Patent Application
20160058890
This invention relates to gene therapy, in particular the treatment of diseases affecting skeletal muscles such as myotubular myopathy, caused by mutations in the MTM1 gene.
In this context, it proposes an expression system comprising a protein-encoding transgene that will ensure the production of a therapeutically effective amount of the protein in the target tissues, preferably the skeletal muscles, and a toxically acceptable amount of the protein in the non-target tissues, especially the heart.
X-linked myotubular myopathy (or XLMTM, OMIN 310400) is the most severe and common form of a group of diseases called centronuclear myopathy. The patients have already been affected during their foetal life, they show reduced mobility during gestation and have myopathy at birth which appears as non-progressive [1,2,3]. They have generalised muscle weakness and hypotonia, leading to respiratory failure and many die during the initial years of life despite intensive medical care. More atypically, less severe forms of myotubular myopathy also exist in male and female subjects with mild symptoms during childhood, which are aggravated during the first or second decades of their life [4].
The skeletal muscles of the affected subjects contain small fibres with an altered distribution of organelles, such as nuclei and mitochondria, which are typically located in the centre of the fibres or, in less severe cases, are arranged in the form of a collar in the subsarcolemmal region [4,5].
The disease results from inactivating mutations in the ubiquitously expressed MTM1 gene, which encodes a phosphoinositide phosphatase called myotubularin [6].
Animal models of the disease are currently seen in zebrafish, mice and dogs [7,8,9,10]. Studies conducted in these models have shown that in the skeletal muscle, myotubularin plays a role in a variety of mechanisms, including the organisation of the T-tubule and the intermediate filament, the excitation-contraction coupling, the transmission of the neuromuscular junction, and the survival and proliferation of satellite cells [11,12,13,14].
The gene replacement therapy by means of a vector represents a potential therapeutic approach for myotubular myopathy. Thus, it was indicated as proof of concept that a single intramuscular injection of a recombinant adeno-associated viral vector (AAV) in mice having a muscle-specific symptomatic impairment of myotubularin (mKO) was capable of improving the pathology and function of the targeted muscles [15].
The question of treatment of muscular disorders remains crucial. Gene transfer, in particular by means of vectors derived from adeno-associated viruses which are found to be tools particularly suitable for muscle transfection, is a particularly promising approach. It involves administration of an intact copy of the gene to the patient, for the production of a functional protein compensating the mutated and inactive protein produced by said subject.
In the case of muscle diseases, the administration may be performed by local injection in the muscles of the vector carrying the transgene. However, systemic administration is preferred clinically, which means that the transgene can be found in the various tissues of the body.
Typically, the transgene is placed under the control of regulatory sequences governing its expression, in particular with regard to the level of expression or the tissue specificity of the expression. Thus and in the case of gene therapy of a muscle disease, a promoter governing an expression more specifically in the muscle may be preferred. For example, a synthetic promoter C5-12 has been developed, which is well known to the person skilled in the art and supposed to promote gene expression in muscles.
However, there is a clear need to develop new tools for gene therapy for treating neuromuscular diseases, leading to the production of effective amounts of protein in the target tissue to compensate for the lack of activity of the mutated proteins, and which are otherwise safe for the treated patients.
This invention is based on the identification by the inventors, that after systemic administration, an expression system intended for the production of a protein in a target tissue, preferably skeletal muscles, can simultaneously lead to an expression in other tissues and organs potentially toxic, rendering said system unsuitable for therapeutic use.
This invention provides technical solutions for this newly identified problem, particularly regarding cardiac leakages related to the skeletal muscle-specific expression of a transgene.
More broadly, this involves the following for a given expression system:
Proteins referred to in this invention are thus those that exhibit toxicity in at least one tissue, particularly in a non-target tissue, when expressed from a given expression system.
Advantageously, the expression system is administered systemically in the body, particularly in an animal, and more preferably in humans.
Preferably, the analysis of toxicity is performed in a body with a defective copy of the sequence encoding the protein, i.e. in a body with the condition being treated, for example, a “Knockout” (KO) animal model. Indeed, if in the context of the invention, cardiac toxicity was observed for expression systems encoding myotubularin or calpain 3, it was detected only in KO mice in the case of myotubularin. In other words, a toxicity analysis that, according to usual practice, would have been performed in a healthy animal, would not have revealed this toxicity.
Thus and in general, this invention relates to an expression system comprising a sequence encoding a protein, the said expression system allowing:
According to the invention, the target tissue is preferably defined as the tissue or organ in which the protein is to play a therapeutic role, especially in cases where the native gene encoding this protein is defective. According to a particular embodiment of the invention, the target tissue designates the striated skeletal muscles, hereafter referred to as skeletal muscles, i.e. all the muscles involved in motor ability including the diaphragm. These muscles are particularly affected in diseases called myopathy. Another potential target tissue is the peripheral nervous tissue, which can also be affected in neuromuscular diseases. Advantageously, the target tissue thus includes skeletal muscles and/or the peripheral nervous tissue.
According to the invention, the non-target tissues are preferably defined as tissues or organs in which the protein has no therapeutic role to play, and optionally, in which the presence of the protein exceeding the endogenous quantity may prove to be harmful or even fatal, and therefore toxic.
In the context of the invention, tissues that are to be protected from this potential toxicity are preferably:
These are vital organs or tissues in which the gene expression systems tend to accumulate.
In the context of the invention, the heart muscle appears to be a tissue of particular interest as demonstrated at least for myotubularin and calpain 3. According to a particular embodiment, the expression system allows an expression at a toxically acceptable level of the protein in the heart.
Thus, and according to a particular aspect, the present invention relates to an expression system comprising a sequence encoding a protein, said expression system allowing:
According to a first characteristic, the expression system of the invention comprises a sequence encoding a protein, corresponding to a transgene. In the context of the invention, the term “transgene” refers to a sequence, preferably an open reading frame, provided in trans using the expression system of the invention.
According to a particular embodiment, this sequence is a copy, identical or equivalent, of an endogenous sequence present in the genome of the body into which the expression system is introduced. According to another particular embodiment, the endogenous sequence has one or more mutations rendering the protein partially or fully non-functional or even absent (lack of expression or activity of the endogenous protein), particularly in target tissues, i.e. skeletal muscles. In other words and preferably, the expression system of the invention is intended to be administered to a subject having a defective copy of the sequence encoding the protein and having an associated pathology. In this context, the protein encoded by the sequence carried by the expression system of the invention can therefore be defined as a protein whose mutation causes a neuromuscular disorder.
