COMBINED THERAPY FOR MUSCULAR DISEASES

FIELD OF THE INVENTION

The present invention relates to the treatment of muscular diseases.

BACKGROUND OF THE INVENTION

Muscular diseases are disorders affecting muscles, which can cause weakness, pain, or even paralysis.

Among muscular diseases, muscular dystrophy is a group of muscular diseases that results in increasing weakening and breakdown of skeletal muscles over time. In particular, dystrophinopathies are pathologies caused by anomalies in the DMD gene that encodes a subsarcolemmal protein called dystrophin. With regard to the large deletions, the most frequent genetic alteration, the severity of the phenotype is primarily conditioned by the impact of the mutation on the protein reading frame of the dystrophin transcript. The dystrophin structure (central rod-domain made of 24 spectrin-like repeats) tolerates large internal deletions (1) which led to the development of two main therapeutic strategies: classical gene therapy with transfer of functional micro-dystrophin cDNAs in muscles, and targeted exon skipping. We also have previously shown in WO2016198676 that a two-step combination therapy of a muscular dystrophy is advantageous over treatment strategies known in the prior art. This combination therapy comprises the administration of an isolated AON suitable for inducing exon skipping within a dystrophin pre-mRNA and inducing muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein. In a second step, the therapy comprises administering to the same subject at least one viral vector encoding a Duchenne muscular dystrophy therapeutic product. The viral vector (or therapeutic viral vector) is designed for restoring a dystrophin function in a muscle cell. For example, the at least one viral vector able to restore a dystrophin function in the muscle cell is a viral vector either (i) coding for an antisense oligonucleotide (which is also referred to as "AON-coding virus" in the following description) able to induce exon-skipping within a dystrophin pre-mRNA and inducing muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein, (ii) designed to introduce into the muscle cell means for correcting the dystrophin gene in the genome of said muscle cell, such as genome-editing means implementing one or more endonucleases specific for the dystrophin gene, or (iii) coding a functional dystrophin protein.

It is herein shown that such strategies, and other strategies for treating muscular dystrophies but also other muscular diseases, benefit from the administration of a GDF5 pathway-activating substance.

SUMMARY OF THE INVENTION In a first aspect, the present invention relates to a GDF5 pathway-activating substance for use in a method for treating a muscular disease by gene therapy, wherein the GDF5 pathway-activating substance is used in combination with another active ingredient suitable for the treatment of the muscular disease.

In a second aspect, the invention relates to a kit comprising (i) a GDF5 pathway-activating substance and (ii) an active ingredient suitable for the treatment of a muscular diseases/. The kit of the present invention is useful for implementing the therapeutic methods described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors herein show that GDF5 overexpression leads to muscle mass increase in both denervated and innervated muscles. This observation can advantageously be implemented in a method for the treatment of a muscular disease, wherein an active ingredient suitable to treat the muscular diseases is administered to a subject in need thereof, and wherein a GDF5 pathway-activating substance is used in combination to said active ingredient to increase or stabilize muscle mass and/or muscle function.

GDF5 pathway-activating substance

In a particular embodiment, the GDF5 pathway-activating substance is a GDF5 peptide, in particular synthetic or recombinant GDF5, more particularly recombinant GDF5, such as recombinant human GDF5. Unprocessed wild-type human GDF-5 (Uniprot Accession No. P43026) has the following sequence:

MRLPKLLTFLLWYLAWLDLEFICTVLGAPDLGQRPQGTRPGLAKAEAKERPPLARNV FRPGG

HSYGGGATNANARAKGGTGQTGGLTQPKKDEPKKLPPRPGGPEPKPGHPPQTRQATA RTVTP

KGQLPGGKAPPKAGSVPSSFLLKKAREPGPPREPKEPFRPPPITPHEYMLSLYRTLS DADRKGG

N S SVKLEAGLANTITSFIDKGQDDRGPVVRKQRYVFDIS ALEKDGLLGAELRILRKKPSDTAK

PAAPGGGRAAQLKLSSCPSGRQPASLLDVRSVPGLDGSGWEVFDIWKLFRNFKNSAQ LCLEL

EAWERGRAVDLRGLGFDRAARQVHEKALFLVFGRTKKRDLFFNEIKARSGQDDKTVY EYLF

SORRKRRAPLATROGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEG LCEFP

LRSHLEPTNHAVIOTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKOYEDM VVESCGC

R

(SEQ ID NO: 8) SEQ ID NO: 8 comprises a signal peptide at amino acid positions 1-27, a propeptide at amino acid positions 28-381 and a part, underlined in the sequence provided above, corresponding to the mature peptide at amino acid positions 382-501.

The mature peptide thus has a sequence as shown in SEQ ID NO:9 below:

APLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLR SHLEP TNHAVIQTFMNSMDPESTPPTCCVPTRFSPISIFFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO:9).

Other recombinant human GDF5 are commercially available, such as the protein having the sequence shown in SEQ ID NO:3, which is available from Thermo Fischer (catalog No. RP-8663): APSATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHL EP TNHAVIQTFMNSMDPESTPPTCCVPTRFSPISIFFIDSANNVVYKQYEDMVVESCGCR (SEQ ID NO: 10)

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 “a reference recombinant human GDF5”.

According to another particular embodiment, the GDF5 pathway-activating substance is a functional derivative of a GDF5 peptide. A functional derivative according to the invention is a peptide having at least one, in particular all, activity of a reference peptide. In the context of the present invention, a functional variant of a GDF5 peptide may have its ability to induce alkaline phosphatase production by ATDC5 mouse chondrogenic cells (Nakamura, K. et al. (1999) Exp. Cell Res. 250:351.) with an ED50 from 0.01 to 10 pg/mL, such as from 0.2 to 4 pg/mL, for example from 0.2 to 1.2 pg/mL. In particular, a functional variant of a GDF5 peptide is a peptide that may treat or prevent sarcopenia in an animal model of the condition, as provided in the experimental part of this application, or in a human subject. GDF5 signaling may also be evaluated by measuring SMAD 1/5/8 phosphorylation, SMAD 4 nuclear translocation and Id-1 transcription as provided in the experimental part below. The activity of the functional variant may be of 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 a particular embodiment, the functional peptide as an activity greater than the activity of the reference GDF5 peptide, such as an activity of at least 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or of at least 150% of the activity of the reference GDF5 peptide. In addition, according to the invention, a functional variant of a GDF5 peptide has at least 80% sequence identity to a 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 to a reference human recombinant GDF5. For example, a functional variant of a GDF5 peptide may comprise from 1 to 20 amino acid modifications (i.e. amino acid addition, deletion or substitution) as compared to a reference recombinant human GDF5, such as from 1 to 15 amino acid modifications, in particular from 1 to 10 amino acid modifications, more particularly from 1 to 6 amino acid modifications, even more particularly 1, 2, 3, 4, 5 or 6 amino acid modifications as compared to a reference recombinant human GDF5. Such a functional variant of recombinant human GDF5 may be a natural variant of GDF5. In a particular aspect, the functional variant is an optimized GDF5 peptide. Optimization may include different changes in the peptide, such as amino acid modifications as provided above, glycosylation, acetylation, phosphorylation and the like, or inclusion of at least one D-amino acid, such as at least 2, at least 3, at least 4 or at least 5 D amino acids. In another aspect, the GDF5 peptide comprises at least one non-natural amino acid, included by insertion, appendage, or substitution for another amino acid of the GDF5 sequence. In yet another aspect, recombinant GDF5 may be fused to another moiety, such as another peptide moiety. Such other moiety may, for example, stabilize the peptide.

