Patent Application
20240228564
The present invention pertains to gene therapy for treating dysferlinopathy. More particularly, the invention relates to a polynucleotide sequence comprising several domains of dysferlin, or functional variants thereof, it also relates to a viral vector for gene therapy comprising at least a polynucleotide sequence of the invention exon 40a of the coding sequence of dysferlin.
Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. Dysferlin is a modular type II transmembrane protein containing seven calcium sensor C2 domains that play a key role in muscle membrane repair. DYSF mutations lead to a wide range of muscular phenotypes, in particular Miyoshi myopathy (MM), that affects distal muscles and limb girdle muscular dystrophy type 2B (LGMD2B or LGMDR2) characterized by proximal weakness. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairment eventually result.
Various therapeutic approaches for treating myopathies or dysferlinopathies have been proposed so far, although no curative treatment for dysferlinopathies exist.
WO 2020/123645 discloses a combination therapy for treating muscular dystrophy, wherein adenovirus gene therapy vectors comprise a polynucleotide encoding a first polypeptide, or a first RNA, and a second polypeptide, or a second RNA. This combination therapy involves both gene restoration and reduction of symptoms associated with a number of secondary cascades such as fibrosis.
WO 2018/170408 discloses an adeno-associated virus vector delivery of muscle specific micro-dystrophin, to treat muscular dystrophy. The gene therapy vectors are used to raise muscular strength and/or prevent fibrosis in patients affected by muscular dystrophy.
US 2020/0010521 discloses a truncated dysferlin nucleic acid and protein designed as “Nanodysferlin”.
WO 2011/054659 discloses an exon-skipping therapy for dysferlinopathies, by a process comprising a step of preventing the splicing of one or more exons coding for amino acid sequences responsible of dysferlin dysfunction. Said exon is in particular exon 32. Complementary antisense oligonucleotides used for correcting said splicing are also disclosed.
Barthelemy et al (“Translational research and therapeutic perspectives in dysferlinopathies”, Mol. Med. 17(9-10) 875-882, sept-oct, 2011) is a review describing therapeutic perspectives in dysferlinopathies. Barthelemy discloses a “mini-dysferlin” comprising the last two C2 domains of dysferlin and the transmembrane domain.
There is therefore a strong need for active therapy of dysferlinopathies.
The inventors have now shown that a protein comprising an amino acid sequence encoded by exon 40a of dysferlin protein is a key factor in the membrane repair process, and participates in other muscle cell functions, like muscle cell membrane protection and protein vesicle trafficking.
The present invention therefore relates to a nucleic acid sequence encoding for essential domains of dysferlin, including a nucleic acid sequence, such as exon 40a (GenBank EF015906), encoding for a cleavage site by calpain. A nucleic acid sequence of the invention is therefore useful to restore muscle cells repairing function in patients with dysferlinopathy.
Krahn et al (“Exclusion of mutations in the dysferlin alternative exons 1 of DYSF-v1, 5a and 40a in a cohort of 26 patients”, Gen. Test. Mol. Biomarkers, 2010, Feb. 14(1); 153-4, DOI 10.1089/gtmb.2009.0131) disclose a screening of mutations causing primary dysferlinopathies in three known alternative exons, DYSF-v1, 5a and 40a. The authors conclude that no disease-causing mutation was identified in said alternative exons, demonstrating a low frequency of disease-causing mutations in these exons.
Redpath et al (“Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair”, MBoC, vol. 25, Oct. 1, 2014) disclose that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a.
The inventors have now developed a nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
A nucleic acid sequence according to the invention codes for a polypeptide which is useful to prevent or abolish symptoms of dysferlinopathy, when injected to a patient in need thereof. A nucleic acid sequence according to the invention codes for a polypeptide useful to restore cell membrane repair, cell membrane protection and/or vesicle trafficking. In particular, a nucleic acid sequence according to the invention codes for a polypeptide able to restore cell membrane repair.
In a particular embodiment of the invention, a nucleic acid sequence of the invention encodes for a protein designated as “Midi-dysferlin” (SEQ ID No 4), a functional fragment thereof or a functional variant thereof.
The inventors have also developed a recombinant expression vector comprising at least a nucleic acid sequence according to the invention. A recombinant expression vector according to the invention can be used as a gene therapy vector. It is submitted that a recombinant expression vector according to the invention at least allows the restoration of the phenotype of a dysferlin deficient cell in an animal model, and may restore the “calpain cleavage activation” of dysferlin in a patient in need thereof.
