WO2022122733A1

WO2022122733A1 - New gene therapy for the treatment of duchenne muscular dystrophy

Info

Publication number: WO2022122733A1
Application number: PCT/EP2021/084602
Authority: WO
Inventors: Kathryn GIBLIN; Frédéric Revah
Original assignee: Genethon
Priority date: 2020-12-08
Filing date: 2021-12-07
Publication date: 2022-06-16

Abstract

The present invention concerns a method of treating Duchenne muscular dystrophy (DMD) in a human, comprising systemically administering by intravascular injection a gene therapy product that comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence encoding a human ΔR4-R23/ΔCT microdystrophin. Advantageously, the gene therapy product is injected up to 1E14 vg/kg.

Description

NEW GENE THERAPY FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY

The present invention provides an efficient gene therapy based on a sequence encoding the microdystrophin encapsulated in an AAV8 vector for the treatment of Duchenne muscular dystrophy (DMD).

BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD) is the most frequent progressive muscle degenerative disease, affecting approximately one in 3,500 to 5000 male births. DMD is caused by deletions or mutations in the gene encoding dystrophin, located on the X chromosome. Dystrophin is required for the assembly of the dystrophin-glycoprotein complex, and provides a mechanical and functional link between the cytoskeleton of the muscle fiber and the extracellular matrix. The absence of functional dystrophin causes fiber degeneration, inflammation, necrosis and replacement of muscle with scar and fat tissue, resulting in progressive muscle weakness and premature death due to respiratory and cardiac failure between the second and fourth decade of life (Moser, H., Hum Genet, 1984. 66(1): p. 17-40).

WO2015/197869 patent application describes a gene therapy product made of 2 components:

- The encapsidated recombinant nucleic acid sequence which defines the expression cassette that provides the therapeutic benefit(s) once expressed in the target cell/tissue; and

- The viral capsid which allows proper gene transfer and to a certain extent, tissue tropism.

The gene therapy product comprises a nucleic acid sequence encoding a functional microdystrophin. Microdystrophin means a peptide or protein, which is shorter than the native or wild type dystrophin. In the context of the invention, the terms “microdystrophin” and “minidystrophin” have the same meaning. In the rest of the application, the term “microdystrophin” will be used, as well as the abbreviations “pdystrophin”, “MD” or “pDys”.

The structure of dystrophin is well documented and active fragments thereof have been disclosed (Athanasopoulos et al., Gene Ther 2004 Suppl 1 : S 109-21). As would be understood in the art, an active fragment is a portion or portions of a full length sequence that retain some biological function of the full length sequence. The full-length dystrophin is characterized by different domains:

- A N-terminal domain which binds to actin;

- 4 hinge domains (Hl to H4);

- 24 spectrin-like repeats or rod domains (1 to 24);

- A cysteine-rich domain;

- A C-terminal domain.

A particularly interesting human microdystrophin has the configuration AR4-R23/ACT, comprising 4 spectrin-like repeats, i.e. spectrin-like repeats 1, 2, 3 and 24 as described in WO2015/197869. More precisely, this sequence comprises deletions of rod domains 4-23 and exons 71-78 of the CT domain of dystrophin, and contains the last three amino acids of exon 79 of dystrophin followed by three stop codons.

Such human microdystrophin noted AR4-R23/ACT or MD1 has e.g. the amino acid sequence described in WO2015/197869.

A particularly interesting viral capsid is an AAV of serotype 8 (AAV8 vector).

A particularly interesting Gene therapy vector is a rAAV2/8-SPc5.12-MD encoding an mRNA sequence-optimized human dystrophin (hMD) under the control of a muscle-specific promoter (Spc5.12) and incorporating the SV40 poly adenylation site. Said Gene therapy vector is described in WO2015/197869.

BRIEF SUMMARY OF THE INVENTION

The present invention aims to alleviating or curing the devastating Duchenne muscular dystrophy (DMD) by improving said Gene therapy vector.

Definitions

Unless otherwise defined, all technical and scientific terms used therein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting. The articles “a” and “an” are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” or “approximately” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a nonnative environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

A “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA or a cDNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

“Identical” or “homologous” refers to the sequence identity or sequence similarity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or identical at that position. The percent of homology/identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched then the two sequences are 60% identical. Generally, a comparison is made when two sequences are aligned to give maximum homology/identity.

A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include nonplasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term “promoter” as used herein is defined as a DNA sequence recognized by the transcriptional machinery of the cell, or introduced transcriptional machinery, required to initiate the specific transcription of a polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence, which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements, which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one, which expresses the gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell preferentially if the cell is a cell of the tissue type corresponding to the promoter.

The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics, which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. A subject can be a mammal, e.g. a human, a dog, but also a mouse, a rat or a nonhuman primate. In certain embodiments, the patient, subject or individual is a human.

A “disease” or a “pathology” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

A disease or disorder is “alleviated” or “ameliorated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced. This also includes halting progression of the disease or disorder. A disease or disorder is “cured” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is eliminated.

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of pathology or has not be diagnosed for the pathology yet, for the purpose of preventing or postponing the occurrence of those signs. As used herein, “treating a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

An “effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase “therapeutically effective amount”, as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a method of treating Duchenne muscular dystrophy (DMD) in a human, comprising systemically administering by intravascular injection a gene therapy product that comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors the nucleic acid sequence SEQ ID NO: 1, encoding a human AR4-R23/ACT microdystrophin.

Thus, the present invention provides a method for treating DMD in a human, wherein:

- a gene therapy product is systemically administered by intravascular injection to said human;

- the gene therapy product comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence SEQ ID NO: 1 encoding a human AR4- R23/ACT microdystrophin.

In other words, the present invention relates to a gene therapy product that comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence SEQ ID NO: 1 encoding a human AR4-R23/ACT microdystrophin, for use in the treatment of DMD in a human, wherein the gene therapy product is systemically administered by intravascular injection. The invention concerns the use of a gene therapy product comprising an adeno- associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence SEQ ID NO: 1 encoding a human AR4-R23/ACT microdystrophin, for the preparation of a medicament for the treatment of DMD in a human, wherein the medicament is systemically administered by intravascular inj ection. The invention concerns an agent for treating DMD in a human consisting of a gene therapy product comprising an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence SEQ ID NO: 1 encoding a human AR4-R23/ACT microdystrophin, systemically administered by intravascular injection.

According to one aspect, the gene therapy product to be used in the frame of the present invention comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence encoding a human AR4-R23/ACT microdystrophin.

As known in the art, a AR4-R23/ACT microdystrophin comprises 4 spectrin-like repeats, i.e. spectrin-like repeats 1, 2, 3 and 24. More precisely, it comprises deletions of rod domains 4-23 and exons 71-78 of the CT domain of dystrophin, and contains the last three amino acids of exon 79 of dystrophin followed by three stop codons.

Such a microdystrophin (named MD1 or hMDl for human MD1) has e.g. the amino acid sequence shown in sequence SEQ ID NO: 3, 4 or 7 of WO2015/197869.

According to a preferred embodiment, the nucleic acid sequence encoding the microdystrophin comprises nucleotides 586 to 4185 of sequence SEQ ID NO: 1.

According to another preferred embodiment, the nucleic acid sequence encoding a human AR4- R23/ACT microdystrophin harbored by the adeno-associated viral (AAV) vector of serotype 8, comprised in the gene therapy product of the invention, comprises or consists of sequence SEQ ID NO: 1.

As mentioned above, the gene therapy product of the invention comprises an adeno-associated viral (AAV) vector of serotype 8.

According to a specific embodiment, the isolated nucleic acid is inserted into said vector. In brief summary, the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

According to the invention, the vector harboring the nucleic acid sequence encoding a human AR4-R23/ACT microdystrophin is an adeno-associated viral (AAV) vector. In one embodiment, the encoding sequence is contained within an AAV vector. More than 100 naturally occurring serotypes of AAV are known. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for dystrophic pathologies. AAV viruses may be engineered using conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus.

In one embodiment, the vectors useful in the compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, i.e. an AAV8 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, i.e. AAV8 rep protein, or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype origin, in the present case all of AAV8 origin. Alternatively, vectors may be used in which the rep sequences are from an AAV serotype, which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector. In some embodiments the AAV vector comprises a genome and a capsid derived from AAVs of different serotypes.

Thus exemplary AAVs, or artificial AAVs, include AAV8 and AAV2/8 (US 7,282,199).

According to the invention, the AAV is of serotype 8. In other words, the vector comprises an AAV8 capsid. Advantageously, the claimed vector is an AAV8 vector or an AAV2/8 vector.

In the AAV vectors used in the present invention, the AAV genome may be either a single stranded (ss) nucleic acid or a double stranded (ds) / self complementary (sc) nucleic acid molecule.