According to another embodiment, this involves a sequence that encodes a protein capable of “compensating” for the failure of a defective protein (regarding its expression or activity) in the subject to whom the expression system of the invention is administered. Thus and by way of example in relation to neuromuscular pathologies:
Thus and more generally, the sequence carried by the expression system of the invention can be defined as encoding a protein having a therapeutic activity in the context of a neuromuscular disease. The concept of therapeutic activity is defined as below in connection with the term “therapeutically acceptable level”.
The sequence encoding the protein is a nucleic acid sequence and may in particular be a DNA (deoxyribonucleic acid), an RNA (ribonucleic acid) or a cDNA (complementary deoxyribonucleic acid).
Advantageously, said sequence encodes a functional protein, i.e. a protein capable of ensuring its native or essential function, especially in the skeletal muscle. For each protein of interest, the desired activity and the sequence necessary for obtaining this activity can be defined.
According to a preferred embodiment, said sequence encodes the native protein, said protein being preferably of human origin. It may also be a derivative or a fragment of this protein, provided that the derivative or fragment retains the desired activity. Preferably, the term “derivative” or “fragment” refers to a protein sequence having at least 60%, preferably 70%, even more preferably 80% or even 90%, 95% or 99% identity with the human sequence of the protein of interest. Proteins with other origins (non-human mammals, etc.) or truncated, or even mutated, but active proteins are for instance designated. Thus and in the context of the invention, the term “protein” is understood as the full-length protein regardless of its origin, as well as functional derivatives and fragments thereof.
In the context of the invention, the proteins allowing the therapeutic treatment of neuromuscular diseases that may affect skeletal muscles and/or the peripheral nervous tissue are encompassed. The proteins enabling the therapeutic treatment of diseases affecting the skeletal muscles, generically called “myopathy”, are more particularly referred to.
In a particular aspect, these diseases are caused by mutations in at least one gene causing non-production of the protein or production of a fully or partially non-functional protein. According to the invention, the expression system helps produce this protein in an active form and in a quantity that at least partially compensates for the absence of the native protein, or another protein capable of compensating for the absence of the native protein. The administration of the expression system thus makes it possible to improve or restore a normal phenotype in the target tissue(s), particularly the skeletal muscles, in terms of mobility and breathing.
A protein having a benefit particularly in the context of the present invention is myotubularin of human origin (SEQ ID NO: 1), murine (SEQ ID NO: 2) or canine (SEQ ID NO: 3). Any sequence encoding these proteins, functional therapeutical derivatives or fragments thereof, can be implemented as part of the expression system of the invention. Thus, by way of example, the corresponding nucleotide sequences (cDNA) are the sequences SEQ ID NO: 4, 5 (or 14) and 6, respectively.
Mutations in the MTM1 gene result, in a known manner, in a muscle disease called myotubular myopathy (MTM or XLMTM). Thus and according to the strategy for replacement or transfer of the gene, the provision in trans of a sequence encoding a therapeutic myotubularin, which is for example native, helps treat this pathology.
In another embodiment, the protein of interest is calpain 3 (CAPN3) whose mutations cause in particular a recessive autosomal genetic disease called type 2A limb-girdle dystrophy (LGMD 2A or calpainopathy, OMIN 253600). For example, human calpain 3 has the sequence SEQ ID NO: 7. Thus and as described above, any sequence that encodes a therapeutic calpain 3, for example that of sequence SEQ ID NO: 7, or a derivative or fragment thereof, may be present in an expression system of the invention. It may, for example, be the cDNA sequence shown in SEQ ID NO: 8, or the nucleotides 307 to 2772 corresponding to the open reading frame thereof.
Given below is a non-exhaustive list of proteins involved in diseases affecting the skeletal muscles and referred to in this invention: Sarcoglycan (α, β, γ, δ), Dystrophin, Dysferlin (DYSF), Selenoprotein 1 (SEPN1), Amphyphisine 2 (BIN1), dynamien 2 (DNM2), cofilin 2 (CFL2), troponin T (TNNT1), tropomyosin 3 (TPM3), ACTA1, contactin 1 (CNTN1), TRIM32, Rapsyn (RASPN), DOK7, Agrin (AGRN), COLQ, CHAT, acetylcholine receptors (CHRNE, CHRNA1, CHRNB1, CHRND), GFPT1, MUSK.
According to a particular embodiment, the sequence contained in the expression system of the invention does not encode a protein of the sarcoglycans family, especially the α-sarcoglycane, or more specifically the sequence described in the paper Mendell et al. (Annals of Neurology, Vol. 68, No 5, pp 629-638, 2010).
In another particular embodiment, the sequence contained in the expression system of the invention does not encode a dystrophin-like protein, including minidystrophin, and in particular that described in the paper Wang et al. (Gene Therapy, Vol. 15, No 22, pp 1489-1499, 2008).
More generally, this invention refers to any protein having therapeutic activity in a neuromuscular disease, for example whose mutation causes a disease in one or more target tissues, if its production from an expression system exhibits toxicity in at least one tissue, preferably a non-target tissue, especially the heart, and more exhaustively in at least one tissue from the following group: heart, liver, brain, lungs, kidney and smooth muscles.
According to the invention and advantageously, the expression system must allow the expression at a therapeutically acceptable level of the protein in the target tissues, preferably in the skeletal muscles and/or the peripheral nervous tissue. Moreover and according to another preferred embodiment, it must allow the expression at a toxically acceptable level of the protein in non-target tissues, particularly the heart.
In the context of this invention, the term “protein expression” may be understood as “protein production”. Thus, the expression system must allow for both transcription and translation of the protein at the levels defined above.
The levels defined in the context of the invention, namely “therapeutically acceptable” and “toxically acceptable” are related to the amount of protein, as well as its activity.
The evaluation of the amount of protein produced in a given tissue can be carried out by immunodetection using an antibody directed against said protein, for example by Western blot or ELISA. Alternatively, the corresponding messenger RNAs may be quantified, for example by PCR or RT-PCR. This quantification can be performed on one sample of the tissue or on several samples. Thus and in the case where the target tissues are skeletal muscles, it may be carried out on a muscular type or several types of muscles (for example quadriceps, diaphragm, tibialis anterior, triceps, etc.).