The invention is based on the surprising discovery that the polypeptide encoded by exon 40a is strongly involved in the dysferlin repairing process of muscle cells membranes through the presence of a cleavage site by calpain, and on the observation that some mutations in said exon 40a sequence do not abolish the functional property of said peptide to form a cleavage site by calpain.
The inventors consequently defined a domain comprising a cleavage site by calpain, wherein said domain is either a polypeptide encoded by exon 40a, a functional fragment thereof or a functional variant thereof, or a polypeptide domain known in the art as susceptible to be cleaved by calpain.
The present invention therefore provides a gene replacement therapy aimed to at least partially restore dysferlin functions. A vector of the present invention therefore helps to reduce or prevent at least one symptom of a dysferlinopathy.
In a first aspect, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
The gene encoding human dysferlin (DYSF, Online Mendelian Inheritance in Man (OMIM) gene number 603009, 2p13, GenBank NM_003494.2) encodes several transcripts, the most common transcript is composed of 55 exons and alternative splicing events generate transcript diversity, such as exon 1, exon 5a and exon 40a (GenBank EF015906).
From its N-terminal to its C-terminal extremity, the human dysferlin protein comprises:
By “a nucleic acid sequence encoding for at least 100 amino acids” of a given nucleotide or amino-acid sequence, it is intended a nucleic acid sequence encoding for at least 100, 150, 200, 250, 280, 290 or 300 amino acids of said amino acid sequence.
By “a nucleic acid sequence encoding for a fragment of at least 100 amino acids” of a given nucleotide or amino-acid sequence, it is intended a nucleic acid sequence encoding for at least a fragment of 100, 150, 200, 250, 280, 290 or 300 amino acids of said amino acid sequence.
By “exhibiting at least 80% identity”, it is meant that said sequences exhibit at least 80% identity after optimal overall alignment, that is to say by global alignment between two sequences giving the highest percentage identity. between them. The optimal global alignment of two sequences can in particular be carried out according to the Needleman-Wunsch algorithm, well known to those skilled in the art (Needleman & Wunsch, “A general method applicable to the search for similarities in the amino acid sequences of two proteins”, J. Mol. Biol., 48 (3): 443-53).
Proteins encoding by a nucleic acid according to the invention comprise, or consist of an amino acid sequence having at least 80%, advantageously at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence SEQ ID No 1 after global alignment optimal. Advantageously, the proteins according to the invention comprise, or consist of an amino acid sequence exhibiting at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity with said SEQ ID No 1
In a particular embodiment of this aspect, a nucleic acid sequence according to the invention encodes for a polypeptide exhibiting, at least partially, functional properties of said polypeptide having an amino acid sequence SEQ ID No 1.
By “exhibiting, at least partially, functional properties of said polypeptide” it is intended a polypeptide exhibiting at least 10%, 15%, 20%, 30%, 40% or 50% of the functional properties of said polypeptide, expressed as a measure or an estimation of the percentage of the functional property of a polypeptide having an amino acid sequence SEQ ID No 1.
By “functional properties” of a polypeptide exhibiting at least 80% identity with SEQ ID No 1, it is intended:
Calpains (code CAPN for Calcium Activated Neutral Proteases) are non-lysosomal cysteine proteases exhibiting a cytosolic papain-like activity controlled by calcium. Calpains are expressed ubiquitously in mammals. Today, 14 different calpains are described, including calpain 1 and calpain 2. The calpain proteolytic system includes the calpain proteases and the small regulatory subunit CAPNS1, also known as CAPN4.
By “a nucleic acid sequence encoding for a polypeptide comprising a cleavage site by calpain” it is intended a nucleic acid sequence encoding for a polypeptide which is cleaved by the calpain proteolytic enzyme, which is naturally present in the cells, under appropriate reaction conditions. Among calpains susceptible to cleave said polypeptide, calpain 1 and/or calpain 2 are preferred.
Appropriate reaction conditions are defined as follows:
Proteins of the treated myoblasts are then extracted and analysed by SDS-PAGE.
It has been shown that the cleavage of dysferlin by calpain leads to the presence of mini-dysferlin. Therefore, the presence of a cleavage site by calpain can be demonstrated by the presence of mini-dysferlin after said cleavage reaction. More generally, the presence of a cleavage site by calpain can be demonstrated by the presence of a protein of the appropriate size after said cleavage reaction.