In another particular embodiment, the substance is a functional variant of a GDF5 peptide corresponding to the GDF5-related proteins as described in WO201308649, having an increased affinity for the BMP receptor IB (BMPR-IB) and/or a reduced affinity for the BMP receptor IA (BMPR-IA). In a particular embodiment, the protein is derived from human wild-type GDF5. In a particular embodiment, the GDF5-related protein is obtained by replacing at least one amino acid residue relating to a BMPR-IB and/or BMPR-IA binding site in the amino acid sequence of the GDF-5 peptide, preferably by genetic engineering technology. In a further embodiment, at least one hydrophobic amino acid in the BMPR-IB and/or BMPR-IA binding site the GDF5 peptide is replaced with a hydrophilic or polar amino acid, such as a hydrophilic amino acid residue 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, such as a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, 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, in particular wherein a hydrophobic amino acid is replaced by a smaller or lager hydrophobic amino acid or wherein a hydrophilic or polar amino acid is replaced by a smaller or lager hydrophilic or polar amino acid. The regions of GDF-5 related proteins which are involved in binding to BMPR-IA and/or BMPR-IB are well known in the art or can easily be determined using methods that are within common knowledge. Referring to the unprocessed, full-length amino acid sequence of wild- type human GDF5 of SEQ ID NO: 8, a particular embodiment provides the replacement of one or more of the following amino acids by any different amino acid:

R399; any one of F409 to W417, in particular M412, G413, W414, and/or W417; any one of E 434 to M 456, in particular F435, P436, L437, R438, S439, H440, P443, N445, V448, 1449, L452, M453, S455, and/or M456;

S475;

1476;

F478; any one of K488 to M493, in particular K488, Y490, and/or D492.

In particular embodiments, referring to the unprocessed, full-length amino acid sequence of SEQ ID NO: 8, one or more of the following amino acids are replaced by the specified amino acid

- R399 is replaced by 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 by R, K, F, Y, H, E or D;

- W417 is replaced by 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 by V, L, I, M, F, Y or W;

- L437 is replaced by D or E;

- R438 is replaced by K, D, H, N, M, E, Q, S, T, Y or W;

- S439 is replaced by 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 by D, Q, H, F, L, R, K, M, S, Y or W;

- V448 is replaced by F, L, I, M, P, Y or W;

- 1449 is replaced by F, L, V, M, P, Y or W;

- L452 is replaced by F, I, V, M, P, Y or W;

- M456 is replaced by F, I, L, P, Y, W, S, T, N, Q, K or D;

- S475 is replaced by M, T, N, Q, Y or W;

- K488 is replaced by R, M, S, T, N, Q, Y or W;

- Y490 is replaced by E, H, K, R, Q, F, T, M, S, N, Q or W;

- D492 is replaced by G, E, M, S, T, N, Q, Y, W, H, K or R;

- 1476 is replaced by G, A, V, L, M, F, Y or W;

- F478 is replaced by G, A, V, L, I, Y or W.

In another particular embodiment, referring to the unprocessed, full-length amino acid sequence of SEQ ID NO: 8, one or more of the following amino acids are replaced by the specified amino acid:

R399 is replaced by M or E;

W414 is replaced by R;

W417 is replaced by R or F;

R438 is replaced by K; S439 is replaced by K or E;

1449 is replaced by V.

The corresponding positions in the mature peptides (such as in SEQ ID NO:9 or SEQ ID NO: 10) will easily be derived from the above information regarding unprocessed, full-length wild-type human GDF- 5.

In a particular embodiment of the invention, the substance is a GDF5 peptide whose amino acid consists of SEQ ID NO:9 or SEQ ID NO: 10. In another particular embodiment, the substance is a GDF5 peptide whose amino sequence consists of SEQ ID NO:9 or SEQ ID NO: 10, with the addition of a methionine residue at its N-terminal end. In another embodiment, the substance is a GDF5 peptide whose amino acid consists of SEQ ID NO:9 or SEQ ID NO: 10, wherein the first alanine residue is replaced by a methionine residue.

In a further particular embodiment, the GDF5 pathway-activating substance is a substance inducing the CaV i-E /GDF5 axis. In this embodiment, a variant comprises the use of a substance that is a small chemical molecule. In a non-limiting variant of this embodiment, the GDF5 pathway-activating substance is an inhibitor of NRSF (Neuron-Restrictive Silencer Factor; also referred to as REST or REl- Silencing Transcription Factor).

In a particular embodiment, the GDF5 pathway-activating substance is the NRSF inhibitor is valproic acid. In a further particular embodiment, the GDF5 pathway-activating substance is selected from the NRSF inhibitors disclosed in Charbord et al., Stem Cells. 2013 Sep;31(9): 1816-28, in particular the 2- (2-Hydroxy-phenyl)-lH-benzoimidazole-5-carboxylic acid allyloxy-amide (X5050), 2-Thiophen-2-yl- lH-benzoimidazole-5-carboxylic acid (2-ethyl-hexyl)-amide (X5917), 3-[l-(3-Bromo-phenyl)-3,5- dimethyl- lH-pyrazol-4-yl] - 1 - {4-[5 -(morpholine-4-carbonyl)-pyridin-2-yl] -2 -phenyl -piperazin- 1 -yl } - propan-l-one (X38210) or 3-[l-(2,5-Difluoro-phenyl)-3,5-dimethyl-lH-pyrazol-4-yl]-l-{ 4-[5- (morpholine-4-carbonyl)-pyridin-2-yl] -2 -phenyl -piperazin- 1 -yl } -propan- 1 -one (X38207) molecule disclosed therein, more particularly the X5050 molecule disclosed therein.

In yet another embodiment, the GDF5 pathway-activating substance is a vector comprising a nucleic acid encoding GDF5, such as human GDF5 or a functional variant thereof. In a particular embodiment, the vector is a plasmid or viral vector, such as an adenoviral vector or an adeno-associated virus (AAV) vector. According to a particular embodiment, the viral vector is suitable for transducing muscle and/or neuronal cells. In a more particular embodiment such viral vector suitable for transducing muscle and/or neuronal cells is an AAV vector, such as an AAV vector having an AAV2/2, AAV2/6, AAV2/8, AAV2/9 or AAV2/10 capsid. In a further particular embodiment. The GDF5 coding sequence may be under the control of regulatory sequences such as promoters, enhancers, repressors and polyadenylation signals. In a particular embodiment, the vector comprises an expression cassette, comprising, in this order, a promoter, the GDF5 coding sequence and a polyadenylation signal. The promoter may be ubiquitous or tissue-selective. In a particular embodiment, the promoter is the natural promoter of the GDF5 gene, such as the promoter of the human GDF5 gene.

In another particular embodiment, the GDF5 pathway-activating substance is a substance that increases the activity or the expression of GDF5.

In a further embodiment, the GDF5 pathway-activating substance is a vector comprising a nucleic acid encoding CaV i-E, such as human CaV i-E. In a particular embodiment, the vector is a plasmid or viral vector, such as an adenoviral vector or an adeno-associated virus vector. Accordingly, the present invention also relates to a vector, such as a viral vector, for example an adenoviral or AAV vector as described above, comprising CaV i-E coding sequence. The CaV i-E coding sequence may be under the control of regulatory sequences such as promoters, enhancers, repressors and polyadenylation signals. In a particular embodiment, the vector comprises an expression cassette, comprising, in this order, a promoter, the CaV i-E coding sequence and a polyadenylation signal. The promoter may be ubiquitous or tissue-selective. In a particular embodiment, the promoter is the natural promoter of the CaV i-E gene, such as the promoter of the human CaV i-E gene.

In a particular embodiment, the GDF5 pathway-activating substance is either (i) a vector comprising a nucleic acid encoding GDF5, such as human GDF5 or a functional variant thereof, such as an AAV vector, 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-activating substance is recombinant GDF5, such as recombinant human GDF5 or a functional variant thereof.

In a particular embodiment, the GDF5 pathway-activating substance is administered separately, sequentially or simultaneously with the other active ingredient(s). For example, administrations can be simultaneous (for example, because the substance and ingredient(s) are comprised within the same composition, or because two different compositions each comprising the substance and the active ingredient(s), respectively, are administered at the same time) or are several hours, days, weeks or months apart. In another particular embodiment, the GDF5 pathway-activating substance is a vector, such as a plasmid vector or a viral vector, in particular an AAV vector, and said substance is administered only once. In yet a further embodiment, the GDF5 pathway-activating substance is a recombinant protein as described above in each of its embodiments, and said recombinant GDF5 protein may be administered only once, for example before, during or after administration of the other active ingredient(s) to increase muscle mass and/or function before, during or after the active ingredient can act. In another embodiment, the GDF5 pathway-activating substance is a recombinant protein as described above in each of its embodiments, and said recombinant GDF5 protein may be administered several times, to maintain suitable muscle mass and/or function throughout the treatment protocol. For example, administration of the GDF5 recombinant protein may be carried out at least once a year, such as at least once every 6 months, such as at least once a quarter, such as at least once a month, such as at least once a week, such as at least once a day.