In the context of the invention, the term “therapeutically acceptable level” refers to the fact that the protein produced from the expression system of the invention helps improve the pathological condition of the patient, particularly in terms of lifespan and quality of life. Thus and in connection with a disease affecting skeletal muscles, this involves improving the muscular condition of the subject affected by the disease or restoring a muscular phenotype similar to that of a healthy subject. As mentioned above, the muscular state, preferably defined by the strength, size, histology and function of the muscles, can be evaluated by one of the following methods: biopsy, measurement of the strength, muscle tone, volume, or mobility of muscles, clinical examination, medical imaging, biomarkers, etc.
Thus, the criteria that help assess a therapeutic benefit as regards skeletal muscles and that can be evaluated at different times after the treatment are in particular:
In the context of the invention, the term “toxically acceptable level” refers to the fact that the protein produced from the expression system of the invention does not cause significant alteration of the non-target tissue, especially histologically, physiologically and/or functionally. In particular, the expression of the protein may not be lethal. Advantageously, the amount of protein produced in the non-target tissue must not exceed the endogenous level of said protein in this tissue, in particular compared to a healthy subject. As already stated, the toxicity in a tissue can be evaluated histologically, physiologically and functionally. In the particular case of the heart and for illustrative purposes, any toxicity of a protein can be evaluated by a study of the morphology and the heart function, by clinical examination, electrophysiology, imaging, biomarkers, monitoring of the life expectancy or by histological analysis, including the detection of fibrosis and/or cellular infiltrates, for example by staining with sirius red or hematoxylin/eosin.
Advantageously, the level of efficacy and/or toxicity of the expression system according to the invention is evaluated in vivo in the animal, even more preferably in an animal having a defective copy of the gene encoding the protein and thus affected by the associated pathology.
Preferably, the expression system is administered systemically, for example by intravenous injection.
According to the invention and preferably, the expression system of the invention comprises at least one sequence that allows to:
According to a particular embodiment, the invention relates to an expression system comprising at least one sequence that allows to:
In the context of the invention, the terminology “prevent the expression” preferably refers to cases where, even in the absence of the said sequence, there is no expression, while the terminology “decrease the level of expression” refers to cases where the expression is decreased (or reduced) by the provision of said sequence.
Similarly, the terminology “maintain the expression” preferably refers to cases where, even in the absence of said sequence, there is a comparable level of expression, while the terminology “increase the level of expression” refers to cases where there is an increase in expression by the provision of said sequence.
In the context of the invention, there are at least three ways, which may be combined, to achieve the desired objective:
Suitably, an expression system of the invention comprises a promoter sequence governing the transcription of the sequence encoding the protein, preferably placed at 5′ of the transgene and functionally linked thereto. Preferably, this ensures a therapeutically acceptable level of expression of the protein in target tissues, particularly in skeletal muscles.
This may include inducible or constitutive, natural or synthetic (artificial) promoters. Similarly, they can be of any origin, including human, of the same origin as the transgene or of another origin.
According to a first embodiment, the promoter sequence corresponds to a ubiquitous or non-selective promoter, that is to say a promoter with low tissue specificity and ensuring a broadly similar level of expression in different tissues, for both target and non target tissues. The following can be cited as examples: the cytomegalovirus promoter (pCMV), the Mtm1 promoter.
According to a particular embodiment, this refers to a promoter suitable for skeletal muscles and/or peripheral nerve tissue but which can be expressed in other tissues, especially in other muscles. The following can be cited as an example: the desmin promoter, preferably of sequence SEQ ID NO: 11, the skeletal alpha-actin promoter, the muscle creatine kinase (MCK) promoter, the C5-12 synthetic promoter, the synapsin I (Syn) promoter or the CK6 promoter. In relation to the skeletal muscles, others can also be cited: troponin, myogenic factor 5 (MyfS), myosin light chain 1/3 fast (MLC1/3f), myogenic differentiation 1 (MyoD1), myogenin (Myog), paired box gene 7 (Pax7), MEF2 promoters. In connection with the peripheral nervous tissue, the PO and MBP (Myelin Basic Protein) promoters can also be cited.
According to a preferred embodiment of the invention, the promoter sequence of the expression system is chosen for its promoter activity which differentiates between target and non-target tissues, in this case superior in the target tissues. In this case, this sequence helps increase the expression of the protein in the target tissues, preferably the skeletal muscles and/or peripheral nervous tissue, while preventing expression in the non-target tissues, particularly those in which the expression of the protein is toxic.
By way of example and in the case where the target tissue is skeletal muscle, the promoter is preferably a muscle-specific promoter. According to another advantageous characteristic, said promoter has low or no promoter activity in the non-target tissues, particularly the heart, enabling a toxically acceptable level of expression of the protein in these tissues.
According to a particular embodiment, said promoter sequence may correspond to the promoter of the calpain 3 gene, preferably of human origin, even more preferably of sequence SEQ ID NO: 12. Another suitable promoter sequence is that of the miRNA 206 (miR206), preferably of human origin, more preferably of sequence SEQ ID NO: 13.
Thus within the framework of the invention, it has been shown at least for Calpain 3, that an expression system comprising the sequence encoding said protein, placed under the control of the calpain 3 or miRNA 206 promoter, was capable of ensuring the expression at a therapeutically acceptable level of the protein in the skeletal muscles, and at a toxically acceptable level of the protein in the heart and liver.
In another aspect, the present invention therefore relates to an expression system comprising a sequence encoding a protein, placed under the control of a promoter having the sequence SEQ ID NO: 12 or SEQ ID NO: 13. Promoter sequences derived from the sequences SEQ ID NO: 12 and SEQ ID NO: 13 or corresponding to a fragment thereof but having a similar promoter activity, particularly in terms of tissue specificity and optionally effectiveness, are also covered under the present invention.
In case this promoter sequence does not allow expression at a toxically acceptable level of the protein in the non-target tissues, it is advantageously associated with a sequence having the function of reducing the level of expression of the protein in the non-target tissue, preferably in non-target tissues where the expression of the protein is toxic.