It is estimated that an ability to being cleaved by calpain, even at a low level, is acceptable for a polypeptide encoded by a nucleic acid sequence of the invention. In particular, an ability to being cleaved by calpain of at least 5%, 10%, 15%, 20%, 30%, 40% or 50% of the property of the wild-type polypeptide encoded by exon 40a is acceptable.
Among nucleic acid sequences encoding for a polypeptide comprising a cleavage site by calpain, a particular embodiment of the invention relates to nucleic acid sequences encoding for the cleavage site by calpain of spectrin (SEQ ID No 6).
Among nucleic acid sequences encoding for a polypeptide comprising a cleavage site by calpain, a particular embodiment of the invention relates to nucleic acid sequences encoding for:
More particularly, nucleic acid sequences encoding for a polypeptide exhibiting the amino acid sequence (SEQ ID No 4) or for an amino acid sequence exhibiting at least 80% identity with SEQ ID No 4 are preferred.
The inventors have shown that some polypeptides exhibiting mutated sequences of SEQ ID No 4 are still able to be cleaved by calpain, these polypeptides exhibit an amino acid sequence chosen among: SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18 and SEQ ID No 19.
Consequently, in a particular embodiment of the invention, a nucleic acid sequence encoding for a polypeptide comprising a cleavage site by calpain encodes for a polypeptide exhibiting an amino acid sequence chosen among: SEQ ID No 4, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18 and SEQ ID No 19.
In a preferred embodiment, a nucleic acid sequence encoding for a polypeptide comprising a cleavage site by calpain encodes for a polypeptide exhibiting an amino acid sequence SEQ ID No 4.
By “a nucleic acid sequence encoding for the dysferlin C2F, C2G and TM domains (SEQ ID No 2) it is intended a nucleic acid sequence encoding for the amino acids No 1563 to 2080 of dysferlin, which comprises domains C2F, C2G and TM of said dysferlin protein. SEQ ID No 2 includes amino acids 2068 to 2080 which are located in the extra-cellular compartment.
According to this aspect, a nucleic acid sequence encoding for at least 100 amino acids of the dysferlin N-terminal amino acids of SEQ ID No 2 or for an amino acid sequence exhibiting at least 80% identity with SEQ ID No 1, exhibits, at least partially, functional properties of SEQ ID No 2.
In a particular embodiment, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
In another particular embodiment, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
In another particular embodiment, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
In a more particular embodiment, said nucleic acid sequence encoding for an amino acid sequence comprising at least 100 amino acids of SEQ ID No 2 is a nucleic acid sequence encoding for an amino acid sequence comprising at least a fragment of at least 200 amino acids of SEQ ID No 2, at least 300, 400 or at least 400 amino acids of SEQ ID No 2.
Said dysferlin C2F, C2G and TM domains (SEQ ID No 2), an amino acid sequence exhibiting at least 80% identity with said SEQ ID No 2 or an amino acid sequence comprising at least 100, at least 200, at least 300, 400 or at least 400 amino acids of SEQ ID No 2 are able to bind to cell membranes.
To show that said protein, or protein fragment, is addressed to the membrane of the tissues in which it is expressed, it is possible to show that it belongs to the protein extract from these tissues which are associated with membranes.
As an example, the following experimental protocol is performed: muscular tissue is homogenized, using a glass-Teflon Potter homogenizer, in ice-cold 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 5 mM MgCl2, inhibitors of proteases and 1 mM EGTA. The homogenates are centrifuged at 100,000 g, at 4° C. for 1 h. The pellets solubilized in 50 mM Tris-HCl buffer containing 0.9% Lubrol-PX and 1 mM EGTA and centrifuged at 100,000 g for 30 min at 4° C. fractions are analyzed on SDS-polyacrylamide (7.5%) gels and transferred to nitrocellulose. The blots are stained with Ponceau red, and the presence of the protein of interest is revealed by using a specific antibody.
In another particular embodiment, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of, from its 5′ to 3′ extremity:
In another particular embodiment, the present invention relates to an isolated nucleic acid sequence comprising, or consisting of the nucleic acid sequence SEQ ID No 21, or a nucleic acid sequence exhibiting at least 80%, 81%, 82%, 83%, 84%, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with SEQ ID No 21.
In a second aspect, the present invention relates to a recombinant vector comprising at least an isolated nucleic acid sequence according to the invention. More particularly, the present invention relates to a recombinant viral vector comprising at least an isolated nucleic acid sequence according to the invention
The present invention relates to a recombinant viral vector comprising at least:
In vivo gene therapy is a direct method of inserting the genetic material into the targeted tissue, and transduction takes place within the transfected cells. A gene therapy vector according to the invention is desirably delivered locally, or systematically.