Muscular diseases and active ingredients suitable for their treatment

The present invention relates to the treatment of a muscular disease. Among muscular diseases that can benefit from the invention are genetic diseases. In a particular embodiment, the muscular disease is a neuromuscular disease or a musculoskeletal disease.

In a particular embodiment, the muscular disease is a motor neuron disease, such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA).

In a particular embodiment, the invention relates to the treatment of ALS, wherein the active ingredient suitable for the treatment of ALS is an AON targeting human SOD 1 pre-mRNA, wherein said AON is suitable to induce exon-skipping in said pre-mRNA. One skilled in the art knows what strategies are relevant to the treatment of ALS. For example, one can refer to the disclosure of WO2016016449. In a particular embodiment, the AON is encoded by a nucleotide sequence of interest introduced within a vector, such as a plasmid or viral vector, for example an AAV vector. General information useful for the design of AONs is available to those skilled in the art, and provided below with respect to AONs targeting dystrophin pre-mRNA, the same principles applying to the design of AONs targeting SOD1 pre-mRNA.

In a further particular embodiment, the 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 for a survival of motor neuron protein, such as the SMN 1 or SMN2 gene.

In yet another embodiment, the muscular disease is an atrophic muscular disorder, such as a muscular dystrophy. Non-limiting examples of muscular dystrophies that can be treated according to the invention include distal myopathies, Glycogen Storage Disease Type VII, limb-girdle muscular dystrophies, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy and myotonic dystrophy. In a particular embodiment, the muscular disease to be treated is a Duchenne muscular dystrophy. In a further particular embodiment, the active ingredients for the treatment of DMD are those disclosed in WO2016198676, wherein a two-step combination therapy of a muscular dystrophy is implemented. In this embodiment, first is administered an isolated AON suitable for inducing exon-skipping within a dystrophin pre-mRNA and inducing muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein. In a second step, the same subject is administered with at least one viral vector encoding a DMD therapeutic product. The viral vector (or therapeutic viral vector) is designed for restoring a dystrophin function in a muscle cell. For example, the at least one viral vector able to restore a dystrophin function in the muscle cell is a viral vector either:

(i) coding for an antisense oligonucleotide (which is also referred to as "AON-coding virus" in the following description) able to induce exon-skipping within a dystrophin pre-mRNA and inducing muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein,

(ii) designed to introduce into the muscle cell means for correcting the dystrophin gene in the genome of said muscle cell, such as genome-editing means implementing one or more endonucleases specific for the dystrophin gene, or

(iii) coding a functional dystrophin protein.

The term "antisense oligonucleotide" and "AON" are used interchangeably and refer to a single stranded nucleic acid sequence, e.g. a DNA or RNA sequence, which is complementary to a part of a pre-mRNA coding the dystrophin protein and is thus able to form, by Watson-Crick base pairing, to a heteroduplex within the target sequence. In particular, the AON of the present invention is designed to block a splice acceptor (SA) site and/or an exon splicing enhancer (ESE) and/or a branch point in the dystrophin pre- mRNA and/or a splice donor (SD) site and/or any sequence which could modulate pre-mRNA splicing, i.e. it is designed to be complementary to apart of the dystrophin pre-mRNA comprising an SA, an ESE, a branch point sequence, an SD, and/or any sequence which could modulate pre-mRNA splicing (14,15). In a particular embodiment, the targeted sequence within the dystrophin pre-mRNA may include a 3' or 5' splice site of a pre-mRNA, or a branch point. The target sequence may be within an exon or within an intron or overlapping an intron-exon or exon-intron junction. The target sequence for a splice site may include an mRNA sequence having its 5' end 1 to about 50 base pairs downstream of a normal splice acceptor junction in a preprocessed mRNA. A preferred target sequence for a splice is any region of a pre-mRNA that includes a splice site and/or is contained entirely within an exon coding sequence and/or spans a splice acceptor and/or donor site. Of course, the target sequence may include several of these sequences that can modulate pre-mRNA splicing, and several such target sequences can be combined to achieve the desired effect.

Tools are available for identifying SA, ESE, SD and branch point sequences in a pre-mRNA of interest. As is well known by those skilled in the art, SA are conserved sequences, they are at the 3' end of the intron and terminate the intron with an almost invariant AG sequence. SD are conserved sequences, they are at the 5’ end of the intron and start the intron with an almost invariant GT sequence. In addition, ESE motifs may be predicted on the exon sequence intended to be skipped using the ESEfmder software tool (http://rulai.cshl. edu/cgi-bin/tools/ESE3/esefmder.cgi?process=home). Design of the AON can then be carried out following the rules published in Aartsma-Rus et al. (16).

The AON of the invention is designed to complement suitable sequences within the dystrophin pre- mRNA which are required for correct splicing of the targeted exon, thereby blocking splicing reactions that would incorporate the targeted exon into mature mRNA.

The mutated human dystrophin genes express no measurable dystrophin at all in muscles of Duchenne muscular dystrophy patients. To remedy this condition, the antisense oligonucleotides of the present invention typically hybridize to selected regions of a pre-mRNA of a mutated human dystrophin gene, induce exon skipping in dystrophin mRNA, and thereby allow muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein. Exon skipping strategy converts an out-of-frame mutation into an in-frame mutation leading to an internally deleted but partially functional dystrophin. Therefore, depending on the skipped exon(s), the rescued proteins will at best improve dystrophic phenotypes toward milder Becker-like phenotypes. In certain embodiments, the resulting dystrophin protein is not necessarily the "wild-type" form of dystrophin, but is rather a truncated, yet functional or semi-functional, form of dystrophin. By increasing the levels of functional dystrophin protein in muscle cells, these and related embodiments may be useful in the prophylaxis and treatment of Duchenne muscular dystrophy. The combined therapy described herein provides significant and practical advantages over alternate methods of treating Duchenne muscular dystrophy.

"Exon skipping" refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-mRNA, and is thereby excluded from being present in the mature mRNA. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein. In certain embodiments, the exon being skipped is an aberrant exon from the human dystrophin gene, which may contain a mutation or other alteration in its sequence. In certain embodiments, the exon being skipped is any one or more of exons 1-79 of the dystrophin gene, though 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 of the localization of the DMD patient mutation, one or several exons are chosen to be skipped to restore the dystrophin coding frame leading to an internally deleted but partially functional dystrophin. "Dystrophin" is a rod-shaped cytoplasmic protein, and a vital part of the protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For instance, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. This large central domain is formed by 24 spectrin-like triple-helical elements of about 109 amino acids, which have homology to alpha-actinin and spectrin. The repeats are typically interrupted by four proline-rich non-repeat segments, also referred to as hinge regions. Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide a major site for proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10-25%. One repeat contains three alpha-helices: 1, 2 and 3. Alpha-helices 1 and 3 are each formed by 7 helix turns, probably interacting as a coiled-coil through a hydrophobic interface. Alpha-helix 2 has a more complex structure and is formed by segments of four and three helix turns, separated by a Glycine or Proline residue. Each repeat is encoded by two exons, typically interrupted by an intron between the nucleic acid position coding amino acids 47 and 48 in the first part of alpha-helix 2. The other intron is found at different positions in the repeat coding region, usually scattered over helix-3. Dystrophin also contains a cystein-rich domain at about amino acids 3080-3360), including a cystein-rich segment (i.e., 15 Cysteins in 280 amino acids) showing homology to the C-terminal domain of the slime mold (Dictyostelium discoideum) alpha-actinin. The carboxy- terminal domain is at about amino acids 3361-3685.