Thus and by way of example, in the case of both myotubularin and calpain 3, it was shown that the use of a desmin promoter presented cardiac toxicity. In contrast and in accordance with the invention, the use of a desmin promoter, preferably of sequence SEQ ID NO: 11, associated with at least one target sequence of the miRNA-208a, preferably of sequence SEQ ID NO: 10, allows both:
As already stated, said sequence is capable of preventing the expression or reducing the level of expression of the protein in non-target tissues, preferably in the non-target tissues where protein expression is toxic. This action may take place according to various mechanisms, particularly:
Such a sequence is preferably a target for a small RNA molecule selected from the following group:
Advantageously, this sequence helps maintain the expression, or even increase the level of expression of the protein in the target tissue(s), preferably in the skeletal muscles.
Preferably, such a sequence is selected for its effectiveness in the non-target tissue wherein the expression of the protein is toxic. Since the effectiveness of this sequence can be variable depending on the tissues, it may be necessary to combine several of these sequences, chosen for their effectiveness in all target tissues where toxicity is proven.
According to a preferred embodiment, this sequence is a target sequence for a microRNA (miRNA). As known, such a judiciously chosen sequence helps to specifically suppress gene expression in selected tissues.
Thus and according to a particular embodiment, the expression system of the invention comprises a target sequence for a microRNA (miRNA) expressed or present in the non-target tissue(s) in which the expression of the protein is toxic, for example in the heart. Suitably, the quantity of this miRNA present in the target tissue, preferably skeletal muscles, is less than that present in the non-target tissue, or this miRNA may not even be expressed in the target tissue. According to a particular embodiment, the target miRNA is expressed specifically in the non-target target tissue, such as heart.
As is known to the person skilled in the art, the presence or level of expression, particularly in a given tissue, of a miRNA may be assessed by PCR, preferably by RT-PCR, or by Northern blot.
Different miRNAs now identified, as well as their target sequence and their tissue specificity, are known to those skilled in the art and are for example described in the document WO 2007/000668.
According to a particular embodiment, the expression system of the invention comprises the target sequence of the miRNA-208a (also noted miR208a, SEQ ID NO: 9). Preferably, this sequence, identical in humans, dogs and mice, has the sequence SEQ ID NO: 10 of 22 pb. Of course, any derived or truncated sequence recognised by the miRNA-208a may be implemented as part of the invention. Thus, it has been shown within the framework of the invention that the use of this target sequence, both in relation to the myotubularin and the calpain 3, makes it possible to solve the problem of their cardiac toxicity, or even hepatic toxicity in the case of calpain 3.
As already stated, a target sequence for a microRNA may be used alone or in combination with other sequences, advantageously target sequences for a microRNA, which may be identical or different. These sequences can be used in tandem or in opposite direction.
According to a preferred embodiment, particularly for the target sequence of the miRNA-208a, one (1) or more, particularly two (2) or four (4) sequences, may be implemented. Preferably, they are used in tandem, that is to say, all in the same direction. In cases where multiple target sequences are implemented, they may be separated by a DNA spacer of random sequence, in a manner known to those skilled in the art.
Preferably, in the case of a target sequence of a miRNA, particularly the miR208a, it is placed at 3′ of the sequence encoding the protein, more advantageously inserted into the 3′ UTR (“Untranslated Region”) region of the expression system, preferably the cDNA encoding the protein. And even more preferably and where the expression system comprises a polyadenylation signal at 3′ of the cDNA encoding the protein, this sequence is inserted between the stop codon of the open reading frame and the polyadenylation signal.
In the context of the invention, it has been demonstrated that at least one target sequence of the miRNA-208a was adapted to obtain a toxically acceptable level of the protein at least in the heart, in particular concerning myotubularin and calpain 3.
According to a particular embodiment, the expression system comprises:
In another particular form of embodiment, the expression system comprises:
Thus, different types of sequences detailed above may be combined in the same expression system.
According to the invention, an expression system or expression cassette comprises the elements necessary for the expression of the transgene present. In addition to sequences such as those defined above to ensure and to modulate transgene expression, such a system may include other sequences such as:
An expression system according to the invention can be introduced in a cell, a tissue or a body, particularly in humans. In a manner known to those skilled in the art, the introduction can be done ex vivo or in vivo, for example by transfection or transduction. According to another aspect, the present invention therefore encompasses a cell or a tissue, preferably of human origin, comprising an expression system of the invention.
The expression system according to the invention, in this case an isolated nucleic acid, can be administered in a subject, namely in the form of a naked DNA. To facilitate the introduction of this nucleic acid in the cells, it can be combined with different chemical means such as colloidal disperse systems (macromolecular complex, nanocapsules, microspheres, beads) or lipid-based systems (oil-in-water emulsions, micelles, liposomes).
Alternatively and according to another preferred embodiment, the expression system of the invention comprises a plasmid or a vector. Advantageously, such a vector is a viral vector. Viral vectors commonly used in gene therapy in mammals, including humans, are known to those skilled in the art. Such viral vectors are preferably chosen from the following list: vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector and adeno-associated viral vector (AAV).
Preferably, it is an adeno-associated viral vector (AAV) corresponding to natural serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9), variants thereof or artificial serotypes. In a manner known to those skilled in the art, chimeric AAV vectors may also be implemented.
Preferably, the expression system of the invention is inserted between two ITR (“Inverted Terminal Repeat”) sequences of the AAV vector.
In connection with a systemic administration to which the expression system of the invention is fully applicable, AAV vectors of serotype 8 or 9 are particularly preferred. This may for example include AAV2/8 or AAV2/9 vectors.
In a manner known to those skilled in the art, recombinant virus particles can be obtained, for example by tri-transfection of HEK 293 cells or by the baculovirus system. The vector titres are conventionally expressed as viral genomes per millilitre (vg/ml).
According to a preferred embodiment, the expression system of the invention includes a vector having a suitable tropism, in this case higher for the target tissue(s) than for the non-target tissues, especially those where the expression of the protein appears toxic. It can be an AAV vector containing a capsid selected for minimum or no targeting/transducing the non-target tissues such as the heart or to specifically target/transduce target tissues, especially skeletal muscles.
As is apparent from the above, the expression systems according to the invention, especially in the form of recombinant AAV vectors or recombinant viral particles, have obvious applications, especially in the field of therapeutics.
Thus and according to another aspect, the invention relates to the use of the expression system described as a medicine. In other words, a pharmaceutical composition comprising such an expression system is also covered. Suitably, it may further comprise a pharmaceutically acceptable and inert carrier, preferably adapted for systemic administration, e.g., intravenous administration. Various excipients, stabilisers, and other known suitable compounds known to those skilled in the art can be added to such a composition.
