8. The combination according to any one of claims 3 to 7, administered to

9. 3. The combination according to claim 1 or 2, for use in the treatment of amyotrophic lateral sclerosis, wherein at least one other active ingredient is an antisense oligonucleotide (AON) capable of inducing exon skipping in SOD1 pre-mRNA, thereby causing incorporation of a premature stop codon into mature mRNA.

10. 3. The combination according to claim 1 or 2, wherein at least one other active ingredient is a vector comprising a gene encoding a motor neuron survival protein, such as the SMN1 or SMN2 gene.

11. - GDF5 pathway activators, and - at least one other active ingredient Kit including:

12. At least one other active ingredient - an isolated AON capable of inducing exon skipping in dystrophin pre-mRNA; and - Viral vector for the treatment of Duchenne muscular dystrophy 12. The kit of claim 11, comprising:

13. Duchenne muscular dystrophy virus vectors - encoding an antisense oligonucleotide capable of inducing exon skipping in the dystrophin pre-mRNA, - encoding a dystrophin gene editing means, or - Encodes a functional dystrophin protein, 13. The kit of claim 12.

14. A kit described in any one of claims 11 to 13, wherein the GDF5 pathway activator is a vector such as a plasmid or a viral vector, particularly a viral vector, more particularly an AAV vector, containing a gene encoding GDF5, particularly human GDF5.

15. 15. A kit according to any one of claims 11 to 14, wherein the GDF5 pathway activator is recombinant GDF5, in particular recombinant human GDF5.

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