The amino-terminus of dystrophin binds to F-actin and the carboxy-terminus binds to the dystrophin- associated protein complex (DAPC) at the sarcolemma. The DAPC includes the dystroglycans, sarcoglycans, integrins and caveolin, and mutations in any of these components cause autosomally inherited muscular dystrophies. The DAPC is destabilized when dystrophin is absent, which results in diminished levels of the member proteins, and in turn leads to progressive fibre damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD), muscle cells produce an altered and functionally defective form of dystrophin, or no dystrophin at all, mainly due to mutations in the gene sequence. The predominant expression of the defective dystrophin protein, or the complete lack of dystrophin or a dystrophin-like protein, leads to rapid progression of muscle degeneration, as noted above. In this regard, a "defective" dystrophin protein may be characterized by the forms of dystrophin that are produced in certain subjects with DMD or BMD, as known in the art, or by the absence of detectable dystrophin.

As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic, or therapeutic function. A "functional" dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or "defective" form of dystrophin protein that is present in certain subjects with DMD or BMD. In certain embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. As one example, dystrophin-related activity in muscle cultures in vitro 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 the pathogenesis of disease, and provide a means to test dystrophin-related activity. Two of the most widely used animal models for DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin negative (see, e.g., Collins & 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. Included are truncated forms of dystrophin, such as those forms that are produced by certain of the exon-skipping antisense compounds of the present invention.

The isolated AON of the invention may be of any suitable type. Representative AON types include oligodeoxyribonucleotides, oligoribonucleotides, morpholinos (such as phosphorodiamidate morpholino (PMO) or peptide-phosphorodiamidate morpholino (PPMO)), 2'-0-methyl- phosphorothioate (2'OMePS), 2’ -0-2 -methoxyethyl -antisense oligonucleotides, tricyclo-DNA- antisense oligonucleotides, tricyclo-phosphorothioate DNA oligonucleotides, LNA, small nuclear RNA-modified such as U7-, Ul- or U6-modified AONs (or other UsnRNPs), or conjugate products thereof such as peptide-conjugated or nanoparticle-complexed AONs.

In particular, for use in vivo, the AONs may be stabilized, for example via phosphate backbone modifications. For example, stabilized AONs of the instant invention may have a modified backbone, e.g. have 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 versions of the AONs also include "Morpholinos" (phosphorodiamidate morpholino oligomers, PMOs), 2'-0-Methyl oligomers, tricyclo-DNAs, tricyclo-DNA- phosphorothioate AON molecules (WO2013/053928) or U small nuclear (sn) RNAs. The latter forms of AONs that may be used to this effect can be coupled to small nuclear RNA molecules such as Ul, U6 or U7 (or other UsnRNPs).

In a particular embodiment, the isolated AON used in the present invention is a 2'OMePS oligonucleotide or a PPMO oligonucleotide. Preferably, the AON is a PPMO oligonucleotide.

AONs employed in the practice of the invention are generally from about 10 to about 40 nucleotides in length, and may be for example, about 10, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40 nucleotides or more in length depending on the targeted sequences within the dystrophin pre-mRNA and the AON chemistry.

A representative AON for practice of the invention effects exon 51 skipping or exon 53 skipping of human dystrophin pre-mRNA. In a more particular embodiment, the AON for practice of the invention effects exon 51 skipping of human dystrophin pre-mRNA. In another particular embodiment, the AON for practice of the invention may be CATTCAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTGTAC (SEQ ID NO: 1), effecting exon 53 skipping of human dystrophin pre-mRNA.

Of course, any AON having the properties described above may be used in the practice of the present invention.

In an embodiment, the isolated AON of the invention has the sequence shown in SEQ ID NO: 1 and is a PPMO oligonucleotide.

For stable and efficient in vivo delivery, the isolated AONs used in the practice of the present invention may also be fused or co-administrated to any cell-penetrating peptides and to signal peptides mediating protein secretion. Cell-penetrating peptides can be RVG peptides (18), PiP (19) such as Pip6a-PMO (20), P28 (21), or protein transduction domains like TAT (22) or VP22 (23). In a particular embodiment, the isolated AON is a PPMO oligonucleotide, i.e. a PMO oligonucleotide fused to a peptide moiety, more particularly a Pip6a-PMO, even more particularly a Pip6a-PMO. In a particular embodiment, the PPMO oligonucleotide moiety comprises or consists of the sequence shown in SEQ ID NO: l. In a further particular embodiment, the isolated AON is a Pip6a-PMO whose oligonucleotide moiety comprises or consists of the sequence shown in SEQ ID NO: 1.

Furthermore, the isolated AON used in the practice of the invention may be administered as a composition further comprising a pharmaceutically acceptable carrier and a reagent that improves oligonucleotide delivery efficiency. Such reagent may include, without limitation, F127 (24).

In a particular embodiment of the invention, the isolated AON of the invention is capable of inducing functional dystrophin expression of at least 10%, preferably of at least 20%, preferably of at least 30%, preferably of at least 40%, preferably 50 %, more preferably at least 51%, 52%, 53%, 54%, 55% or at least 56 %, as compared to the normal expression level of wild-type dystrophin. Of course, higher functional dystrophin expression is also preferable, such as an expression of at least 60%, 70%, 80% or even of at least 90%, as compared to the normal expression level of wild-type dystrophin.

In the second step of the method invention, a viral vector coding a therapeutic product is also administered to the same patient in need of the treatment. In the context of the present invention, the therapeutic product is able to restore a dystrophin function in a muscle cell in need thereof.

In a particular embodiment, the viral vector coding a therapeutic product is an AON-coding virus. The AON coded by this virus is as defined above, and is able to induce exon-skipping within a dystrophin pre-mRNA and to induce muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein.

In another specific embodiment, the viral vector, or several viral vectors, encodes means for correcting the dystrophin gene in the genome of a muscle cell. In this embodiment, the viral vector able to restore dystrophin function in a muscle cell is designed for correcting a mutant dystrophin gene in a subject by introducing into the genome of said cell a genome-editing system. For example, a site-specific nuclease may be encoded by the viral vector, which may restore the expression of a full-functional or partially- functional dystrophin protein with a repair template or donor DNA, which can replace the entire dystrophin gene or the region containing the mutation. The site-specific nuclease may be used to introduce site-specific double strand breaks at targeted genomic loci. Site-specific double-strand breaks are created when the site-specific nuclease binds to a target DNA sequence, thereby permitting cleavage of the target DNA. This DNA cleavage may stimulate the natural DNA-repair machinery, leading to one of two possible repair pathways: homology-directed repair (HDR) or the non-homologous end joining (NHEJ) pathway. This embodiment may comprise introducing into the muscle cell a genome-editing means implementing one or more endonucleases (for example one or more meganuclease (s), TALEN(s), ZFN(s) or a CRISPR/Cas9 endonuclease) specific for the dystrophin gene and one or more repair matrices. Such systems are described and known to those skilled in the art, for example in WO 11036640, W013163628, W014009567 and W014197748.

In another embodiment, the therapeutic product coded by the viral vector is a functional dystrophin. As mentioned above, a "functional" dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or "defective" form of dystrophin protein that is present in certain subjects with DMD or BMD. In certain embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. The functional dystrophin protein may be a truncated dystrophin protein, such as a mini- or micro-dystrophin. Such mini- and micro dystrophin are known in the art, for example in W002/29056, WOO 1/83695, WO08/088895 and Foster et ah, 2008 (37). In a particular embodiment, the micro-dystrophin is a AAB/R3-R18/ACT or AR4- R23/ACT micro-dystrophin (more particularly a AR4-R23/ACT micro-dystrophin), such as the AAB/R3-R18/ACT or AR4-R23/ACT micro-dystrophin described in Foster et ah, 2008, more particularly the AR4-R23/ACT micro-dystrophin. In a further particular embodiment, the mini- or micro dystrophin coding gene is codon-optimized. In a further particular embodiment, the mini- or micro dystrophin coding gene is codon optimized and encodes the AAB/R3-R18/ACT or AR4-R23/ACT micro- dystrophin, in particular the AR4-R23MCT micro-dystrophin described in Foster et al., 2008. The corresponding coding sequences are shown in SEQ ID NO: 6 and 7 respectively.