The present invention has demonstrated the benefit of the expression system described in cases where administration is not done locally in the target tissues, but instead generally in the whole body, resulting in its delivery in the non-target tissues.
Thus, and preferably, an expression system according to the invention is administered by one of the following routes: enteral, parenteral, oral, intravenous, intraarterial and by inhalation.
Preferably, it is a systemic administration, and more preferably an intravenous injection. Note that a systemic administration may be performed near a treatment area, for example near a skeletal muscle.
According to a particular form of embodiment, it is not a loco-regional administration. More specifically, the following can thus be excluded: an intravascular, particularly intravenous, injection (performed under pressure and in the presence of a tourniquet, close to the target muscle), as described for example by Petrov et al. (Methods Mol Biol 2011; 709: 277-86), or an intra-arterial injection (catheter in an artery and venous clamping near the artery, upstream, to prevent diffusion) as described for example by Gonin et al. (J Gene Med 2005; 7: 782-791).
When the composition of the invention is to be injected, it is preferably in liquid form. The active concentration, in this case the expression system of the invention, the quantity to be injected and the frequency of injections are determined by a person skilled in the art. A single administration may be sufficient. A therapeutic effect is preferably observed for a period of at least 1 month, 3 months, 6 months, 1 year, 5 years or more.
Such medicines are intended for gene therapy, particularly for the treatment of neuromuscular disorders and more particularly diseases mainly affecting skeletal muscles (myopathy). More generally, the invention helps improve muscle function in a subject.
Patients to be treated are preferably mammals, particularly humans.
A disease particularly referred to in the context of the invention is centronuclear myopathy, more precisely X-linked myotubular myopathy (XLMTM). Furthermore, other centronuclear myopathy and neuromuscular diseases associated with myotubularin, such as some forms of the Charcot-Marie-Tooth disease, can be treated.
Type 2A limb-girdle dystrophy (LGMD2A) may also be treated with an expression system of the invention.
More generally, a non-exhaustive list of diseases covered by this invention is as follows: congenital muscular dystrophy with selenoprotein N deficiency, congenital muscular dystrophy with primary merosin deficiency, Ullrich congenital muscular dystrophy, Duchenne (DMD) or Becker (BMD) muscular dystrophy, central core congenital myopathy, multi-minicore congenital myopathy, centronuclear autosomal myopathy, myopathy with fibre dysproportion, nemaline myopathy, congenital myasthenic syndromes, other neuromuscular diseases associated with myotubularin, Type 2B or 2D limb-girdle dystrophy, miyoshi distal myopathy, dysferlinopathies, sarcoglycanopathies.
According to a particular form of embodiment, the pathology is not the Duchenne (DMD) or the Becker (BMD) muscular dystrophy or even the LGMD2D.
Therefore, both an improvement of the condition, and thus the quality of life and longevity of the patient are expected of the medicine according to the invention, while avoiding potential side effects in other tissues of such a treatment.
As will be demonstrated by way of example, the present invention has demonstrated the potential cardiac toxicity of the gene therapy treatments for muscle diseases and offers technical solutions to overcome this problem.
The invention and the advantages resulting from it will be better understood with the examples of realisation given below and with the help of the figures annexed. However, these are not exhaustive.
The present invention is illustrated in connection with the myotubularin (MTM1) and calpain 3 (CAPN3) gene. However, the strategy described can be applied to any transgene encoding a protein of interest in the skeletal muscles whose cardiac toxicity is demonstrated.
A/ Mtm1 expression cassette, devoid of target sequences for miRNA-208a;
B/ expression cassette containing 1, 2 or 4 target sequences for miRNA-208a (box) at 3′ of the Mtm1 gene.
Top: distribution of the vector in skeletal muscles (tibialis anterior=TA; quadriceps=QUA; triceps=TRI) and in the heart of a wild mouse (WT), 1 month after the intravenous administration of vectors (vg/diploid genome).
Bottom: level of MTM 1 protein in skeletal muscles and the heart of a wild mouse (WT), one month after administration of the vectors. The values indicate the multiplication rate in relation to the endogenous levels. As controls, mice were injected with either PBS or empty AAV8 vector (AAV-MCS).
A/ Histological analysis of the heart muscle after injection of PBS or the vectors AAV2/9-desm-CAPN3 (pdes.C3), AAV2/9-pC3-CAPN3 (pC3.C3), AAV2/9-pmiR206-CAPN3 (p206.C3) and Sirius red staining (top, scale=500 μm) or Hematoxylin Phloxine Saffron (HPS) (bottom, scale=100 μm).
B/ Evaluation of the vector DNA level by qPCR in the heart of WT wild mice after injection.
C/ Evaluation of the CAPN3 mRNA level by qPCR in the heart of WT wild mice after injection. The line “H” corresponds to the CAPN3 endogenous mRNA level in the heart of WT wild mice.
D/ Analysis of serum enzymes. The alanine aminotransferase tests (ALT) were carried out on sera of WT mice treated with pC3.C3 to the left, or p206.C3 to the right. The standard deviation and mean (SEM) for each condition are indicated by a circle and a vertical bar, respectively.
A/ Histological analysis of the heart muscle, 35 days after injection of PBS or identical doses of the vectors AAV2/9-desm-CAPN3 (pdes.C3) or AAV2/9-desmin-CAPN3-miR208aT (pdes.C3-T) and Sirius red staining (top, scale=500 1,μm) or HPS (bottom, scale=100 μm).
B/ Evaluation of the vector DNA level by qPCR in the heart of WT wild mice after injection (top) and the mRNA level of CAPN3 transgene (bottom). The line “H” corresponds to the CAPN3 endogenous mRNA level in the heart of WT wild mice.
C/ Analysis of the expression of calpain 3 by Western blot in the skeletal muscle and heart of WT mice injected with PBS or the vectors AAV2/9-desmin-CAPN3 (pdes.C3) or AAV2/9-desm-CAPN3-miR208aT (pdes.C3-T). The entire protein is indicated by an arrow and its cleavage products (60, 58 and 55 kDa) by a hook.
D/ Quantification of mRNA levels of miR-208a (miR208a), HOP (Hop) and connexin 40 (Cnx40) in the heart of WT mice injected with AAV2/9-desmin-CAPN3 (pdes.C3) or AAV2/9 desmin-CAPN3-miR208aT (pdes.C3-T). The quantity of RNA in the pdes.C3-T condition is given as a percentage of the RNA level in the pdes.C3 condition.