Among possible gene therapy vectors appropriate for the invention, viral vectors are particularly indicated. More particularly, the present invention relates to a recombinant viral vector comprising at least an isolated nucleic acid sequence according to the invention, for gene therapy.
Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single stranded DNA genome of which is about 4.7 kb in length, including 145 nucleotides inverted terminal repeat (ITR).
AAV possesses many features that make it attractive for delivering foreign DNA to cells, in particular for gene therapy. Multiple studies have demonstrated long-term recombinant AAV-mediated protein expression in muscle and that the muscle is capable of stable expression of secreted protein therapeutics.
As known by a person skilled in the art of designing and manipulating vectors for gene therapy, and particularly AAV, grows only in cells in which certain functions are provided by a co-infecting helper virus.
Recombinant AAV genomes of the invention comprises nucleic acid molecules of the invention and one or more AAV ITRs flanking a nucleic acid molecule.
Reported clinical doses for AAV-based viral vectors range from 1011 to 1014 vector genomes per patient, depending on therapeutic area.
In a particular embodiment, a recombinant vector of the invention is a viral vector. In a more particular embodiment, a recombinant vector of the invention is a recombinant AAV (rAAV) vector or a recombinant lentiviral vector.
In a more particular embodiment, the vector is of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 serotype, or a derivative of a known serotype. In certain embodiment, the vector exhibits a desired tissue specific or tropism or other desirable properties for a pharmaceutical composition or gene therapy for various indications.
In another particular embodiment, the muscle-specific control element is human skeletal actin gene element, a human myosin kinase promoter or a synthetic muscle specific promoter C5.12.
In certain embodiments, a vector of the invention comprises a polyadenylation (polyA) sequence for inserting a polyA sequence into a transcribed mRNA.
In a third aspect, the present invention relates to a composition comprising a recombinant vector according to the invention and a therapeutically acceptable carrier, diluent or excipient.
In a forth aspect, the present invention relates to a recombinant vector comprising at least an isolated nucleic acid sequence according to the invention or a composition comprising a recombinant vector according to the invention, for its use as a medicament.
More particularly, the present invention relates to a recombinant vector comprising at least an isolated nucleic acid sequence according to the invention or a composition comprising a recombinant vector according to the invention, for its use as a medicament for the treatment of a dysferlinopathy.
Vectors suitable for the invention can be produced by using any the methods known in the art. Viral particles can be produced by using any the methods known in the art such as using stable mammalian cell lines.
Another aspect of the invention provides a method of producing a viral vector of the invention, comprising culturing a cell that has been transfected with a viral vector of the invention and recovering the viral particles from the supernatant of the transfected cells.
Another aspect of the invention provides viral particles comprising any of the viral vectors of the invention.
Another aspect of the invention provides a composition comprising a vector according to the invention.
According to an embodiment, a composition according to the invention is a pharmaceutical composition further comprising a therapeutically compatible carrier, diluent or excipient.
For administration, effective amounts and therapeutically effective amounts, also referred as doses, may be initially estimated based on results from in vitro assays and/or animal model studies. A vector according to the present invention, when injected to a mouse model, such as for example a deficient-dysferlin mouse model (Dysf Y1159X/Y1159X), leads to the production of a recombinant protein; analysis of expressed protein and the quantification of dystrophic features lead to the characterization of the polypeptide encoded by said vector according to the invention.
The actual dose of vector according to the invention will vary according to the particular vector used, the mode of administration, the treatment goal, the individual and the cell types being targeted, and may be determined by methods standard in the art. The actual dose of vector according to the invention may also be determined by a physician taking into account physical and physiological factors, severity of condition and/or route of administration. Exemplary doses may range from about 1×1010 to about 1×1015 vector genomes per kilogram of body weight.
According to an embodiment, a pharmaceutical composition according to the invention is in a dosage form comprising at least 5×1011 vector genomes, more preferably from about 5×1011 to about 1015 vector genomes.
The present invention also provides a vector according to the invention, or a composition according to the invention, for its use as a medicament.
The present invention also provides a vector according to the invention, or a composition according to the invention, for its use as a medicament for treating dysferlinopathy.
Even more particularly, the present invention relates to a recombinant vector comprising at least an isolated nucleic acid sequence according to the invention or a composition comprising a recombinant vector according to the invention, for its use as a medicament for the treatment of a dysferlinopathy chosen among Miyoshi myopathy (MM), limb girdle muscular dystrophy (LGMD) and limb girdle muscular dystrophy type 2B (LGMD2B or LGMDR2).