Adeno-associated viral vector (AAV)-mediated delivery of microdystrophins into dystrophin-deficient mice with DMD has shown remarkable efficiency (25, 26, 27) which has led to the initiation of an early- phase clinical trial (28).

Viral vectors include, but are not limited to, non-integrating viral vectors (or vectors integrating the genome of the target cell with low efficacy) such as episomally-maintained vectors, including adenoviruses; parvoviruses such as adeno-associated viruses; SV40-type viruses. One can readily employ other vectors not named but known in the art. Among the vectors that have been validated for clinical applications and that can be used to deliver the antisense sequences AAVs show a greater potential for exon skipping strategy.

In a preferred embodiment, the viral vector is a parvovirus, in particular an AAV vector. The parvovirus Adeno-Associated Virus (AAV) is a dependent virus that is naturally defective for replication and which is able to integrate into the genome of the infected cell to establish a latent infection. The last property appears to be unique among mammalian viruses because the integration occurs at a specific site in the human genome, called AAVS1, located on chromosome 19 (19ql3.3-qter). AAV-based recombinant vectors lack the Rep protein and integrate with low efficacy and are mainly present as stable circular episomes that can persist for months and maybe years in the target cells. Therefore AAV has aroused considerable interest as a potential vector for human gene therapy. Among the favorable properties of the virus are its lack of association with any human disease and the wide range of cell lines derived from different tissues that can be infected. Actually 12 AAV serotypes (AAV1 to 12) and up to 120 variants are known (29; 30), each with different tissue tropisms. Accordingly, the present invention relates to an AAV vector coding the AON described above, targeting a dystrophin pre-mRNA and adapted to induce exon-skipping in said human pre-mRNA and to induce the production of a functional dystrophin protein in muscle cells. According to a particular embodiment, the AAV genome is derived from an AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 serotype. In a preferred embodiment, the AAV capsid is derived from an AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 serotype or AAV variants. In a further particular embodiment, the AAV vector is a pseudotyped vector, i.e. its genome and capsid are derived from AAVs of different serotypes. For example, the pseudotyped AAV vector may be a vector whose genome is derived from the AAV2 serotype, and whose capsid is derived from the AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 serotype AAVs or AAV variants. In addition, the genome of the AAV vector may either 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 genome is half the length of the wild type AAV genome have the tendency to package DNA dimers.

Preferably, the AAV vector implemented in the practice of the present invention is a vector targeting muscle cells. In particular, AAV1, 6, 8 and 9 exhibit a high tropism for striated muscles (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, this vector being optionally pseudotyped.

In a particular embodiment, the AON coded by the AON-coding vector as described above is linked to a small nuclear RNA molecule such as a Ul, U2, U6, U7 or any other small nuclear RNA (snRNA), or chimeric small nuclear RNA (32; 33). Information on U7 modification can in particular be found in Goyenvalle, et al. (34); WO2011/113889; and W02006/021724. In a particular embodiment, the U7 cassette described by D. Schumperli is used (35). It comprises the natural U7-promoter (position -267 to +1), the U7smOpt snRNA and the downstream sequence down to position 116. The 18 nt natural sequence complementary to histone pre-mRNAs in U7 snRNA is replaced by one or two (either the same sequence used twice, or two different sequences) or more repeats of the selected AON sequences using, for example, PCR-mediated mutagenesis, as already described (34).

In a particular embodiment, the U7-modified AON comprises the sequence shown in SEQ ID NO:2: CATTCAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTGTAC (SEQ ID NO: 2), effect exon 53 skipping in dystrophin pre-mRNA. More particularly, the U7-modified AON effecting exon 53 skipping in dystrophin pre-mRNA is as disclosed in WO2017098187.

In a particular embodiment, the small nuclear RNA-modified AONs, in particular the U7-modified AONs, are vectorized in an AAV vector.

Typically, the viral vector, notably the functional dystrophin-coding vector or the AON-coding vector may also comprise regulatory sequences allowing expression of the encoded functional dystrophin or AONs, such as e.g., a promoter, enhancer internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTD), and the like. In this regard, the vector most preferably comprises a promoter region, operably linked to the coding sequence, to cause or improve expression of the AON. Such a promoter may be ubiquitous, tissue-specific, strong, weak, regulated, chimeric, etc., to allow efficient and suitable production of the AON. The promoter may be a cellular, viral, fungal, plant or synthetic promoter. Most preferred promoters for use in the present invention shall be 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 its aspect relating to the expression of an AON, promoters may be selected from small nuclear RNA promoters such as Ul, U2, U6, U7 or other small nuclear RNA promoters, or chimeric small nuclear RNA promoters. Other representative promoters include RNA polymerase Ill-dependent promoters, such as the HI promoter, or RNA polymerase II-dependent promoters. Examples of regulated promoters include, without limitation, Tet on/off element-containing promoters, rapamycin-inducible promoters and metallothionein promoters. Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, the RSV promoter, the SV40 promoter, hybrid CBA (Chicken beta actin / CMV) promoter, etc. and cellular promoters such as the PGK (phosphoglycerate kinase) or EFlalpha (Elongation Factor 1 alpha) promoters.

The practitioner can use compositions comprising the isolated AON or the viral vector, as described above, in a pharmaceutically acceptable carrier. In addition to the AON or to the virus, a pharmaceutical composition of the present invention may also include a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc. The composition will generally be in the form of a liquid, although this need not always be the case. Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, mineral oil, etc. The formulation can also include lubricating agents, wetting agents, emulsifying agents, preservatives, buffering agents, etc. In particular, the present invention involves the administration of an isolated AON or AON-coding virus and is thus somewhat akin to gene therapy. Those of skill in the art will recognize that nucleic acids are often delivered in conjunction with lipids (e.g. cationic lipids or neutral lipids, ormixtures ofthese), frequently in the form of liposomes or other suitable micro- or nano-structured material (e.g. micelles, lipocomplexes, dendrimers, emulsions, cubic phases, etc.).

The compositions of the invention are generally administered via enteral or parenteral routes, e.g. intravenously (i.v.), intra-arterially, subcutaneously, intramuscularly (i.m.), intracerebrally, intracerebroventricularly (i.c.v.), intrathecally (i.t), intraperitoneally (i.p.), although other types of administration are not precluded.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispensing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. While delivery may be either local (i.e. in situ, directly into tissue such as muscle tissue) or systemic, usually delivery will be local to affected muscle tissue, e.g. to skeletal muscle, smooth muscle, heart muscle, etc. Depending on the form of the AON or the viral vector that is administered and the tissue or cell type that is targeted, techniques such as electroporation, sonoporation, a “gene gun” (delivering nucleic acid- coated gold particles), etc. may be employed.

One skilled in the art will recognize that the amount of an isolated AON or of a viral vector to be administered will be an amount that is sufficient to induce amelioration of unwanted muscular dystrophy symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition of the patient, etc. and may be determined on a case by case basis. The amount may also vary according to other components of a treatment protocol (e.g. administration of other medicaments, etc.). Generally, a suitable dose is in the range of from about 1 mg/kg to about 100 mg/kg, and more usually from about 2 mg/kg/day to about 10 mg/kg. In the case of viral -based delivery of AON, suitable doses will depend on different factors such as the virus that is employed, the route of delivery (intramuscular, intravenous, intra-arterial or other), but may typically range from 10e9 to 10el5 viral particles/kg. Those of skill in the art will recognize that such parameters are normally worked out during clinical trials. Further, those of skill 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 a partial or intermittent relief of symptoms may be of great benefit to the recipient. In addition, treatment of the patient may be a single event, or the patient is administered with the AON and/or the viral vector on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart.

Combination therapy with a GDF5 pathway-activating substance

As mentioned above, the present invention relates to a GDF5 pathway-activating substance, for use in a method for the treatment of a muscular disease by gene therapy, in combination with another active ingredient.