A/ Transverse sections of the TA muscles of C3KO mice were stained with the HPS, 4 months after injection either with PBS or vectors (1.2×1013 vg/kg) AAV2/9-desmin-CAPN3-miR208aT (pdes.C3-T), AAV2/9-PC3-CAPN3-miR208aT (pC3.C3-T), AAV2/9-pmiR206-CAPN3-miR208aT (P206.C3-T). Scale=100 μm.
B/ Number of centronuclear fibres (CNF/mm2) measured in the stained sections with the HPS in TA (left) and PSO (right) muscles of C3KO mice injected either with PBS or vectors (1.2×1013 vg/kg) AAV2/9-desmin-CAPN3-miR208aT (pdes.C3-T) AAV2/9-PC3-CAPN3-miR208aT (pC3.C3-T), AAV2/9-pmiR206-CAPN3-miR208aT (p206.C3-T). A difference with a P value<0.05 is indicated by an asterisk. TA: tibialis anterior; PSO: Psoas muscle.
1) Generation of Recombinant AAV Vectors:
The vector rAAV-Des-Mtm1 was constructed by cloning the open reading frame of the murine Mtm1 gene (SEQ ID NO: 14) downstream of the human desmin promoter (SEQ ID NO: 11) in a vector serotype 2 AAV. Target sequences (1, 2 or 4 sequences, miRHT1, miRTH2 and miRHT4 respectively) of the miRNA-208a of 22 pb (SEQ ID NO: 10), each separated by DNA spacers, have been added in the 3′UTR region of the Mtm1 cDNA. An empty vector (rAAV-Des-MCS) was also generated as a control. Recombinant viral particles of serotype 8 (AAV8) were obtained using a tri-transfection protocol of the HEK 293 cells as described previously (15). The vector titres are expressed in terms of viral genomes per ml (vg/ml).
Similarly, the vector rAAV-desm-CAPN3 (or AAV-desmin-CAPN3 or AAV-pDes-CAPN3) was constructed using the cDNA of human calpain 3 (SEQ ID NO: 8) under the control of the human desmin promoter (SEQ ID NO: 11). RAAV-PC3-CAPN3 and rAAV-pmiR206-CAPN3 vectors were obtained by replacing this promoter by the promoter region of the human calpain 3 (SEQ ID NO: 12) or that of the miARN206 (SEQ ID NO: 13), respectively. The vectors AAV-desm-CAPN3-miR208aT, AAV-PC3-CAPN3-miR208aT and AAV-pmiR206-CAPN3-miR208aT were obtained by adding 2 target sequences for the miARN208a (SEQ ID NO: 9) in tandem (miR208aT), at 3′ of the calpain gene 3. Recombinant viral particles of serotype 1 (AAV1), 8 (AAV8) and/or 9 (AAV9) were produced.
2) In Vivo Experiments:
The mice were treated according to the French and European legislation regarding animal testing. In this study, WT C57B1/6 wild mice (Charles River Laboratories) and a mouse strain constitutively inactivated for myotubularin (knockout) KO-MtMi, also called BS53d4-129pas, were used. For calpain 3, the C3KO murine model, described by Laure et al. (Febs J., 2010, 277: 4322-4337), was used.
Recombinant vectors, as per the indicated doses were injected into the tail vein of the mice as indicated (aged 3 weeks to 2 months). An equivalent volume of saline buffer (PBS) was administered as a control. The clinical status and animal weight were monitored weekly for WT animals and three times per week for the mutant mice. The mice were sacrificed at the indicated times.
3) Western Blot:
Muscles frozen in isopentane were cut in cross-sections of 30 μm and lysed on ice in a buffer containing 150 mM NaCl, 10 mM Tris HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 100 mM sodium fluoride, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% Triton X100 and 0.5% IGEPAL supplemented with a complete cocktail of protease inhibitors (Roche). The muscle extracts were incubated for 1 h and centrifuged at 4° C. at 12,000×g for 30 min. The protein concentrations in the supernatant were determined using the Bio-Rad “protein assay kit”. Proteins were subjected to migration to SDS-PAGE and, after transfer to a nitrocellulose membrane, incubated with polyclonal antibodies directed against the myotubularin (p2348 [15]) and GAPDH (#MAB374, Millipore). The protein bands were viewed by infrared fluorescence using the “Odyssey Imaging System” (LICOR Biotechnology Inc.) and quantified using the program “Odyssey Infrared Imaging System Software” (software application, version 1.2, 2003).
For detection of calpain 3, a similar protocol was used:
The muscles were homogenized by FastPrep using the lysis buffer according to [20 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 2 mM E64 (Sigma)] and protease inhibitors (Complete Mini protease inhibitor cocktail; Roche Applied Science, 25 μl per mg of tissue). The samples were treated with 250U/100 μl of Benzonase (Calbiochem) for 30 min at 4° C. to digest the DNA. The muscle lysates were mixed with the load buffer [NuPage LDS (Invitrogen), TNT 3M (Sigma)], denatured for 10 minutes at 70° C. and centrifuged briefly. The supernatants were separated by polyacrylamide gel NuPAGE Bis-Tris in 4-12% gradient (Invitrogen). After the transfer, the membranes were hybridised with antibodies against calpain 3 (mouse monoclonal antibody, Novocastra NCL-CALP-12A2, 1/200 dilution), at 4° C. overnight or at room temperature for 2-3 hours. Finally, the membranes were incubated with IRDye® in order to be revealed on the Odyssey infrared scanner (LI-COR Biosciences, Lincoln, Nebr., USA).