According to an embodiment, the present invention provides a vector according to the invention, or a composition according to the invention, for its use as a medicament for treating Miyoshi myopathy (MM) or limb girdle muscular dystrophy type 2B (LGMD2B or LGMDR2).
In a particular embodiment, a vector according to the invention or a composition is in a form suitable for intramuscular injection, intravenous injection, parental delivery or systemic administration.
The present invention also provides a method for the treatment of dysferlinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to the invention or a composition according to the invention.
The following examples illustrates, without limiting it, the key role played by a polynucleotide encoding for exon 40a in cellular processes of dysferlinopathy, supporting the medical solution provided by a gene therapy vector according to the invention and its use for treating dysferlinopathy.
Any one embodiment described herein can be combined with any one or more other embodiments of the invention, unless such combination is expressly disclaimed or improper.
Characteristics, advantages and uses of the subject-matter of the present invention are more detailed hereunder, in an illustrated and non-limiting way. When present, the disclosure of the ranges expressed as “from . . . to . . . ” mean that the limits are included in said ranges.
WT (C25) and DYSF-null (AB320) human myoblasts cells lines were kindly given by Vincent Mouly from the Myology Research Center (Paris, France). Cells were grown in a humidified environment at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium, supplemented with 15% medium 199, 15% fetal bovine serum, 25 μg/mL fetuin, 5 ng/ml hEGF, 0.5 ng/mL bFGF, 5 μg/mL insulin and 0.2 μg/mL dexamethasone.
C2C12 murine myoblasts and HEK cells lines were bought at ATCC. Cells were grown in a humidified environment at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium (ThermoFisher), supplemented with 20% fetal bovine serum and 100 μg/mL antibiotic antimycotic (GE Healthcare, ref P11-002).
GFP-dysferlin1 plasmid was a generous gift from Dr. Kate Bushby, it contains the transcript of the main isoform of dysferlin (exon 1 to 55). mCherry-dysferlin1 plasmid was generated by GFP switching to mCherry (Shaner et al, Nature Biotech., 21 Nov. 2004) using the EcoRI and KpnI restriction enzymatic sites.
Coding sequence of GFP-dysferlin11 was generated by Cliniscience by inserting the alternative exon 40a between exon 40 and exon 41 into the GFP-dysferlin1 plasmid to reproduce the transcript 11 of the dysferlin. mCherry-dysferlin11 plasmid was generated by GFP switching to mCherry using the EcoRI and KpnI restriction enzymatic sites. Dysferlin11 constructs to identify the cleavage site by calpain in DYSF exon 40a were created by PCR fusion. Table 1 summarizes the primers used to create dysferlin11 constructs by PCR fusion.
HEK cells were plated at 70% confluence in 6-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's directions. After 24 h, cells were injured with 30 μM ionomycin and cell scrappers, then they were pelleted at 300 g for 5 min and the cell pellet was solubilized in RIPA buffer (Life technologies) and protease inhibitor cocktail (Life technologies). Samples were separated by SDS-PAGE on 3-8% NuPAGE Tris-Acetate gels (Life technologies) using Chameleon Duo as a size marker and transferred onto nitrocellulose membranes (at 100V for 3 h at 4° C.). Membranes were blocked using fluorescent WB blocking buffer (tebu-bio) in TBS 1× for 1 hour at room temperature. Primary antibodies (Hamlet, 1:150, abcam 75571) were then diluted in blocking buffer and incubated overnight at 4° C. After washing in TBS-T, membranes were then incubated with secondary antibodies (IRDye 800CW Donkey anti-mouse, Li-Cor, ref 926-32212), which were diluted 1:10000 in blocking buffer for 45 min at room temperature. The membranes were washed in TBS-T and developed using NIR Fluorescence LI-COR. Actin (1:5000, Merck MA1501r) was detected using a dilution of 1:10000 of secondary antibodies (IRDye 680RD Donkey anti-mouse, Li-Cor, ref 925-68072).