Therefore, the invention defines a combination product, comprising a GDF5 pathway-activating substance and another active ingredient suitable for the treatment of a muscular disease.

In a particular embodiment, the combination product comprises a vector, such as a viral vector, in particular an AAV vector, encoding GDF5, such as one of the GDF5 proteins disclosed above.

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

In another embodiment, the combination product comprises an active ingredient suitable for the treatment of a muscular diseases, such as: - an AON suitable for exon-skipping within the SOD1 pre-mRNA, for the treatment of ALS;

- a vector, such as a viral vector, in particular an AAV vector, comprising a gene encoding a 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 within the dystrophin pre-mRNA, or a vector encoding the same, as described above, and (ii) 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.

In a further particular embodiment, the components of the combination product are for sequential, separate or simultaneous use. For example, the GDF5 pathway-activating substance may be administered before, during, or after the administration of a gene therapy vector or of an AON.

For example, the GDF5 pathway-activating substance may be administered before administration of an AON, such as an AON for use in the treatment of ALS or DMD.

For example, the GDF5 pathway-activating substance may be administered during administration of an AON, such as an AON for use in the treatment of ALS or DMD. In this embodiment, the GDF5 pathway activating substance and the AON may be either in the same composition, or in different compositions. In another example, the GDF5 pathway-activating substance may be administered after administration of an AON, such as an AON for use in the treatment of ALS or DMD.

According to another embodiment, the GDF5 pathway-activating substance may be administered before administration of a gene therapy vector for use in the treatment of a muscular disease, such as a gene therapy vector for the treatment of SMA or DMD as described above.

In a particular embodiment, the GDF5 pathway-activating substance may be administered during administration of a gene therapy vector, such as a gene therapy vector for use in the treatment of a muscular disease, such as a gene therapy vector for the treatment of SMA or DMD as described above. In this embodiment, the GDF5 pathway-activating substance and the gene therapy vector may be either in the same composition, or in different compositions.

In another particular embodiment, the GDF5 pathway-activating substance may be administered after administration of a gene therapy vector for use in the treatment of a muscular disease, such as a gene therapy vector for the treatment of SMA or DMD as described above.

Below are provided details for the treatment of DMD according to the method in two steps disclosed above. However, one skilled in the art will readily adapt the treatment strategy to other active ingredients and diseases thanks to the information provided herein and the common general knowledge in the field of gene therapy.

The treatment of Duchenne muscular dystrophy, may comprise a two-step administration of: - first an isolated antisense oligonucleotide which is complementary to a part of the dystrophin pre- mRNA and is able to induce exon-skipping during processing of this pre-mRNA to a mRNA; and

- secondly, a viral vector encoding a Duchenne muscular dystrophy therapeutic product, such as either

(i) coding an antisense oligonucleotide able to induce exon-skipping within a dystrophin pre-mRNA,

(ii) encoding dystrophin gene-editing means, or (iii) coding a functional dystrophin.

In a particular embodiment, the GDF5 pathway-activating substance may be administered before the first step.

In a further particular embodiment, the GDF5 pathway-activating substance may be administered during the first step.

In another embodiment, the GDF5 pathway-activating substance may be administered between the first step and the second step.

In another embodiment, the GDF5 pathway-activating substance may be administered after the second step.

In a further particular embodiment, the GDF5 pathway-activating substance may be during the second step.

In a particular embodiment, the therapeutic product is an U7-modified AON, in particular an U7- modified AON comprising the sequence shown in SEQ ID NO:2, targeting exon 53 of the dystrophin gene.

As described above, it is believed that the first administration of the isolated AON induces sufficient functional dystrophin expression in muscle cells to secure membrane integrity and therefore limits loss of viral vectors, in particular AON-coding viruses (e.g. AAVs), dystrophin-correcting viruses (e.g. AAVs), or functional dystrophin-coding viruses (e.g. AAVs), due to otherwise repeated cycles of necrosis-regeneration of the dystrophic myofibers.

The time period between the injection of the isolated AON and the viral vector may vary depending on a number of factors, such as the stage of the disease, the age or condition of the patient, and the dosage of the therapy. In any event, the time between the first and second steps of the present method is sufficient for providing a long-lasting benefit of the viral vector treatment. The present method allows the maintenance of high viral therapeutic genome content and improved transgene expression in dystrophic muscles. The consequences of these initial events are a therapeutic benefit stronger of the AAV based therapy lasting longer than the non-combined therapy. Accordingly, the time between the first and second step of the method of the invention may be of from 1 to 40 days, such as at least one or more days, such of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days; or of at least one or more weeks, such as at least 1, 2, 3, 4 or 5 weeks. In a particular embodiment, the time period between both administrations is of two weeks (i.e. of about 12 to 16 days, such as of about 12, 13, 14, 15 or 16 days), about three weeks (i.e. of about 19 to 23 days, such as of about 19, 20, 21, 22 or 23 days) or about four weeks (i.e. of about 26-30 days, such as of about 26, 27, 28, 29 or 30 days). More particularly, this time period is comprised between 14 and 28 days, and is more particularly of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In another embodiment, the time period between both administrations is of about 14 (i.e 13, 14 or 15 days, more specifically 14 days), 21 (i.e 20, 21 or 22 days, more specifically 21 days) or 28 days (i.e 27, 28 or 29 days, more specifically 28 days).

Further aspects and advantages of the present inventions will be disclosed in the following experimental section, which shall be considered as illustrative only, and not limiting the scope of this application.

LEGEND OF THE FIGURES

Figure 1. Viral genomes are efficiently maintained in Pip6a-PMO rescued mdx muscles

(a) TAs from mdx and wt mice were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before the injection of lE+11 vg of the non-therapeutic AAV1-U7scr vector (day 0, dO). Control mdx and wt TAs were injected with AAV1-U7scr vector alone. Four TAs were injected per group. The mice were sacrificed 3 weeks later (3w). (b) Dystrophin rescue monitored by immunostaining with the NCL-DYS2 monoclonal antibody on transverse sections of TA muscles. One representative immuno-stained section is shown per condition (c) Dystrophin restoration evaluated by western blotting with NCL-DYS1 monoclonal antibodies (upper panel) on whole protein extracts from the PPMO-treated muscles (lower panel: a-actinin). Dystrophin restoration was quantified by ImageJ software and expressed as the percentage of dystrophin expression in wt muscle (d) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as the AAV genome number relative to the value obtained for the non PPMO-treated mdx muscles. The data represent the mean values of 4 muscles per group ± SEM. n. s.: non-significant, ***p < 0.001, Student’s t-test. One of two representative experiments is shown.

Figure 2. Pip6a-PMO pre-treatment allows important dystrophin rescue at low AAV-U7ex23 dose after 6 months

(a) Mdx TAs were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before the injection of lE+10 vg of therapeutic AAV1-U7ex23 vector (day 0, dO). Control mdx TAs were injected with PPMO or AAV1-U7ex23 vector alone. Four TAs were injected per group. The mice were sacrificed 6 months later (6m). (b) Level of exon 23 skipping estimated by nested RT-PCR. The 901 bp PCR product corresponds to full-length dystrophin transcripts whereas the 688 bp product corresponds to transcripts lacking exon 23. (c) Quantification of exon 23 skipping performed by relative TaqMan qPCR and expressed as a percentage of total dystrophin transcripts (d) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as the AAV genome number relative to the value obtained for the non PPMO-treated mdx muscles. The data presented in (c) and (d) represent the mean values of the four TAs per group ± SEM. *p < 0.05, ***p < 0.001, Student’s t-test. (e) Dystrophin restoration evaluated by western blotting with NCL-DYS1 monoclonal antibodies (upper panel) on whole protein extracts from the treated muscles (lower panel: a-actinin). Dystrophin restoration was quantified by Image J software and expressed as the percentage of dystrophin expression in wt muscle.