Advantageously, the gene of interest or transgene is inserted between the ITR (« Inverted Terminal Repeat ») sequences of the AAV vector. Typically, ITR sequences originate from AAV2. Advantageously, the typical ITR sequences correspond to: nucleotides 1 to 128 of sequence SEQ ID NO: 1 (5TTR sequences); nucleotides 4511 to 4640 of sequence SEQ ID NO: 1 (3TTR sequences). In one embodiment, the AAV vector comprises regulatory sequences, especially a promoter sequence. Such promoters can be natural or synthetic (artificial) promoters, inducible or constitutive.

In a preferred embodiment, the promoter sequence is chosen in order to adequately govern the expression of the nucleic acid sequence placed under its control, in terms of expression level, but also of tissue specificity. In one embodiment, the expression vector comprises a muscle specific promoter. Such a promoter allows a robust expression in the skeletal muscles, and possibly in the cardiac muscle as well as in the diaphragm. A preferred promoter is the synthetic promoter C5-12 (spC5-12) as shown in sequence SEQ ID NO: 1 (nucleotides 215 to 537), which allows a robust expression in skeletal and cardiac muscles.

A non-exhaustive list of other possible regulatory sequences is:

- a polyadenylation signal, e.g. the polyA of the gene of interest, the polyA of SV40 or of beta hemoglobin (HBB2), advantageously in 3’ of the sequence encoding the microdystrophin ; The poly A of SV40 is disclosed in sequence SEQ ID NO: 1 (nucleotides 4223 to 4353);

- sequences for transcript stabilization, e.g. intron 1 of hemoglobin (HBB2);

- enhancer sequences ;

- miRNA target sequences, which can inhibit the expression of the sequence encoding the functional dystrophin in non target tissues, in which said expression is not desired, for example where it can be toxic. Preferably, the corresponding miRNA is not present in the skeletal muscles, and possibly not in the diaphragm nor in the heart.

According to one embodiment, the gene therapy product comprises an AAV vector harboring the sequence SEQ ID NO: 1.

Recombinant viral particles can be obtained by any method known to the one skilled in the art, e.g. by co-transfection of 293 HEK cells, by the herpes simplex virus system and by the baculovirus system. The vector titers are usually expressed as viral genomes per mL (vg/mL).

According to a preferred embodiment of the invention, the gene therapy product is produced using a three-plasmid transfection of HEK293T cells, as known by the skilled person.

According to a further preferred embodiment of the invention, the gene therapy product is purified using separation on CsCl gradient. According to a specific embodiment, the gene therapy product is purified using affinity chromatography, e.g. on an AVB column, followed by separation of empty/full particles on CsCl gradient. According to a further aspect, the gene therapy product is formulated in a pharmaceutical composition. Such a composition comprises a therapeutically effective amount of the gene therapy product, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to release pain at the site of the injection.

In one embodiment, the composition according to the invention is suitable for administration in humans. The composition is preferably in a liquid form, advantageously a saline composition, more advantageously a phosphate buffered saline (PBS) composition or a Ringer-Lactate solution.

The amount of the gene therapy product which will be effective in the treatment of Duchenne muscular disease (DMD) can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, the weight and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, as shown in the examples below and in an unexpected manner, the efficient doses are significantly below the one disclosed in WO2015/197869 as efficient for intravenous administration in mutant dogs (1E14 vg/kg). As known by the skilled person, a dose as low as possible given a satisfying result in term of efficiency is preferred in order to avoid potential toxicity and/or immune reactions.

Suitable administration should allow the delivery of a therapeutically effective amount of the gene therapy product to the target tissues, especially skeletal muscles and possibly smooth muscles (e.g. esophagus), diaphragm and heart. In the context of the invention, the therapeutic dose is defined as the quantity of viral particles (vg for viral genomes) containing the microdystrophin sequence, administered per kilogram (kg) of the human.

Available routes of administration are topical (local), enteral (system-wide effect, but delivered through the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered by routes other than the GI tract). The preferred route of administration of the compositions disclosed herein is parenteral which includes systemic administration (i.e. into the circulating system). In this context, the term “injection” (or “perfusion” or “infusion”) encompasses intravascular, in particular intravenous (IV) administration. Injections are usually performed using syringes.

In one embodiment, the composition is injected in a limb of the subject. In one embodiment, the subject is a mammal, preferably a human. When the subject is a human, the limb can be the arm or the leg. According to one embodiment, the composition is administered in the lower part of the body of the subject, e.g. below the knee, or in the upper part of the body of the subject, e.g., below the elbow. Advantageously, the gene therapy product is administered in the forearm.

A preferred method of administration according to the invention is systemic administration. Systemic injection opens the way to an injection of the whole body, in order to reach the entire muscles of the body of the subject including the heart and the diaphragm and then a real treatment of these systemic and still incurable diseases. In certain embodiments, systemic delivery comprises delivery of the composition to the subject such that composition is accessible throughout the body of the subject.

According to a preferred embodiment, systemic administration occurs via injection of the composition in a blood vessel, i.e. intravascular (intravenous or intra-arterial) administration. According to one embodiment, the composition is administered by intravenous injection, through a peripheral vein. The systemic administration is typically performed in the following conditions: a flow rate of between 1 to 10 mL/kg/min, advantageously between 1 to 5 mL/kg/min, e.g. 3 mL/kg/min;

- the total injected volume can vary between 1 and 20 mL, preferably 5 mL of vector preparation per kg of the subject. The injected volume should not represent more than 10% of total blood volume, preferably around 6%.

When systemically delivered, the composition is preferably administered with a dose less than or equal to 10¹⁴ vg/kg (1E14 vg/kg), advantageously with a dose less than or equal to 1, 2, 3, 4, 5, 6, 7, 8 or 9.10¹³ vg/kg.

According to specific embodiments, the gene therapy product is injected up to 1E14 vg/kg, advantageously up to 6E13 vg/kg, more advantageously up to 3E13 vg/kg. Preferred doses are 6E13 vg/kg, advantageously 3E13 vg/kg or IE 13 vg/kg.

This significant and unexpected improvement at a lower dose is demonstrated in the Examples below in relation to a rat model (see Example 2). It is likely to be further demonstrated in an in vivo study such as the clinical study of Example 3 where patients are treated with the gene therapy product as disclosed above at a dose of 1E13 vg/kg, 3E13 vg/kg or 6E13 vg/kg.

Useful criteria for selecting the appropriate dose, besides safety consideration, are e.g. (see Example 3 for details):

At a molecular level:

Vector shedding quantification in blood, urine, saliva, feces (DNA level) Creatine kinase CK (blood)

- Muscular Biopsy of biceps brachii (at baseline and in the contralateral muscle) Quantification of hMDl (and endogenous dystrophin) positive fibers (and location) Quantification of hMDl (and endogenous dystrophin) protein expression Quantification of vector copy number VCN (DNA)

General histology (necrosis, infiltration, etc)

- DAPC staining

- Regeneration index

Sarcolemnal permeability

At a clinical level:

- NorthStar Ambulatory Assessment (NSAA)

Time to 10 Meters Walk/Run Test (10MWRT)

Time to Rise From Floor (RFF)

- 6-Minutes Walk Test (6MWT) MyoTools (Grip, Pinch) ActiMyo (Wearable Device - Sysnav®: Stride velocity 95th centile measured at the wrist and the ankle, SV95C)

- Respiratory function tests (Forced Vital Capacity (FVC); PEF (Peak Expiratory Flow); Forced Expiratory Volume in the first second of exhalation (FEV1); Peak Cough Flow (PCF); Vital Capacity (VC); Respiratory frequency (fR))

- Muscle Magnetic Resonance Imaging (MRI), especially qNMRI, i.e. % of Fat and contractile cross-sectional area (Dixon) in glutei and thighs

Patient questionnaire (ACTIVLIM, EQ-5D) Other efficacy biomarkers.

In a specific embodiment, the treatment comprises a single administration of the gene therapy product.

In one embodiment, the presence of the gene therapy product and/or the expression of the microdystrophin, as well as the associated therapeutic benefits, are observed for up to 1 month, or 3 months or 6 months or even 1 year, 2 years, 5 years, 10 years, or even the whole life of the subject.

Said gene therapy product is intended to treat dystrophic diseases. “Dystrophic disease” means a disease linked to a defect in the dystrophin gene. This defect can be deletions or mutations leading to low level of expression or absence of expression, introduction of a premature stop codon in the open reading frame, or the production of an inactive protein. Preferred dystrophic diseases are Duchenne and Becker muscular dystrophy (DMD/BMD) caused by mutations of the dystrophin gene, advantageously DMD. Said mutations can result in the absence or a low level of dystrophin expression, or in the production of a partially or fully inactive, possibly truncated protein.

Subjects that could benefit from said gene therapy include all patients diagnosed with a muscular dystrophy or at risk of developing such a muscular dystrophy. A subject to be treated can then be selected based on the identification of mutations or deletions in the dystrophin gene by any method known to the one skilled in the art, including for example sequencing of the dystrophin gene, and/or through the evaluation of the dystrophin level of expression or activity by any method known to the one skilled in the art. Therefore, said subjects include both subjects already exhibiting symptoms of a dystrophic disease and subjects at risk of developing said disease. In one embodiment, said subjects include subjects already exhibiting symptoms of a dystrophic disease. In another embodiment, said subjects are ambulatory patients and early non- ambulant patients. A first target of the invention is to provide a safe (not toxic) treatment. A further aim is to provide an efficient treatment which allows to postpone, slow down or prevent the development of the disease, and possibly to ameliorate the phenotype of the patient which can be easily monitored at the clinical level.