4) PCR:
4-1-Myotubularin:
The isolation of DNA from the muscles was performed using the “Gentra Puregene Tissue Kit” (Qiagen), in accordance with the manufacturer's instructions. The total DNA concentration was determined using a ND-8000 Nanodrop spectrophotometer (Nanodrop Technologies, France), and 80 ng of DNA for each sample was used as matrix for the PCR in real time. The Taqman real-time PCR was performed on each sample for both a part of the skeleton common to the rAAV2/X vector to identify copies of the viral genome, and the murine gene of the titin, to standardise the number of murine genomes present in each sample. The primers used for amplification of the rAAV vectors were: 5′-CTCCATCACTAGGGGTTCCTTG-3′ (forward; SEQ ID NO: 15), 5′-GTAGATAAGTAGCATGGC-3′ (reverse; SEQ ID NO: 16). The MGB probes were double-labelled (FAM-NFQ): 5′-TAGTTAATGATTAACCC -3′ (probe; SEQ ID NO: 17). Primers and a probe used for the titin were: 5′-AAAACGAGCAGTGACGTGAGC-3′ (forward; SEQ ID NO: 18), 5′-TTCAGTCATGCTGCTAGCGC-3′ (reverse; SEQ ID NO: 19), and 5′-TGCACGGAAGCGTCTCGTCTCAGTC-3′ (probe; SEQ ID NO: 20) (Applied Biosystem). The amplifications of the titin were performed using 80 ng of DNA diluted in an “Absolute QPCR ROX Mix” (Thermo Fischer Scientific), 0.1 μM of Taqman probes and 0.2 μM of primers (forward and reverse), in a final volume of 25 μM. The cycle conditions consisted of: an activation step for the Thermo-Start DNA polymerase at 95° C. for 15 min, followed by 40 two-step cycles, 15 seconds of denaturation at 95° C. and 60 seconds of hybridisation and extension at 60° C. The amplification of the rAAVs was performed using 0.1 μM of Taqman probes, 0.3 μM of reverse primer and 0.05 μM of forward primer in a final volume of 25 μl. The cycle conditions consisted of: an activation step for the Thermo-Start DNA polymerase at 95° C. for 15 min, followed by 40 two-step cycles, 15 seconds of denaturation at 95° C. and 60 seconds of hybridisation and extension at 54° C. The PCR was performed on a 7900 HT thermocycler (Applied Biosystem). A standard dilution series of a plasmid containing the sequences of a rAAV skeleton and the titin was used in each PCR plate in real time as control of the number of copies. All samples and controls were duplicated. The data are expressed as number of copies of the viral genome per diploid genome.
4-2-Calpain 3:
The muscles were extracted using the Trizol method (Invitrogen). During extraction, a sample fraction was preserved for DNA extraction for quantification by quantitative PCR. The total RNA was extracted from the remaining extract treated with the “DNA-Free” kit (Ambion) to remove residual DNA. For quantification of the expression of endogenous microRNAs, a total of 20 ng RNA were subjected to a reverse transcription using the “reverse transcription TaqMan MicroRNA” kit (Applied Biosystems) and analysed by the microRNA ID511 Taqman assay for miR-208a (Applied Biosystems). The standardisation of the samples was carried out with the expression of snoRNA202 with test ID 1232 (Applied Biosystems).
For the amplification of mRNAs of the endogenous or transgenic calpain 3, one μg of RNA was reverse transcribed using random hexamers and oligodT and the cDNA Verso kit (Abgene) or “RevertAid H Minus First Strand cDNA Synthesis” kit (Fermentas). The real-time PCR was performed using the TaqMant method applying the ABI PRISM 7700 (Applied Biosystems) system and the “Absolute QPCR Rox Mix” solution (ABgene) with the help of the primer pairs (.f and .r) and Taqman probe (.p) below: for the quantification of transgenic calpain: CAPN3sfr.f (SEQ ID NO: 21) 5′_CGCCTCCAAGGCCCGT—3′; CAPN3sfr.r (SEQ ID NO: 22) 5′_GGCGGAAGCGCTGGCT—3′; MGBTUCAPN3.p (SEQ ID NO: 23) 5′_CTACATCAACATGAGAGAGGT—3 ; for quantification of human calpain: CAPN3.f (SEQ ID NO: 24) 5′_CGCCTCCAAGGCCAGG—3′, CAPN3.r (SEQ ID NO: 25) 5′_GGCGGAAGCGCTGGGA—3 et CAPN3.p (SEQ ID NO: 26) 5′_TACATCAACATGCGGGAGGT—3. A serial dilution of a control RNA was used in each experiment and treated with the experimental samples to avoid the variability in the efficiency of the cDNA preparation and the PCR in order to be able to compare the different experiments. This RNA was prepared by an in vitro transcription reaction from a plasmid carrying a cDNA calpain 3 mutated and amplifiable by all the pairs of primers.
The analysis of the expression of the connexin 40 and HOP was performed using the TaqMan® Gene Expression tests (Applied Biosystens) given below: for Cnx40; Gja-5 [Mus Musculus]: Mm00433619_s1 and hop: HOP homeobox [Mus musculus]: Mm00558630_m1. The qRT-PCR results are expressed in arbitrary units related to the expression of the ubiquitous ribosomal phosphoprotein acid murine gene (P0 G1: 15029771; MH181PO.F (SEQ ID NO: 27): 5′_CTCCAAGCAGATGCAGCAGA—3′/M267PO.R (SEQ ID NO: 28): 5′_ACCATGATGCGCAAGGCTAT—3′/M225PO.p (SEQ ID NO: 29): 5 <<_CCGTGGTGCTGATGGGCAAGAA—3′).
5) Histology:
Cross cryosections (8 μm thickness) of the cardiac, hepatic or skeletal muscles were stained with hematoxylin eosin (HE), sirius red or Hematoxylin Phloxine Saffron (HFS) using standard protocols.
The sections were mounted with the Eukitt medium (LABONORD). The digital images were captured using a CCD camera (Sony). The morphometric analyses of the skeletal muscles to define the number of centronuclear fibres (CNF/mm2) were performed using the Histolab software (Microvision, Evry).
6) Measurement of ALT Activity:
Blood samples were collected without coagulation. After centrifugation (8000 g, 10 min, 4° C.), the sera were analysed using the VITROS DT60 device (Ortho Clinical Diagnostics, UK) using the “Vitros ALT DT slides” cassettes for the determination of the alanine aminotransferase (ALT) rate.