Figure 3. Effect of Pip6a-PMO pre-treatment on AAV1 mediated micro-dystrophin gene therapy

(a) Mdx TAs were injected with 1 nmole of Pip6a-PMO two weeks (-2w) before injection of lE+10 vg of AAV1-MD1 micro-dystrophin expressing vector (day 0, dO). Control mdx TAs were injected with PPMO or AAV1-MD1 vector alone. Five TAs were injected per group. The mice were sacrificed 4 weeks later (4w). (b) Quantification of AAV genomes by absolute Taqman qPCR. AAV genome content is expressed as the AAV genome number relative to the value obtained for the non PPMO-treated mdx muscles. The data represent the mean values of the 5 muscles per group ± SEM. *p < 0.05, Student’s t- test. (c) Expression of PPMO-induced dystrophin (DYS, 427kDa) and micro-dystrophin (pDYS, 132kDa) evaluated by western blotting with MANEX1011B monoclonal antibodies (upper panel) on whole protein extracts from the treated muscles (lower panel: a-actinin).

Figure 4. GDF5 overexpression in young mice muscle.

A-C: RT-qPCR for (A) Gdf5, (B) Cacnbl-E (Ex2-3) and (C) Cacnbl-D in adult TAs innervated (Inn) or denervated for 15 days (Den) treated with Sera or GDF5. A: minimum medium axis=150; minimum top axis =3000.

D, E: Immunofluorescence images of TA Inn (top) or Den (bottom) treated with (D) Sera or (E) GDF5, stained with GDF5 (Magenta), CaV iE (Y ellow). Bar: 10 pm.

F, G: RT-qPCR for (F) Idl and (G) Id2 in TAs Inn or Den treated with Sera or GDF5.

H, I: Haematoxylin and eosin (El/E) staining of TAs Inn (top) or Den (bottom) treated with (H) Sera or (I) GDF5. Bar 100 pm.

J, K: Sirius red (SR) staining of TAs Inn (top) or Den (bottom) treated with (J) Sera or (K) GDF5. Bar 100 pm.

L: Muscle/body-weight ratio of Inn or Den adult TAs treated with Sera or GDF5.

A: Means ± s.e.m. (n=6) *P < 0.05, ***P < 0.001, *P < 0.05, ** P < 0.01, ***P < 0.001; (two stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. Each row was analyzed individually, without assuming a consistent SD).

B, C, F-G, L: Means ± s.e.m. (n=6) *P < 0.05, ***P < 0.001, *P < 0.05, ** P < 0.01, ***P <0.001, (ordinary one-way Anova - by Sidak’s test). EXAMPLES

Materials and Methods

Viral vector production and animal experiments

A three-plasmid transfection protocol was used with pAAV(U7smOPT-SD23/BP22), pAAV(U7smOPT-scr) and codon optimized pAR4-R23/ACT (MD1) plasmids for generation of single strand AAV1-U7ex23 (7), AAV1-U7scr (13) and AAV1-MD1 (37) vectors. AAV-GDF5 has been generated by direct cloning of Gdf5 ORF (NM_008109.2), flanked by EcoRI and Nhel sites (GeneArt string; ThermoFisher), in pSMD2 AAV2 vectors backbones, under CMV promoter. pSUPER retro puro Scr ShRNA (SCRA) was a gift from John Gurdon (Addgene plasmid #30520). BamHI site has been inserted by PCR and the Hl-SCRA cassette has been cloned in pSMD2-sh through BamHI and Sail sites. The final viral preparations were kept in PBS solution at -80°C. Vector titers were determined by real-time PCR and expressed as vector genomes per ml (vg/ml). Three-month-old mdx mice were injected into the Tibialis anterior (TA) muscles with 1 nmole of Pip6a-PMO oligonucleotides (GGCCAAACCTCGGCTTACCTGAAAT - SEQ ID NO: 11) (20). Additionally, 50m1 of AAV1-U7scr, AAV1-U7ex23 or AAV1-MD1 containing lE+10 or lE+11 vg were injected into C57BL/6 (wt) or mdx TAs. AAV-GDF5 was injected into 8 week-old C57BL/6 TAs at 5E+10. As control, 8-week-old C57/BL6 mice were injected using the same procedure with SCRA AAV vector. Mice were sacrificed 10 or 12 weeks after the injection. These animal experiments were performed at the Myology Research Center, Paris, France, according to the guidelines and protocols approved by the Institutional Review Board. A minimum of four mice were injected per group for each experiments. At sacrifice, muscles were collected, snap-frozen in liquid nitrogen-cooled isopentane and stored at -80°C.

Denervation experiments

Ten weeks after injection of mice with AAV, the sciatic nerve was neuroectomized (ablation of a 5 -mm segment of the sciatic nerve) under general anesthesia (Isofluorane, 3% induction, 2% maintenance) with Buprenorphine (vetergesic lmg/Kg, subcutaneous). Mice were sacrificed 1, 3, 7 or 15 days after denervation, and TA were dissected, weighed and thereafter frozen in isopentane precooled in liquid nitrogen and stored at -80 °C until histology or molecular analysis.

Viral genome quantification

Genomic DNA was extracted from mouse muscles using Puregene Blood kit (Qiagen). Copy number of AAV genomes and genomic DNA were measured on lOOng of genomic DNA by absolute quantitative real-time PCR on a StepOnePlusTM (Applied Biosystems) using the TaqmanR Universal Master Mix (Applied Biosystems). Primers (forward: CTCCATCACTAGGGGTTCCTTG (SEQ ID NO:3) and reverse: GTAGATAAGTAGCATGGC (SEQ ID NO: 4)) and probe (TAGTTAATGATTAACCC (SEQ ID NO:5)) were used to specifically amplify the viral genome sequence. As a reference sample, a pAAV plasmid was 10-fold serially diluted (from 10 ⁷ to 10 ¹ copies). All genomic DNA samples were analyzed in duplicates.

RT-PCR analysis

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

Gene expression analysis by RT-qPCR

Total RNA was prepared from TA cryosections using TRizol (Life Technologies) following the manufacturer’s instructions. Complementary DNA was generated with Superscript II Reverse transcriptase (Life Technologies), amplified using PCR Master Mix (M7505, Promega) for RT-PCR oranalyzed by real-time qPCR. Real-time qPCR was performed on StepOne Plus Real-Time PCR System (Applied Biosystems) using Power SyberGreen PCR MasterMix (Applied Biosystems). All data were analyzed using the AACT method and normalized to PO (mouse acidic ribosomal phosphoprotein) mRNA expression levels. The sample reference to calculate mRNA fold change is indicated in each panel. Primers used are listed

Western blot analysis

Protein extracts were obtained from pooled muscle sections treated with 125 mM sucrose, 5 mM Tris- HC1 pH 6.4, 6% of XT Tricine Running Buffer (Bio-Rad), 10% SDS, 10% Glycerol, 5% b- mercaptoethanol. The samples were purified with the Pierce Compat-Able™ Protein Assay preparation Reagent Set (Thermo Scientific) and the total protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were denatured at 95 °C for 5 minutes and 100 pg of protein were loaded onto Criterion XT Tris-acetate precast gel 3-8% (Bio-Rad). Membrane was probed with primary monoclonal antibodies directed against dystrophin (NCL-DYS1, 1:50, Leica Biosystems; MANEX101 IB, 1:50, kindly gifted by The Muscular Dystrophy Association Monoclonal Antibody Resource (38)) and a-actinin (1: 1000, Sigma- Aldrich), followed by incubation with a sheep anti-mouse secondary antibody (horseradish peroxidase conjugated; 1: 15000) and Pierce ECL Western Blotting Substrate (Thermo Scientific).

Immunohistochemistry and Histology

TA sections of 12 pm were cut and examined for dystrophin expression using the NCL-DYS2 monoclonal antibody (Leica Biosystems). Rabbit polyclonal antibody for O'hnb 1 C-terminus (API 6144b) was purchased from AbGent and the mouse monoclonal to GDF5 was obtained by Santa Cruz Biotechnologies (SC-373744). Fluorescent secondary antibodies goat anti-rabbit and goat anti mouse were purchased from Fife technologies.