In a subject, the gene therapy product according to the invention can be used: for ameliorating muscular function. Of particular interest are the skeletal muscles, but also the cardiac muscle and the diaphragm; for ameliorating gait; for ameliorating cardiac function; for ameliorating respiratory function; for ameliorating digestive function; and/or for prolonging survival, more generally to ameliorate the quality and the expectancy of life.

According to one aspect, the invention concerns a method for ameliorating muscular function, gait, digestive function, cardiac function and/or respiratory function, and/or for prolonging survival, advantageously without adverse effects (cellular and/or humoral immune response), comprising administering to a subject in need thereof a therapeutic quantity of a gene therapy product as disclosed above.

Advantageously, said ameliorations are observed for up to 1 month after administration, or 3 months or 6 months or 9 months, more advantageously for up to 1 year after administration, 2 years, 5 years, 10 years, or even for the whole life of the subject.

In one embodiment, said ameliorations results in reduced symptom severity and/or frequency and/or delayed appearance, wherein said symptom is chosen within the group consisting of frequent fall, inability to walk, dysphagia, cardiomyopathy, ptyalism, reduced motor skills (running, hopping, jumping), breathing abnormalities, pseudohypertrophy, lumbar hyperlodosis, and muscle stiffness.

An amelioration of said functions can be evaluated based on methods known in the art, e.g.: assessment of the percentage of muscle fibers expressing the dystrophin protein;

- walking tests; assessment of strength by dynamometer measurements; assessment of motor function of a precise limb by motor function measurements; assessment of global activity using a movement monitor; assessment of gait by accelerometric recording in 3 axes; assessment of cardiac function by echocardiographic, Doppler analyses and Speckle tracking analysis; assessment of respiratory function by evaluation of diaphragm kinetics; assessment of vital functions, especially cardiac, respiratory and digestive functions, by clinical follow-up; assessment of quality and expectancy of life by clinical score.

As illustrated in the examples below, the claimed treatment allows improving the clinical state and the various parameters disclosed above in comparison with an untreated subject.

As known in the art, the level of dystrophin expression in muscles is easily determined by the skilled person, advantageously by immunohistochemistry, e.g. by immunostaining of muscular biopsies with an anti-Dystrophin antibody as disclosed above. The calculation of clinical scores is also routine for the skilled person. Concerning patients, Bushby and Connor have e.g. listed clinical outcome measures for trials in Duchenne muscular dystrophy (Clin Investig (Lond). 2011; 1(9): 1217-1235).

According to a particular embodiment, the therapy comprises at least said gene therapy product, and possibly other active molecules (other gene therapy products, chemical molecules, peptides, proteins. . .), dedicated to the treatment of the same disease or another disease.

As it will be illustrated in the examples below, the administration of the gene therapy product according to the invention is not believed to be associated with adverse immune reactions. Therefore, and according to one embodiment, said administration is not combined with any further or extra immunosuppressive treatment (immunosuppression).

Alternatively, the human receiving the administration of the gene therapy product undergoes a prophylactic systematic immunosuppressive therapy, e.g. using prednisolone (or prednisone) and/or methylprednisolone. A protocol can be as follows:

1 mg/kg/day per oral (PO) of prednisolone (or prednisone) the calendar day before infusion of the gene therapy product

- 1 mg/kg/day per oral (PO) of prednisolone (or prednisone) the day infusion (at least 3 hours prior to the infusion)

- 100 mg intravenous bolus of methylprednisolone administered in 15 minutes the day of infusion (60 minutes prior to the start of infusion)

- 2 mg/kg/day PO of prednisolone (or prednisone) for 5 calendar days starting the day after infusion, before tapering the dose

- Then Img/kg/day PO of prednisolone (or prednisone) for 8 weeks before tapering the dose - Tapering dose: step down acceptable range of dose change is 25% of the previous dose (a window of at least one week at stable dosing is recommended between any steps down of dose)

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting.

The results presented below have been obtained with the gene therapy product disclosed n WO2015/197869 (SEQ ID NO: 1 harbored by an AAV8, thereafter named rAAV8-MDl or rAAV8-hMDl or rAAV2/8-Spc5.12-hMDl-spA or GNT0004) in the DMDmdx rat model (see EXAMPLES 1 and 2), a powerful animal model for DMD, and provide proof-of-concept for the ongoing clinical trials in DMD patients (see EXAMPLE 3). 1. EXAMPLE 1: STUDY IN ANIMALS

Twenty-eight (28) rats were included in a pilot study, which goals were to evaluate:

- The immunogenicity and the efficacy of the human microdystrophin (hMDl) protein after rAAV8-mediated gene transfer in the DMDmdx and the non affected rat models;

- The impact of the manufacturing process (Sf9/baculovirus platform vs 293/transfection platform) on the efficacy of rAAV8-MDl gene transfer in the DMDmdx rat model.

Results:

- The different rAAV8-MDl vectors have the ability to transduce the target organs for DMD treatment, i.e. the skeletal muscles, the heart and the diaphragm to some degree. This allows expression of MD1 (of human or of canine origin) mRNA and of MD1 protein.

- The hMDl transgene is efficient in DMDmdx rats, to restore tissue pathology (fibrosis) in skeletal muscles and in heart, to restore muscle function (grip force), but also to restore cardiac function.

- The manufacturing process (Sf9/baculovirus +AVB/CsCl platform vs 293/transfection + CsCl platform) has a surprising impact on the efficacy of rAAV8-hMDl gene transfer in the DMDmdx rat model, the 293/transfection + CsCl platform being the most efficient. These results correlate with the infectious titers seen in vitro (vg/pi ratio and in vitro functionality) for the different rAAV batches.

- Hematology and clinical biochemistry parameters, assessment of force parameters, molecular analyses (quantification of transgene copy number, MD1 transcript) and histopathological analyses (MD1 expression in muscles) are surprisingly better with rAAV8-hMD 1/293 vector compare with rAAV8-hMDl/Sf9 vector.

2. EXAMPLE 2: DOSE FINDING STUDY OF GNT0004 AFTER IV ADMINISTRATION IN DMD^MDX RATS

2.1. AIM OF THE STUDY

The aim of this study was to determine the pharmacologically effective dose range (i.e., minimally effective dose (MED)) of the GNT0004 product, a recombinant vector derived from a serotype-8 adeno-associated virus (rAAV2/8) encoding human p-dystrophin (hMDl) under the control of the muscle-specific promoter Spc5.12 (rAAV2/8-Spc5.12-hMDl-spA). The GNT0004 product is well described in the WO2015/197869. This dose-finding study was performed in the DMDmdx rat model.

A second objective of this dose-finding study was to confirm that the GNT0004 product reaches the target anatomic site/tissue/cell, when administered intravenously (IV).

It is expected that data derived from this dose-finding study will guide the design of the GLP toxicology study and planned phase I/II clinical trials in DMD patients, and will help demonstrate that the GNT0004 product is safe in the intended patient population.

2.2. EXPERIMENTAL STUDY PLAN

This preclinical batch 17DB204 was produced at 200L scale by using a three-plasmid transfection of HEK293T cells. A purification process using immunoaffinity step was optimized by using Poros AAV8 resin. The purified product was then concentrated and formulated in Ringer Lactate + 0,001% Pluronic F68®.

Material

Species/strain: Rats - Sprague Dawley. DMDmdx or healthy (littermates).

The DMDmdx rat model was described in 2014 (Larcher et al., PLosOne) and is considered one of the best animal models for Duchenne muscular dystrophy (DMD), as it mimics both the skeletal and cardiac alterations observed in DMD patients. By contrast, the mdx mouse and GRMD dog models are characterized by late onset and inconsistent cardiac alterations, making these animal models unreliable for assessment of the therapeutic effects of a test item on both parameters.

Another feature of this model is the rapid onset of a complex muscular pathology, characterized by a marked reduction in strength associated with Phase I (necrosis, cellular infiltration, regeneration) and Phase II (fibrosis and adiposis) histopathological lesions. Finally, this model offers high phenotypic reproducibility and stability, with little or no phenotypic variation between animals, a feature critical for the generation of robust data. In summary, the DMDmdx rat model provides a means of assessing therapeutic outcome measures after treatment.

Supplier: Boisbonne Center (internal breeding, with genotyping performed by PAC, UMR 1089 Gene Therapy Laboratory)

Sex: male Number of animals: 90 (76 DMDmdx rats + 14 healthy rats)

Approximate age at initiation of treatment: ~ 8 weeks (specifically, between 7.0 and 8.9 weeks)

Animal identification: after genotyping, each animal was identified with an electronic chip. Each sample generated in the Boisbonne Center during the study was assigned an internal code (ID number) linked to the electronic chip number.