A-Myotubularin
1) Cardiac Toxicity of the Construction AAV-pDES-Mtm1:
The beneficial effect of a single intramuscular injection of the myotubularin (Mtm1) gene under the control of the CMV promoter in a vector AAV2/1 was known from the paper Buj-Bello et al. [15]
A gene therapy approach by systemic route in Mtm1 knockout mice was attempted and it has been shown that administration of an AAV8 vector (rAAV-Des-Mtm1) expressing myotubularin under the control of human desmin promoter (
However, following systemic administration of the vector AAV8-DES-Mtm1 in Mtm1 KO mice, it was observed that the level of myotubularin protein was very high in the heart compared to the skeletal muscles (results not shown). In addition, the presence of inflammatory infiltrates and fibrosis in the heart of XLMTM mice treated with AAV at different times following the viral injection (
2) Developments of Expression Systems Without Cardiac Toxicity
Given the difficulty to predict the biodistribution and transgene expression from a vector AAV8 after systemic administration, particularly in humans, new vectors carrying regulatory sequences increasing the muscle specificity have been developed in order to avoid potential side effects affecting the heart.
Three viral constructs (rAAV-Des-Mtm1-miRHT1; rAAV-Des-Mtm1-miRHT2 and rAAV-Des-MTM1-miRHT4) were developed, as shown in
3) Muscle and Heart Production of MTM1 after Injection in a WT Mouse
In order to select the expression vector that is most suitable for MTM1, a single dose of 3×1013 viral genomes (vg)/kg of these vectors was administered in the tail vein of wild-type mice aged 3 weeks. An empty vector (AAV-Des-MCS) and PBS (“Phosphate Buffered Saline”) were used as internal controls.
The vector distribution and protein level in myotubularin in the heart and in different skeletal muscles (anterior tibial=TA; quadriceps=QUA, triceps=TRI) were assessed 1 month after the injection. Western blot results showed that these vectors are able to decrease the level of myotubularin produced from vectors specifically in the heart. In addition, a single target sequence of the miRNA208a is sufficient to reduce expression in this tissue (
4) Validation of the Vector Construction after Injecting an Mtm1 Mutated Mouse
Based on previous results, the construct rAAV-Des-Mtm1-miRHT1 was selected for further experiments. WT wild mice mutated in the MTM1 gene (KO for “Knock Out”) received 3×1013 vg/kg of AAV-Des-Mtm1, rAAV-Des-Mtm1-miRHT1 and rAAV-Des-MCS, respectively, or PBS at the age of 3 weeks, and were clinically monitored for 1 month.
All mutant mice that received AAV8-Des-Mtm1-miRHT1 survived until the end of the study, with a growth curve similar to that of KO mice treated with AAV8-Des-Mtm1 showing that the inclusion of the miRHT1 sequence does not affect the therapeutic efficacy of the transgene (
The histology of the heart of WT and KO mice was analysed one month after treatment, with hematoxylin-eosin and Sirius red staining. Fibrotic areas were observed in the heart of 7 KO mice out of 9 treated with 9-AAV8-Des-Mtm1, but not in the KO mice treated with AAV8-Des-Mtm1-miRHT1 (n=10). The administration of the vector AAV8-Des-Mtm1 did not cause fibrosis in WT animals 1 month after injection (n=8).
In conclusion, these results indicate that the inclusion of a single target sequence of miARN208a is sufficient to reduce the cardiac toxicity of an AAV8-Des-Mtm1 construct.
Similar experiments were conducted with regard to calpain 3 (CAPN3):
B-CALPAIN 3
The paper Bartoli et al. (Molecular Therapy, 2006, Vol. 13, No. 2, 250-259) indicates a beneficial effect and non-toxicity of AAV type of constructs carrying the calpain 3 gene under the control of muscle-specific promoters, after intramuscular or local administration. However, the experiments carried out in connection with the invention have revealed toxicity in such constructs after systemic administration:
1) Cardiac Toxicity of AAV-desm-CAPN3 Constructs:
The condition of WT mice was monitored, following intravenous injection of different constructs, and is presented in Table 2 below:
For all tested AAVs, a destruction of heart tissue is observed in case of systemic administration, excluding the use for therapeutic purposes of these gene expression systems.
2) Reduction of Cardiac Toxicity of the Constructs AAV-desm-CAPN3 by Replacing the Promoter:
Two vectors were constructed by exchanging the desmin promoter with that of CAPN3(AAV2/9-pC3-CAPN3) or miR-206 (AAV2/9-pmiR206-CAPN3). After viral preparation of vectors, the in vivo consequences of the changes introduced by intravenous injection (6×1012 vg/kg) were analysed in C57BL/6 mice (WT) aged 2 months.
35 days after injection, no cardiac fibrosis was observed in mice treated with the vectors AAV2/9-pC3-CAPN3 and AAV2/9-pmiR206-CAPN3, unlike the mice injected with AAV2/9-desm-CAPN3 (
Finally, the promoters CAPN3 and miR-206 reduce the cardiac toxicity of the transgene CAPN3 without causing liver toxicity.
3) Reduction of the Cardiac Toxicity of the AAV-desm-CAPN3 Constructs by Addition of Two Target Sequences of miR208a:
Two target sequences of MiARN208a (SEQ ID NO: 10) were cloned in tandem in a miR208aT cassette. This was then inserted into the 3′UTR area of the construct AAV2/9-desm-CAPN3 to produce the construct AAV2/9-desm-CAPN3-miR208aT.
After injecting a dose of 6×1012 vg/kg, no cardiac fibrosis was observed in the treated mice, unlike the mice injected with AAV2/9-desm-CAPN3 (
4) Combination of Two Strategies:
New vectors were constructed by combining the promoters CAPN3 and miR-206 and 2 copies of the target sequence of miR-208a: AAV2/9-pC3-CAPN3-miR208aT and AAV2/9-pmiR206-CAPN3-miR208aT. C3KO mice (knockout for calpain 3) received an injection of 1.2×1013 vg/kg of these vectors.
As previously observed in the wild mice, none of the three vectors (AAV2/9-desm-CAPN3-miR208aT, AAV2/9-pC3-CAPN3-miR208aT and AAV2/9-pmiR206-CAPN3-miR208aT) proved to be toxic for the heart, 3 months after the injection (results not shown).
In contrast, a histological and morphological examination of skeletal muscles of C3KO mice aged 4 weeks and injected with these vectors has shown a positive effect of the expression of calpain 3 on the pathological signs of the murine model. The anterior tibialis (TA) muscles injected with these vectors showed improved histological features compared to those injected with PBS (
In conclusion, these results indicate that the expression of calpain 3 in skeletal muscles transduced with these recombinant vectors can correct the pathological signs of a mouse deficient in calpain 3, without presenting cardiac toxicity.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1353306 | Apr 2013 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2014/050866 | 4/10/2014 | WO | 00 |