For H&E staining, sections were fixed in 4% PFA for 10 min, washed in PBS and then stained in haematoxylin for 5 min and eosin for 30 sec. The muscle sections were further dried in gradually increasing concentration of ethanol/water solutions and, after fixation in 100% xylene, were mounted in Vectamount (Vector Faboratories). Sirius Red staining was performed to analyze total collagen I and III content. Muscle cryosections were fixed in PFA 4% for 10 min, washed in water and dried in 100% ethanol for 5 min. Sections were then stained in Picro-Sirius Red (0.3%) solution for 1 h while protected from light. After a washing in acidified water (5 min in acetic acid 0.5% vol/vol), sections were fixed in 100% ethanol (3 washes for 5 min) and the final dehydration was performed in xylene 100%, mounted in Vectamount and visualized using a macroscope Nikon AZ100. Confocal images were taken with Feica SPE or a Nikon Ti2 microscope equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometries).

Results

Effect of dystrophin restoration by AON pre-treatment on non-therapeutic viral genome maintenance

In order to induce temporary dystrophin expression at the sarcolemma of Mdx myofibers, Mdx Tibialis anterior (TA) muscles were injected with llpg of Pip6a-PMO (20), a peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotide that is particularly efficient for mdx exon skipping. The non-therapeutic AAV-U7scr vectors (carrying a scrambled, non-specific sequence) were injected in the same muscles two weeks later (Fig. la), when dystrophin rescue was optimal, at a high dose (lE+11 viral genomes). We had previously shown that these U7scr vectors, which are unable to induce exon skipping and thereby to rescue dystrophin expression, are drastically lost within three weeks from dystrophin-deficient mdx muscle (13).

Following AON pre-treatment inducing exon skipping, three weeks after AAV1-U7scr injection, immunofluorescence staining revealed a strong dystrophin restoration and its correct localization at the sarcolemma in mdx injected muscles (Fig. lb). Dystrophin levels were quantified in mdx muscles by western blotting and showed that the AON pre-treatment resulted in 56 to 98% of quasi-dystrophin restoration compared to normal levels (Fig. lc). As expected (13), the viral genome content analyzed by quantitative PCR (qPCR) was 6 times higher in wild-type muscles (wt) than in non AON treated mdx muscles. Interestingly, the viral genome content analyzed by qPCR in the mdx AON treated group is similar to the one observed in wt muscles (Fig. Id). Therefore, a significant dystrophin expression induced by PPMO pre-treatment at the time of AAV1-U7scr injection protects against the rapid loss of AAV1-U7scr genomes in mdx muscles comparable to what was observed in wt muscles.

Effect of Pip6a-PMO pre-treatment on dystrophin rescue at low dose of therapeutic AAV-U7ex23

AAV1 vectors encoding the U7ex23 (AAV1-U7ex23) allow efficient exon 23 skipping and therefore quasi-dystrophin rescue in the mdx muscles. To evaluate the benefit of an AON pre-treatment on the quasi-dystrophin rescue via AAV1-U7ex23, we injected llpg of Pip6a-PMO antisense oligonucleotides into mdx TAs two weeks before injection of AAV1-U7ex23 vectors (Fig.2A). A low vector dose (lE+10 viral genomes) was chosen as this dose allows a weak quasi-dystrophin rescue (less than 5% of the normal levels) (13).

The benefit of AAV1-U7ex23 injection was analysed six months later when dystrophin rescue induced by the single PPMO injection was nearly abolished. Levels of exon 23 skipping analyzed by nested RT- PCR (Fig. 2b) and quantified by qPCR (Fig. 2c) in mdx TAs treated with AAV1-U7ex23 or PPMO alone were low as expected, respectively 9 and 6% of skipped transcripts, leading to the synthesis of rescued dystrophin around 2% of the normal level (Fig. 2e). Conversely, TAs treated sequentially with PPMOs and AAV1-U7ex23 showed 54% of skipped transcripts (Fig. 2c) and 20% of the normal levels of dystrophin (Fig. 2e). AAV genome copy number quantified by absolute qPCR was 8 fold higher in the dual PPM0/AAV1-U7ex23 treated muscles than in AAV1-U7ex23 only injected muscles (Fig. 2D). These data demonstrate that the PPMO pre-treatment allowed a better maintenance of the therapeutic U7ex23 genomes in the mdx muscles six months after the AAV-U7 injections and remarkably resulted in a 10 fold improvement of the rescued dystrophin amount.

Pip6a-PMO pre-treatment significantly increases the efficacy of AAV1 mediated micro dystrophin gene therapy

To evaluate the efficacy of an AON pre-treatment on AAV-micro-dystrophin gene therapy, we injected Pip6a-PMO AONs into mdx TAs two weeks before injection of AAV1-MD1 vector (lE+10 vg) expressing a murine micro-dystrophin (MD1) (37) (Fig.3a). Four weeks later, a strong dystrophin restoration was observed in PPMO-treated mdx TAs induced by the PPMO pre-treatment (Fig. 3c). AAV genome copy number and micro-dystrophin expression were 3-fold higher in the PPMO/AAV1- MD1 treated muscles than in AAV1-MD1 only treated muscles (Fig. 3b&c), illustrating the PPMO pre treatment benefit on AAV-micro-dystrophin gene therapy. This experiment establishes the proof of concept that the AON pre-treatment is capable of enhancing all AAV-based gene therapies for DMD. Effect of GDF5 overexpression

We overexpressed GDF5 in young TAs (Fig 4 A, D, E) which induced Cacnbl- E transcription in innervated TAs compared to scrambled (Fig 4 B, D, E) without affecting Cacnbl -D expression (Fig 4 C). Nevertheless, GDF5 over-expression and its activated signaling checked by Id-1 and Id-2 transcription (Fig 4 F, G) increased mostly innervated muscle mass (Fig 4 H-L).

Discussion

AAV genomes are rapidly lost from dystrophic muscles during AAV-U7-mediated exon-skipping therapy, certainly because of their episomal nature and the fragility of the dystrophic muscle fibers that undergo cycles of necrosis/generation, show abnormally leaky membranes, and in addition are characterized by increased excretion of exosomes and microparticles (36). We showed here that a significant (>60%) quasi-dystrophin rescue following PPMO pre-treatment at the moment of AAV-U7 injections allows an efficient maintenance of the viral genomes in mdx muscles three weeks later. Additionally, this initial maintenance of viral genomes increases quasi-dystrophin restoration by AAV- U7, around 6 fold at RNA level and around 10 fold at protein level six months later.

The PPMO pre-treatment resulted in substantial dystrophin expression at the time of AAV-U7 injection. This likely reduces, like in normal control muscle, the membrane abnormalities leading to AAV genome loss before AAV-U7 induced quasi-dystrophin expression occurs. Once established, a AAV-U7 mediated high quasi-dystrophin expression will be maintained because it will by itself prevent transgene loss. Hence, by allowing the maintenance of high viral genome content in the critical period between AAV injection and AAV-mediated transgene expression in the treated dystrophic muscles, PPMO- mediated quasi-dystrophin restoration guarantees a long-lasting benefit of AAV-U7 treatment.

This pre-treatment could be induced by any AONs allowing substantial quasi-dystrophin rescue (i.e. using different skippable mutations and different target sequences and different AON chemistries such as tricyclo-DNA (36)) using the principle demonstrated here with the PPMO chemistry.

This AON pre-treatment is applicable to all therapeutic approaches for Duchenne myopathy using AAV vectors, in particular AAV-U7-mediated exon skipping and classical gene therapy with transfer of functional micro-dystrophin cDNAs into muscles, as demonstrated thanks to the data presented herein.

In addition, it is herein shown that muscle mass is increased when GDF5 is overexpressed, meaning that muscles to be treated can further be protected during the application of the above therapeutic strategy thanks to the administration of either a vector expressing GDF5, or a recombinant GDF5 protein. On the eve of clinical trials using AAV-based therapies for DMD patients, this study underscores the strong impact of combined approaches to improve the benefit of AAV-based therapies allowing the use of lower and thus safer vector doses for a larger level of dystrophin expression in the long term.

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