AAV8 serological status: pre-inj ection anti-AAV8 serological status was not evaluated in the animals included in this study. However, to ensure that the colony of DMDmdx rats in the Boisbonne center was seronegative for AAV8, serum samples from 10 rats from the colony (that were not included in the study) were analyzed for anti-AAV8 neutralizing factors at the Gene Therapy Immunology core (UMR 1089, Nantes) before the study began. The results confirmed that these animals were either seronegative or expressed low levels of anti-AAV8 antibodies (maximum titer = 1/20) that are unlikely to interfere with gene transfer efficacy. These results confirm the serological status of the colony and the validity of using animals from this colony in the present study.

Experimental Design

The study involved a total of 90 animals:

- 76 DMDmdx rats (70 experimental + 6 “baseline pathological status”)

- 14 Healthy (WT) rats

DMDmdx rats (aged ~8 weeks) received IV injections of 1 of 4 different doses of GNT0004. The wild type (Sprague Dawley) and DMDmdx control groups were injected with formulation buffer (vehicle) only. For each experimental condition, 6 rats were euthanized 3 months postinjection (p.i.) and 8 rats at 6 months p.i.. Groups are presented in the Table below:

Method of administration: GNT0004 and the corresponding vehicle were administered on injection day (DO) as single infusion, by intravenous injection into the penile vein. In adult rats, the dorsal penile vein injection is much simpler, faster, more reproducible, and easier to accomplish than tail vein injection. In-house validation has determined that the minimal age for intravenous injection into the penile vein is ~ 8 weeks for DMDmdx rats. Importantly, substances into this vein are carried to the general circulation and a first-pass effect on metabolism is not expected (Nightingale & Mouravieff, 1973).

The anesthesia protocol has been adapted given the possibility of malignant hyperthermia resulting in death when using isoflurane as an anesthetic agent in DMDmdx rats (Larcher e/ al., unpublished data). The etomidate anesthesia protocol used has not been associated with a specific morbidity to date. Briefly, before GNT0004 or vehicle delivery, the analgesic buprenorphine (0.04 mg/kg) was administered subcutaneously between 30 min and 6 hours before anesthesia with etomidate (16-32 mg/kg), administered intraperitoneally in 2 to 4 injections separated by 3 min.

Vehicle or GNT0004 were administered in a single injection in anesthetized animals. The flow rate was set at ~1 mL/min and a total volume of 2.84-7.05 mL per animal was administered depending on animal’s weight (177.4-360.4 g for DMDmdx rats and 247.0-440.4 g for WT rats). The total volume injected was the same in all animals (in both experimental and control groups), adjusted to 16 mL/kg (20mL/kg being the maximum volume authorized in the rat). Practically, while GNT0004 dose varied depending on the experimental group. The total volume injected per kg remained constant.

DMDmdx rat observations

Clinical follow-up consisted of observing the animals twice daily in their home cages for global activity and mortality/morbidity.

Body weight was measured and plotted on day 0 (the day of injection), daily during the first week after injection and subsequently every week until sacrifice.

Muscle function was assessed using the open field test and grip test (performed at least 3 days after the open field test), ~ 1 week before sacrifice for the “3-month” follow-up groups. For the “6-month” follow-up groups, these tests were performed at ~ 3 months p.i. and ~ 1 week before sacrifice. Thus, for each group a total of 14 animals were evaluated at ~ 3 months p.i. and a total of 8 animals were evaluated at ~ 6 months p.i.

- Motor behavior was examined using the open field test. Rats were individually placed for 5 min in an open-field arena (70 cm x 70 cm x 70 cm) with a camera placed above. Recordings were subsequently analyzed using the Smart software (Planlab, Bioseb), which determines resting and ambulatory time (s), speed (cm/min) distance traveled (cm) and the number of rearing movements. To avoid bias, experiments were conducted and analyzed by personal who were blinded to both treatment and genotype.

- For the grip test, rats were placed with their forepaws on a grid and were gently pulled backward until they released their grip. A grip meter (Bio-GT3. BIOSEB. France), attached to a force transducer, measured the peak force generated. Five tests were performed in sequence with a short latency (~ 20 seconds) between each test, and the reduction in strength between the first and the last test was taken as an index of fatigue (De Luca, Treat NMD report, 2008). Results were expressed in grams (g) and normalized to the body weight (g/g). To avoid bias, experiments were conducted and analyzed by personal who were blinded to both treatment and genotype.

Cardiac function was assessed in anesthetized animals by EKG, 2D-echocardiography, and pulsed Doppler. For all animals, these tests were performed at least 3 days after the grip test, and just before sacrifice for the “3-month” follow-up groups. For the “6-month” follow-up groups, these tests were performed at ~ 3 months p.i. and just before sacrifice. Thus, for each group a total of 14 animals were evaluated at ~ 3 months p.i. and a total of 8 animals were evaluated at ~ 6 months p.i.. Measurements were taken in anesthetized rats (etomidate: one single dose of 16 mg/kg delivered IP in two injections separated by 3 to 5 min).

- EKG allows the recording of the electrical activity of the heart over a period of time using electrodes placed on the skin. These electrodes detect the tiny electrical changes on the skin that arise from the heart muscle's electrophysiological pattern of depolarization/repolarization during each heartbeat. Six-lead EKGs were recorded using 25-gauge subcutaneous electrodes and an analog-digital converter (IOX 1.585. EMKA Technologies, France) for monitoring and off-line analysis (ECG Auto v3.2.0.2, EMKA Technologies). EKG data provide information about the function of the electrical conduction system of the heart. Specifically, EKG was used to measure the rate and rhythm of heartbeats and to detect the presence of any damage to heart muscle cells or the conduction system. Classical intervals and segments (PR interval, PR segment, QT interval, QRS interval, ST segment) were measured and compared between the different experimental groups. To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype. 2D-echocardiography and pulsed Doppler were performed using a Vivid 7 ultrasound unit (GE. Waukesha, WI). To detect possible structural remodeling, left ventricular end-diastolic diameter and free wall end-diastolic thickness were measured during diastole from long- and short-axis images obtained by M-mode echocardiography. Systolic function was assessed by measuring the ejection fraction, while transmitral flow measurements of ventricular filling velocity were obtained using pulsed Doppler, with an apical four-chamber orientation. Diastolic dysfunction was assessed based on Doppler-derived early (E) and late diastolic (A) velocities, the E/A ratio, and isovolumetric relaxation and deceleration times. To avoid bias, experiments were conducted by personnel who were blinded to both treatment and genotype.

Blood sampling and laboratory Assays

For the “baseline pathological status” animals: Just before sacrifice, about 2 to 3 mL of whole blood were obtained from each animal, from which serum was obtained and aliquoted for subsequent storage at <-70°C in aliquots of ~ 200pL. These samples were used for

(i) Troponin Tc measurement; and

(ii) storage for subsequent analysis if required.

Another fraction of whole blood was reserved for the measurement of the following clinical hematology and biochemistry parameters:

- Hematology (~ 500pL of whole blood in EDTA-coated tubes): white blood cells, red blood cells, hemoglobin, hematocrit, eosinophils, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, platelets, reticulocytes, neutrophils, lymphocytes, monocytes, basophils.

- Biochemistry (~ 600pL of whole blood in heparin lithium-coated tubes): total serum proteins, urea, creatinine, total bilirubin, total creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), lactate dehydrogenase (LDH).

Injected animals:

Just before injection, about 1 mL of whole blood was obtained from each animal to obtain serum. Sera were frozen at <-70°C in aliquots of ~ 200pL. Samples were analyzed for detection of anti-MDl antibodies. The remaining samples were stored for subsequent analysis if necessary.

Just before sacrifice, 3-5 mL of whole blood was obtained from each animal. A fraction (at least 2 x ~ 300 pL) was directly frozen for the biodistribution (qPCR) study. Serum was obtained from whole blood and aliquoted for subsequent storage at <-70°C in aliquots of ~ 200 pL. Samples were used for (i) the determination of anti-MDl antibodies,

(ii) Troponin Tc measurement, and

(iii) storage for subsequent analysis if required.

Finally, another fraction of whole blood was reserved for the measurement of the following clinical hematology and biochemistry parameters:

- Biochemistry] (~ 600pL of whole blood in heparin lithium-coated tubes): total serum proteins, urea, creatinine, total bilirubin, total creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), lactate dehydrogenase (LDH).

Immunohistochemistry assessment

For all animals (“baseline pathological status” and injected animals), tissue samples obtained during necropsy were snap frozen for immunohistochemistry analyses. These included biceps femoris muscle, triceps brachii muscle, heart, diaphragm, and liver.

To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype.

Analysis of hMDl expression: hMDl expression was assessed by quantifying the number of dystrophin-positive fibers in biceps femoris muscle, triceps brachii muscle, heart, and diaphragm in frozen sections from all animals incubated with NCL-DYSB, a mouse monoclonal antibody that recognizes dystrophin (including hMDl) from different species (Novocastra Laboratories, Newcastle upon Tyne, UK).

Expression of hMDl was also stained (but not quantified) in liver samples from the “DMDmdx + vehicle” groups (3 and 6 months p.i.) and the “DMDmdx + 1E14 vg/kg" groups (at 3 and 6 months p.i.).

Analysis of fibrosis: Fibrotic tissue was stained (using wheat germ agglutinin conjugate for frozen skeletal muscle sections and picrosirius for fixed heart muscle sections) and quantified in biceps femoris, diaphragm, and heart samples from all animals. Analysis of regenerative activity: Regeneration was assessed by staining (without quantification) biceps femoris samples from 3, randomly chosen animals from each experimental group for the developmental myosin heavy chain isoform (MyoHCDev).

Analysis of dystrophin-associated proteins: Staining (without quantification) for dystrophin- associated proteins (e.g. a-sarcoglycan) was performed in biceps femoris samples from 1 animal in which high levels of hMDl expression were detected, and from 1 DMDmdx control animal and 1 WT control animal.

Molecular biology and western Blot analysis

Absolute quantification of transgene copy numbers by qPCR: Absolute vector genome quantification was performed by qPCR analysis on genomic DNA obtained from whole blood, biceps femoris muscle, triceps brachii muscle, longissimus muscle, heart, diaphragm, spleen and liver for all injected animals. To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype.

Western blot analysis: hMDl protein expression was analyzed by Western blot using the MANEX 1011C antibody. As no quantification was performed, this analysis was performed on 3 rats from each of the “3-months” follow up groups and 3 rats from each of the “6-months” follow-up groups, randomly chosen in each group. Tissues selected were biceps femoris muscle, triceps brachii muscle, heart, and diaphragm. To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype.

Immunological analysis

Anti -hMDl humoral immunity: Anti-hMDl IgG antibodies in serum were detected using an immune-Western-blot. Because the production of recombinant hMDl protein was not feasible, cell extracts expressing the hMDl transgene product after transfection with a CMV-hMDl plasmid were used. Protein cell extracts were subjected to precast polyacrylamide gel electrophoresis, and subsequently blotted. After an overnight saturation, the membranes were incubated with rat sera collected before and after rAAV administration. After hybridization with peroxidase-conjugated anti-rat IgG antibody, proteins were visualized by enhanced chemiluminescence. For each Western-blot membrane, an anti -Dystrophin antibody (MANEX1011C) was used as a positive control. To validate the specificity of the assay (anti- hMDl IgG detection in rat sera), all sera were tested on untransfected (hMDl -negative) cell extracts, and the results compared with those obtained for transfected (hMDl -positive) cell extracts. These analyses were performed using serum samples obtained before injection and at sacrifice for all injected animals. To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype.

Anti-hMDl cellular immunity: The anti-hMDl T-cell response was monitored using a ratspecific IFNy-ELISpot assay, using an overlapping peptide library that covers the entire sequence of the hMDl protein (length, 15 mers; overlap, 10 amino acids). T-cell responses were evaluated for all injected animals by analysis of splenocytes harvested at sacrifice. For each experimental sample, negative and positive controls consisted of unstimulated cells (Medium) and cells stimulated with mitogenic Concanavalin A (ConA), respectively. To avoid bias, experiments were conducted by personnel who were blind to both treatment and genotype.

2.3. RESULTS

2.3.1. DMD^MDX RATS OBSERVATIONS

Global activity: No adverse effects on global activity were associated with the injection and/or the GNT0004 product itself.

Morbidity/mortality: One animal from the “DMDmdx + 1E14 vg/kg” group was found dead in its cage during the first observation round of the day on week +17 p.i.. The case history of this animal was unremarkable. No clinical signs were observed during the preceding days. No blood or biochemical tests had been scheduled for this animal at this stage of the study. A few hours (<4 hours) after discovery, an autopsy was conducted under the supervision of Dr Thibaut Larcher, PhD, DVM and European Board certified pathologist, and several tissues (lungs, liver, heart and spleen) were obtained for histopathology analysis and determination of the cause of death.

2.3.2. BODY WEIGHT

Data showed that although there were some variability within the different experimental groups, GNT0004 administration had no negative effects on growth. Moreover, regardless of the injected dose, the weight curve for GNT0004-treated DMDmdx rats was superior to that of vehicle-treated counterparts. Expression of the percentage variation in body weight of each experimental group relative to pre-inj ection values revealed a dose effect, with the greatest improvements observed in animals treated with the 3 highest GNT0004 doses (3^E13, 6^E13 and 1^E14 vg/kg). Notably, because vehicle-treated WT rats were significantly heavier than DMDmdx rats at the moment of injection (301 ,7+/-13.6g for WT rats versus between 232.4+/- 8.0g and 264.4+/-11.2g for DMDmdx rats), weight-gain over the course of the study was less efficient in these animals than in DMDmdx rats.

2.3.3. ASSESSMENT OF MUSCLE FUNCTION

Grip test: Mean absolute and relative forelimb grip force values recorded at 3 and 6 months

A reduction in absolute and relative muscle strength was observed in vehicle-treated DMDmdx rats versus vehicle-treated WT animals, at both 3 and 6 months p.i. Moreover, in contrast to WT + vehicle rats, DMDmdx + vehicle rats showed a progressive and marked decrease in relative forelimb strength over the course of the test. This observation is typical of the DMDmdx model, which is characterized by marked muscle weakness and an inability to maintain consistent force over 5 successive trials, as previously described by Larcher et al. (PlosOne, 2014).

- At 3 months p.i., the increases in absolute forelimb grip force (with respect to the DMDmdx + Vehicle group, mean value of 1588.7+/-72.5g) that were observed for the DMDmdx + 1E13, + 3E13 and + 6E13 vg/kg groups (respectively 2085.7+/-69.5g, 2117. l+/-58.3g and 2081.5+/- 57.5g) were higher than that observed for the DMDmdx + 1E14 vg/kg group (1971.9+Z-48.0g), and a significant improvement in relative grip force was observed for the 1E13, 3E13 and 6E13 vg/kg GNT0004 dose groups (respectively 3.69+/-0.13g/g BW, 3.75+/-0.10g/g BW and 3.83+/- 0.07g/g BW, versus 3.11+/-0.14g/g BW for vehicle-treated DMDmdx rats) , but not with the 1E14 vg/kg GNT0004 dose (3.49+/-0.08g/g BW).

- At 6 months p.i., when compared to DMDmdx + Vehicle group (2331. l+/-84.0g), absolute forelimb grip forces were significantly higher in the DMDmdx + 1E13 and +3E13 vg/kg groups (respectively 2448.8+/-110.8g and 2482.5+/-82.1g) but not in the DMDmdx + 6E13 and 1E14 vg/kg groups (respectively 2192.4+/-68.1g and 2231.0+/- 100.4g). The same results were obtained when considering relative forelimb grip force (respectively 3.62+/-0.70g/g BW, 3.74+/-0.13g/g BW, 3.42g/g BW and 3.24+/-0.12g/g BW for GNT0004-treated DMDmdx rats, versus 3.18+/-0.13g/g BW for vehicle-treated DMDmdx rats).

Analysis of forelimb strength over the course of the test revealed a dose-independent improvement in the 4 GNT0004-treated DMDmdx groups, as evidenced by a cross-trial evolution identical to that of the WT + Vehicle group. At 3 months p.i., no decrease in relative forelimb grip force was observed at the end (Trial 5) versus the beginning (Trial 1) of the test, regardless of the dose injected. However, at 6 months p.i., although the same beneficial effect of the 1E13, 3E13 and 6E13 vg/kg GNT0004 doses were observed in DMDmdx rats, a significant decrease in forelimb grip force was observed at the end (Trial 5) versus the beginning (Trial 1) of the test for the DMDmdx rats treated with the 1E14 vg/kg GNT0004 dose.

2.3.4. IMMUNOHISTOCHEMISTRY ASSESSMENTS

Analysis of hMDl expression in Muscle

Mean values obtained from quantitative analysis of positive fibers, which expressed hMDl protein in the biceps femoris, triceps brachii, diaphragm and cardiac muscles.

The results revealed the following:

- In muscles sampled from WT + vehicle rats, all muscle fibers displayed intense and homogeneous subsarcolemmal labeling with the DYSB antibody. In muscles from DMDmdx + vehicle rats, similar intense and homogeneous subsarcolemmal labeling was observed only in a very small percentage of fibers (with a maximum of 6.5% in analyzed muscles). These fibers correspond to revertant fibers. A slightly lower percentage of revertant fibers was observed in Baseline Pathological Status animals sacrificed at 2 months of age (with a maximum of 1.2% in analyzed muscle). This observation was in line with the gradual, progressive increase of the number of revertant fibers in aging muscles.

- In GNT0004-treated DMDmdx rats, the percentage of DYSB-positive fibers was dramatically increased in all muscles analyzed, with fibers showing intense subsarcolemmal labeling that was predominantly continuous or, less frequently, discontinuous.

In certain skeletal muscle fibers from several rats from the DMDmdx + 3E13, 6E13, and 1E14 vg/kg groups and at both 3 and 6 months p.i., some intense continuous perinuclear labeling was observed in the cytoplasm. These fibers did not exhibit any alterations.

- For the purposes of quantification, labeling of two thirds of the fiber was considered necessary to categorize the fiber as DYSB-positive. Briefly, in all muscles analyzed we observed a significant effect of the GNT0004 product with respect to DMDmdx + vehicle rats.

More specifically:

In the biceps femoris, no differences in dystrophin expression were observed at either 3 or 6 months p.i. between WT + vehicle rats and DMDmdx rats treated with the 3E13 vg/kg dose or higher, with levels hMDl+ fibers reaching 92.7+/-8.1% to 98.2+/-1.5%. In the triceps bracchii, no differences in dystrophin expression were observed at either 3 or 6 months p.i. between WT + vehicle rats and any of the GNT0004-treated groups, with levels hMDl+ fibers reaching 79.7+7-22.7% to 99.5+/-0.6%. In the diaphragm, the levels of dystrophin expression were somewhat lower that those observed in the other muscle types, i.e. dystrophin expression levels comparable to those of the WT + vehicle group were observed at 3 or 6 months p.i. only in DMDmdx rats treated with the highest dose of GNT0004, with levels hMDl+ fibers reaching 86.2+/-7.0%. At lower doses, a dose-dependent increase in the number of DYSB- positive fibers was observed up to the 1E14 vg/kg dose.

In cardiac muscle, no differences in dystrophin expression were observed between WT + vehicle rats and DMDmdx rats treated with the 3E13 vg/kg dose and higher (at 3 months p.i., with levels hMDl+ fibers reaching 98.1+/-1.4% to 98.8+7-1.7%) and the 6E13 vg/kg dose and higher (6 months p.i., with levels hMDl+ fibers reaching 95.0+/-3.4% and 98.0+/-0.6%).

2.3.5. ANALYSIS OF REGENERATIVE ACTIVITY

While no regeneration was observed in muscle samples from the WT + vehicle groups, some randomly scattered clusters of newly regenerated fibers were observed in the muscles of DMDmdx + vehicle rats, in which we observed a slight decrease in the total number of MyoHCDev-positive fibers between 3 and 6 months p.i.. At these same time points in GNT0004-treated DMDmdx rats, the number of newly regenerated fibers decreased with increasing GNT0004 dose. A few clusters of positive fibers were detected in rats treated with the 1E13 vg/kg dose, while only isolated positive fibers were observed in those treated with the 3E13 vg/kg and 6E13 vg/kg doses. Few if any positive fibers were evident in rats treated with the 1E14 vg/kg dose.

2.3.6. ANALYSIS OF HUMORAL IMMUNITY

Anti-hMDl humoral immunity

Anti-hMDl humoral immunity (IgG antibody) was assessed by Western blot of serum samples obtained before injection and after injection (i.e., at euthanasia).

As expected, vehicle-treated WT and DMDmdx rats were negative for anti-hMDl IgG both before and after vehicle administration.

All GNT0004-treated DMDmdx rats were negative for anti-hMDl IgG before injection. Pooling of 3- and 6-month follow-up data showed that after GNT0004 administration, 85% of GNT0004-treated DMDmdx rats were positive for anti-transgene IgG antibodies. The proportion of positive rats in each group is shown in Table below. The proportion of rats positive for anti-hMDl IgG increased with GNT0004 dose, with the highest proportion (100%) observed for the 6E13 vg/kg and 1E14 vg/kg doses at 3 and 6 months p.i., respectively.

2.4. CONCLUSION

2.4.1. SAFETY ASSESSEMENT

Finally, this report provides evidence that the GNT0004 product is detected by the host immune system. Nonetheless, the observed humoral response to the hMDl protein had not evidenced significant impact on the therapeutic efficacy of the GNT0004 product, during the 6 months of follow up described in this study. Systemic delivery of an AAV vector encoding a foreign polypeptide does not induce destruction of transduced cells if it does not elicit a cellular response (which, in the present case, was indeed not observed).

2.4.2. EFFICACY ASSESSEMENT

Taken together, the results of this study reveal the following:

In skeletal muscles (except diaphragm)

At both 3 and 6 months p.i., dose-dependent increase in hMDl-positive fibers in rats treated with doses of up to 3E13 vg/kg: this dose resulted in high level of expression levels as detected by Western-blot and >90% of hMDl-positive fibers, even if hMDl-positive fibers in GNT0004-treated DMDmdx rats were not homogeneously distributed across different muscles, something that is expected and previously published (see for example: Le Guiner et al., Mol Ther, 2014; Yue et al. Hum Mol Genet, 2015; Mack et a.l, Mol Ther, 2017). Normal subsarcolemmal hMDl expression was observed and was associated with the restoration of DAPC expression.

At both 3 and 6 months p.i., increases in grip force and reduced fatigue in rats treated with doses of 1E13 vg/kg and higher, with maximum improvement (results comparable to those of vehicle- treated WT rats) observed for the 1E13, 3E13, and 6E13 vg/kg doses, and partial improvement observed for the 1E14 vg/kg dose.

In heart

At both 3 and 6 months p.i., a dose-dependent increase in hMDl-positive fibers in DMDmdx rats treated with doses of up to 3E13 vg/kg, which resulted in >95% hMDl-positive fibers.

At 6 months p.i., a significant decrease in fibrosis in GNT0004-treated DMDmdx rats (2.6% versus 7% in the vehicle-treated DMDmdx rats) regardless of the dose injected, with levels similar to those of vehicle-treated WT rats were observed in DMDmdx rats treated with all doses. At 3 months p.i., no significant difference with respect to vehicle-treated DMDmdx rats was observed.

At both 3 and 6 months p.i., improvements in EKG abnormalities (PR and QTc intervals) and in diastolic function (E/A ratio, IVRT, DT) in rats treated with doses of 1E13 vg/kg and higher. At 6 months p.i., structural remodeling (LV free-wall thickness) was also partially prevented by all GNT0004 doses. Notably, compared with lower GNT0004 doses, the lE14vg/kg GNT0004 dose was often associated with lesser improvements.

2.4.3. THERAPEUTIC DOSE

The minimum effective dose (MED) is the dose resulting in partial to complete normalization of the aforementioned outcome measures, and varied depending on the outcome measure in question.

More specifically:

- High levels of hMDl expression were obtained from the lowest dose (1E13 vg/kg) in heart, and from the low-intermediate dose (3E13 vg/kg) in the biceps femoris, triceps brachii and diaphragm. - Analysis of the number of muscle fibers expressing hMDl revealed a linear relationship between the 4 doses of GNT0004 and the number of muscle fibers expressing hMDl only in the diaphragm. For all other muscle types, a plateau effect was observed, whereby further dose escalation had no further effect on the number of hMDl -expressing fibers. Surprisingly, a significant effect, therefore, was observed from the lowest dose (1E13 vg/kg) in triceps brachii and from the low intermediate dose (3E13 vg/kg) in the biceps and heart.

- Again surprisingly, for forelimb grip force, significant improvement was observed for the 2 lowest doses (1E13 and 3E13 vg/kg). A reduced fatigue was observed for all doses of the testitem at 3 months p.i., and for all doses except the 1E14 vg/kg dose at 6 months p.i..

- No clear correlation was observed between the dose of GNT0004 and the improvement of cardiac function.

- Finally, for body weight, improvement was observed with all the doses.

In conclusion, based on these data obtained at 3 and 6 months p.i., the Minimal Effective Dose (MED) can be considered to be in the dose range from 1E13 to 3E13 vg/kg. This was a very surprising result. It has to be highlighted that better efficacy results are surprisingly obtained with the 2 lowest doses (1E13 and 3E13 vg/kg) compared to the highest dose 1E14 vg/kg.

This significant unexpected efficacy demonstrated above is likely to be further demonstrated in an in vivo comparative test such as the clinical study protocol described. In fact, the clinical study may demonstrate an even more profound result than the one demonstrated in this example.

3. EXAMPLE 3: CLINICAL STUDY PROTOCOL

Title: Microdystrophin (GNT0004) Gene Therapy Clinical Trial in Duchenne Muscular Dystrophy

A phase I/II/III study with a dose determination part followed by an efficacy and safety evaluation, quadruple blind placebo-controlled part and then by a long term safety follow up part, in ambulant boys.

Study Design

- Phase EIEIII

- Multi center

- Prospective - Part 1: Dose determination (cohorts Cl and C2, and optionally C3)

- Multicenter, open-label, with sequential enrolment

- Part 2: One-year evaluation of efficacy and safety of the selected dose of the tested IMP (pivotal cohort) o Quadruple blind, placebo-controlled o Randomized (ratio 1 : 1) to either IMP/placebo or placebo/IMP (patients being treated by the second treatment after the one-year pivotal parallel evaluation)

- Part 3: Long term safety and efficacy follow-up for all cohorts of patients enrolled in parts 1 and 2

Duration of participation per patient

Duration:

- Dose determination cohorts (Parts 1 and 3): o Up to week 12 after IMP infusion to assess dose effect (Part 1) o Up to 5 years after IMP infusion to assess long term safety and efficacy (Part 3)

- Randomized placebo-controlled cohort at selected dose (Parts 2 and 3): o 5 years for patients randomized in active IMP arm first

- 1 year up to primary efficacy endpoint after IMP infusion (Part 2)

- Plus 4 years for long term safety and efficacy follow-up (Part 3) o 6 years for patients randomized in placebo arm first

- 1 year follow-up in placebo arm up to primary efficacy endpoint (Part 2)

- 1 year follow-up after IMP infusion (Part 2)

- Plus 4 years for long term safety and efficacy follow-up (Part 3)

Study objectives

Primary objectives:

Part 1:

- To determine the dose of IMP: a safe and tolerable dose with acceptable gene expression, to carry over to part 2

Part 2:

With the selected dose of IMP:

- To demonstrate the clinical efficacy versus placebo at one year after inclusion

- To assess the safety and tolerability versus placebo at one year after inclusion

Part 3:

- To assess the long-term safety and tolerability of IMP

Secondary objectives:

Part 2:

- To assess the biodistribution of IMP - To demonstrate the pharmacodynamic activity of IMP

- To assess the immunogenicity of IMP: o Antibodies to AAV8 virus vector o Antibodies to the microdystrophin transgene protein product

- To compare the efficacy on the disease course after 2 years after inclusion, between patients treated with active IMP at first and patients treated after a delay of one year (ie patients treated at first with placebo)

Part 3:

- To evaluate the long-term efficacy of IMP

Number of Patients

Part 1: 6 patients will be enrolled in Cl (n=3) and C2 (n=3) cohorts of dose.

An optional cohort C3 (n=3) could be added after review of data by the Data Monitoring Committee (DMC) to possibly assess a third higher dose.

(refer to study protocol for criteria that trigger to recruit a further cohort)

Part 2: 42 patients randomized to active IMP (n=21) or Placebo (n=21), ratio 1 : 1, will be included.

- Randomization will be stratified by region and class of age

- Patient allocated to placebo first will be treated subsequently with IMP after one year and patients treated by IMP first, by placebo

- The cohort of patients (n = 3) enrolled in part 1 at the final selected dose will contribute to the sensitivity efficacy analyses of part 2 (secondary analyses of the main endpoint)

- All patients treated in part 1 and in part 2 will be included in the safety cohort (part 3)

IMP / Dosing

IMP (Investigational Medicinal Product): GNT0004

A replication-defective adeno-associated virus (AAV) vector with AAV8 serotype capsid and carrying a recombinant sequence-optimised human microdystrophin cDNA gene expressed using a promoter intending to be specific for human skeletal and cardiac muscles (see details in the study protocol).

Doses:

Up to 3 single doses to be tested in 3 successive cohorts of 3 patients:

- Cl at first dose: 1.0x10¹³ vg/kg

- C2 at second dose: 3.0xl0¹³ vg/kg

- Optional C3 dose : 6.0xl0¹³ vg/kg

Selected dose*: i.e. a safe and tolerable dose with acceptable gene expression to carry over to part 2 (21 IMP treated + 21 placebo patients)** Administration procedure:

- Parenteral route: single peripheral intravenous (IV) infusion (preferably the forearm of the contralateral arm of muscular biopsy site)

- Reconstituted IMP (GNT0004) or Placebo: o Total dose calculated according to the body weight o To be thawed at room temperature during 1 hour and diluted with saline and HSA up to 200 ml in an infusion bag o Stable for 26h maximum at room temperature until the end of the IMP infusion

- Infusion rate: o To be administered according to pediatric infusion rate clinical guide (https://reference.medscape.com/calculator/526/maintenancefluid-calculations, accessed May 31, 2020) (e.g. 60mL/kg of bodyweight/hour, if body -weight = 20kg) Placebo control composition: Same formulation of buffer that is used for IMP

Concomitant Therapy(ies)

Prophylactic systematic immunosuppressive therapy:

- 1 mg/kg/day per oral (PO) of prednisolone (or prednisone) the calendar day before IMP infusion

- 1 mg/kg/day per oral (PO) of prednisolone (or prednisone) the day of IMP infusion (at least 3 hours prior to the IMP infusion)

- 100 mg intravenous bolus of methylprednisolone administered in 15 minutes the day of IMP infusion (60 minutes prior to the start of IMP infusion)

- 2 mg/kg/day PO of prednisolone (or prednisone) for 5 calendar days starting the day after IMP infusion, before tapering the dose

- Then Img/kg/day PO of prednisolone (or prednisone) for 8 weeks before tapering the dose

- Tapering dose: step down acceptable range of dose change is 25% of the previous dose (a window of at least one week at stable dosing is recommended between any steps down of dose) Medications for the management of adverse event/safety signal

- Isolated elevated transaminases 1: o Prednisolone (or prednisone) will be increased up to 2 mg/kg/day

PO as long as the liver enzymes ALT or AST or GGT or GLDH: □ are above 2 x ULN

□ or above 2 x baseline, value if the baseline value was above ULN o As soon as all above liver enzymes return below 2x ULN, tapering dose can be started

- HUS (Hemolytic Uremic Syndrome) o Eculizumab (Soliris®), intravenous infusion o Dosing according to commercial label (USPI/SmPC)

- Troponin I elevation

Rapid local cardiological expertise to discuss indication for: o CMRI (Cardiac Magnetic Resonance Imaging) o Further diagnostic assessments (troponin I, ECG, echocardiography) o Permanent cardiac rhythm monitoring o Cardiac treatments

Selection criteria

Main Inclusion criteria

1. Ambulant male

2. 6 to 10 years (inclusive)

3. Body weight <75th percentile of the BMI scale (validated chart in force in country site)

4. Positive gene testing with detailed genotyping confirmation of Duchenne Muscular Dystrophy (DMD), i.e. DMD mutations expected to abolish the production of dystrophin

5. Able to achieve a score in the NSAA (North Star Ambulatory Assessment) scale > 18 at screening visit

6. Stable or descending evolution in the NSAA total score during the last assessment periods recorded (evolution inferior or equal to 1 during the 2 last consecutive assessment-periods)

7. Gowers test (Time to Rise From Floor) <7 sec

1. In case of significant elevation, transaminases could be monitored twice a week until event resolves or returns to baseline

2. Sustained evolution inferior or equal to 1 during the 2 last consecutive available NSAA evaluation periods (scores recorded in the GNT-014-MDYF study) OR Evolution inferior or equal to 1 compared to the last available NSAA score assessment (recorded in the GNT-014- MDYF study) AND relevant medical history, which clearly confirms that the patient's Duchenne disease is in stable or declining condition within the past months.

8. Ongoing corticosteroid therapy with prednisolone (or prednisone) according to standard of care:

- For at least 6 months before the anticipated first administration of study medication

- At stable dosing except for body weight adjusting

9. Up to date vaccination record including hepatitis A and B, PCV pneumococcal (13 serotypes), DTaP/IPV (diphtheria, tetanus, pertussis, polio), Haemophilus influenzae type b (Hib), measles, mumps, rubella, chickenpox, meningococcal group A, Y, W135 and B, C (Neisseria meningitidis incl)

10. Willing and able to comply with all protocol requirements and procedures

11. Signed informed consent and assent as required by local law

12. Affiliated/beneficiary of an insurance health care scheme or National Health service (if required by local law)

Main Exclusion criteria

1. Presence of neutralizing antibodies against AAV8

2. Cardiomyopathy based on physical/cardiological examination and echocardiography with Left Ventricular Ejection Fraction (LVEF) below 55% and/or fractional shortening (SF) below 28%

3. Any respiratory assistance needed including non-invasive daytime or nocturnal ventilation

4. Inability to perform the planned respiratory functions tests

5. Any co-morbidity(ies) and/or previous or planned surgical event(s), which may interfere with DMD evolution /or evaluation of patient’s safety or IMP’s tolerability, e.g., chronic infective (e.g. pulmonary, eamose-throat, urine, cutaneous) or immunological conditions, other neurologic disease or relevant somatic disorders that are not related to DMD etc ... that could preclude safety

6. Acute infection that is not expected to be fully resolved for a sufficient time before gene therapy administration

7. Clinically significant laboratory abnormality values that is either not expected or is of a greater severity than what is expected in DMD patients, considered clinically significant by the investigator, including but not limited to:

- White blood cell count > 18,500 per microL

- Platelets < 150,000 per microL

- Direct bilirubin > 0.3 mg/dL (5 micromol/L)*

- Gamma-Glutamyl Transferase(GGT) > 2/ upper limit of normal (ULN)*

- GLDH upper ULN*

*(to be confirmed by 2 consecutive lab tests)

8. Inability to cooperate with muscle strength and functional assessments, i.e. NSAA, Myotools, 6MWT, timed 10MWRT and RFF, Actimyo

9. Nuclear Magnetic Resonance Imaging (NMRI) contraindications: Metal implants in regions of interest for the study (namely skeletal muscles, heart), or other contraindication for NMRI such as severe phobia

10. Unwilling and/or unable to comply with all the study protocol requirements and/or procedures

11. Chronic hepatitis B or C, ie, positive hepatitis B surface antigen or hepatitis C RNA viral load positive 12. Need or planned vaccination with alive attenuate vaccine during the 4 week-period prior to IMP administration or for the 3-month period, after IMP infusion

13. Need for anticoagulants, antithrombotic, or platelet aggregation inhibiting drugs

14. Contraindication to Eculizumab (Soliris®)

15. Previous inclusion to another clinical trial with any non-gene therapy related within either the previous 6 months or within an IMP washout period, prior to the screening period for the study

16. Previously or currently treated with any drug(s) that may interfere with either natural disease course and/or IMP evaluation (except for corticosteroid therapy) including hormonal therapy, systemic antiviral and/or Interferon therapy

17. Within the wash out period (7 half-life) of an approved exon skipping or stop codon read- through agents for DMD

18. Previous gene therapy for DMD

Conduct of the study

Three parts:

- Part 1: Dose determination in Cl, C2, and optional C3 cohorts

- Part 2: Randomized cohort to demonstrate efficacy and to assess safety of the selected dose vs placebo

- Part 3: Long term safety follow-up (all cohorts of patients)

Part 1:

- At least 3 patients to be enrolled within each cohort with an interval of 4 weeks after IMP infusion between each patient

- In the absence of relevant safety issue during a 4-week period after IMP infusion with patient #1, then patient #2 then # 3 could be enrolled and treated with IMP

- Once the cohort has been completed, i.e. 3 patients* dosed with at least a complete 12-week follow-up period after IMP infusion, the Data Monitoring Committee (DMC) will review the safety and efficacy 12-week follow up data and provide go/no go recommendations to proceed or not to start enrolment and dosing in the higher dose (next cohort)

* Any patients discontinued from the study prior to the first 12-week follow-up in either Cl, C2, or C3 cohorts, for any non-safety related reasons, will be replaced

Part 2:

- 42 patients will be randomized at the selected dose or placebo (21 patients per group) and followed up to primary efficacy timepoint, one year after IMP infusion (or placebo)

- The 21 patients enrolled in the placebo group will be treated by active IMP once they reached the primary efficacy timepoint (after the first year of follow-up) Part 3

- All patients will be followed for long-term safety and efficacy assessments during 4 additional years after the one-year post-infusion of the active drug assessments

Study Endpoints

Part 1:

- Safety Endpoints: o Body -weight and height o Vital signs o Physical examination (body systems specified in study protocol) o ECG, Echocardiography, cardiac MRI o Troponin I o Blood: Full Blood Count (FBC), glucose, albumin, Blood Urea Nitrogen (BUN), creatinine, electrolytes, eGFR (estimated glomerular filtration rate) o LDH, ALP, AST, ALT, GGT, GLDH, Bilirubin o aPTT (activated partial thromboplastin time), Prothrombin time o TMA (thrombotic microangiopathy, ie FI, CH50, C3, etc) test o Dipstick for protein/blood (urine) o Humoral immune biomarkers: Antibodies (neutralizing/binding)

• to AAV8

• to recombinant dystrophin (hMDl) o Cellular immune biomarkers (ELISPOT(s) tests) o Incidence of adverse event(s) (AE), Serious Adverse Event(s) (SAE)

- Main PK/PD Endpoints: o Vector shedding quantification in blood, urine, saliva, feces (DNA level) o CK (blood): Change from Baseline in serum creatine kinase (CK). Changes in the circulating levels of CK. o Muscular Biopsy of biceps brachii (at baseline and in the contralateral muscle at week 8) Main endpoints for dose selection:

• quantification of hMDl (and endogenous dystrophin) positive fibers (and location)

• quantification of hMDl (and endogenous dystrophin) protein expression Microdystrophin protein expression level from a muscle biopsy will be assessed by capillarybased immunoassay (Simple Western assay). Microdystrophin positive myofibres from a muscle biopsy will be detected and quantified by immunofluorescence.

Other endpoints:

• VCN= quantification of vector copy number (DNA)

• general histology (necrosis, infiltration, etc) • DAPC staining

• Regeneration index

• Sarcolemnal permeability

- Clinical Efficacy Endpoints: o NSAA: Change from Baseline in North Star Ambulatory Assessment (NSAA). The NSAA is a 17-item test that measures gross motor function in children with Duchenne. o Time to 10 Meters Walk/Run Test (10MWRT): Change from Baseline in the 10-meter run/walk test velocity. Velocity is calculated based on the time that it takes to complete the IOmeter run/walk test. o Time to Rise From Floor (RFF): Change from Baseline in the rise from floor velocity. Velocity is calculated based on the time that it takes to the rise from floor. o 6-Minutes Walk Test (6MWT) o MyoTools (Grip, Pinch): Change from baseline of MyoPinch and MyoGrip tool o ActiMyo (Wearable Device - Sysnav®: Stride velocity 95th centile measured at the wrist and the ankle, SV95C) o Respiratory function tests (see details in part 2) o Muscle MRI:

• qNMRI: % of Fat and contractile cross-sectional area (Dixon) in glutei and thighs

• for other outcome refer to § efficacy biomarker in part 2

- Patient Reported Outcome and Quality of Life (QoL) questionnaire: o ACTIVLIM o EQ-5D: Change from baseline of quality of life (EQ-5D scale)

- Emerging DMD Biomarkers: o Biobank to search for potential gene modifiers and biomarkers of efficacy in blood and urine samples

Part 2:

- Safety Endpoints: o Refer to § in part 1 (same outcomes)

- PK/PD Endpoints: o Refer to § part 1 : same outcomes, but muscular biopsies are performed at baseline and in the contralateral muscle either at week 52 or 104 (according to a specific biopsy -timepoints randomization)

- Efficacy Endpoints: o Primary Efficacy Endpoint:

□ NSAA: change from baseline at week 52 o Main Secondary Efficacy Endpoints:

□ ActiMyo Wearable Device - Sysnav®: Stride velocity 95^th centile measured at the ankle (SV95C)

□ Myotools (Pinch, Grip)

□ ACTIVLIM

□ EQ-5D

□ qNMRI : % of Fat and contractile cross sectional area (Dixon) in glutei and thighs o Other Efficacy Biomarkers:

□ Muscle Imaging Nuclear Magnetic Resonance (qNMRVS):

□ Water T2 (inflammation/oedema) by T2map in glutei and thighs (quantitative)

□ Water T1 by Tlmap in glutei and thighs (quantitative)

□ pH, Phosphodiester (PDE) content using 3 IP NMRS in the leg (semi-quantitative)

□ Water T2 by T2map in the leg (quantitative)

□ Water T1 by Tlmap in the leg (quantitative)

□ Restricted diffusion (myocytes permeability) by NMRI in the leg (quantitative) o Other Efficacy Clinical Endpoints:

□ Muscle Strength Assessments:

□ Time to Rise From Floor (RFF)

□ Time to 10MW/RT

□ NSAA sub scores

□ 6MWT

□ Respiratory Functions tests:

□ Forced Vital Capacity (FVC)

□ PEF (Peak Expiratory Flow)

□ Forced Expiratory Volume in the first second of exhalation (FEV1)

□ Peak Cough Flow (PCF)

□ Vital Capacity (VC)

□ Respiratory frequency (fR)

- Emerging DMD Biomarkers: o Biobank - to search for potential gene modifiers and biomarkers for efficacy in blood and urine samples

Part 3

- Safety Endpoints: o Body weight and height o Vital signs o Physical examination o ECG, Echocardiography, cardiac MRI o Standard lab tests o Incidence of adverse event(s) (AE), Serious Adverse Event(s) (SAE)

- PD Endpoint: o CK (Creatine kinase) - Efficacy Endpoints: o NSAA o Timed 10MW/RT o Timed RFF

06MWT o Myotools (Grip, Pinch) o ACTIVLIM o EQ-5D o Respiratory tests (same endpoint as in parts 1 and 2 ) o Muscles Imaging (qNMRI) (same outcomes as in parts 1 and 2) - Emerging DMD Biomarkers: o Biobank to search for potential gene modifiers and biomarkers for efficacy in blood and urine samples

Claims

1. A method of treating Duchenne muscular dystrophy (DMD) in a human, comprising systemically administering by intravascular injection a gene therapy product that comprises an adeno-associated viral (AAV) vector of serotype 8, which harbors a nucleic acid sequence SEQ ID NO: 1 encoding a human AR4-R23/ACT microdystrophin.

2. The method of claim 1, wherein the gene therapy product is injected up to 1E14 vg/kg.

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