C2C12 myoblasts were grown on Lab-TEK II™ (Fisher Scientific) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's directions. After 24 h, cells were injured with glass beads, fixed with 4% paraformaldehyde for 10 minutes and then washed in PBS. Cells were then incubated for 10 minutes with a permeabilization solution (200 μL of PBS 1×+0.5% triton X-100+protease inhibitors cocktail) (Roche). From there, cells were exposed to a blocking buffer (PBS+1% BSA+protease inhibitors cocktail) for 30 minutes. The primary antibody (Hamlet, 1:40, abcam 75571) was applied in blocking buffer for 3 hours at room temperature, followed by a wash in PBS and 1 hour of contact with the secondary antibody (Dylight 550 donkey anti mouse IgG, 1:100, abcam 96876) in blocking buffer. After washing in PBS, Lab-TEK II™ were mounted with Vectashield-Dapi 25 ng/ml and kept at 4° C. until pictures were taken. Observation was performed using a Zeiss Axio Imager Z2 microscope (40× objective), and images were processed with ZEN software and/or ImageJ software.
WT (C25) and DYSF-null (AB320) human myoblasts were plated at 50% confluence on 6-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's directions. After 24 h, cells were visualized in the presence of Ca2+ (1 mM) and membrane-impermeable dye FM 1-43 (2.5 μM, Molecular Probes) with a confocal microscope (LSM 800, 63× objective, ZEISS). Membrane damage was applied to myoblast using a UV laser to irradiate a 0.33 μm2 area at maximum power for 15 seconds at t=10 sec. Images were captured every second for 5 min, and the mean fluorescence intensity of FM1-43 was measured on a 13.5 μm2 area around the damage with the Zeiss LSM 800 imaging software.
WT (C25) and DYSF-null (AB320) human myoblasts were plated at 70% confluence on 12-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's directions. After 24 h, myoblasts were washed with distilled water and hypo-osmotic shock was performed by incubating cells with distilled water. Cell fluorescence was followed with Fast calcium imaging observer (Axio observer. Z1/7, 10× objective, ZEISS). Images were captured every minute for 30 minutes.
WT (C25) and DYSF-null (AB320) human myoblasts were plated at 70% confluence on Lab-TEK II™ (Fisher Scientific) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer's directions. After 24 h, myoblasts were incubated in Dulbecco's modified Eagle's medium, supplemented with 1% L-glutamine and 0.5% bovine serum albumin at 37° C. and 5% CO2 for 30 minutes. Then, cells were incubated in the same medium with 25 μg/mL transferrin from human serum (coupled with Alexa Fluor 488, Thermo Scientific) at 37° C. and 5% CO2 for 30 minutes. For transferrin recycling assays, cells were washed in cold PBS and incubated in Dulbecco's modified Eagle's medium, supplemented with 15% medium 199, 15% fetal bovine serum, 25 μg/mL fetuin, 5 ng/ml hEGF, 0.5 ng/ml bFGF, 5 μg/mL insulin and 0.2 μg/mL dexamethasone. Then, myoblasts were washed in PBS and incubated in stripping buffer (NaCl, acid acetic) during a few seconds. After that, cells were washed in PBS, fixed with 4% paraformaldehyde for 10 minutes and washed again in PBS. Lab-TEK II™ were mounted with Vectashield-Dapi 25 ng/ml and kept at 4° C. until pictures were taken. Observation was performed using a Zeiss Axio Imager Z2 microscope (20× objective), and images were processed with ZEN software and/or ImageJ software.
Individual means were compared using the non-parametric Mann-Whitney test. The power of the tests was strictly superior at 92%. Differences were considered to be statistically significant if p<0.05 (*) or very significant if p<0.01 (**).
Dysferlinopathy encompasses muscular dystrophies caused by mutations in the DYSF gene. Dysferlin gene encodes a sarcolemmal protein required for repairing muscle cell damage. It consists of calcium-dependent lipid binding domains and a transmembrane domain. Dysferlin-deficient muscle fibers have a defect in membrane repair.
Several publications demonstrated that membrane injury causes calcium influx at injury sites which induces local activation of calpains. This activation led to dysferlin cleavage by calpains. In 2014, it has been shown that the cleavage site by calpain resides in the DYSF exon 40a. To identify the exact location of cleavage site by calpain in dysferlin exon 40a, four plasmids containing the dysferlin transcript 11 with deletions of fragments in exon 40a were constructed: Dysf11deIN, Dysf11delC, Dysf11 delN1, Dysf11deIN2 (
When exon 40a is deleted in two parts (one on the N-terminal DeIN side and one on the C-terminal DelC side), deletion of the N-terminal part had a hardly effect on the intensity of the band corresponding to the mini-dysferlin. Thus, the N-terminal part would be more important than the C-terminal part for the recognition and cleavage of this region by calpains.
Mutations and small deletions were performed in DYSF exon 40a. Surprisingly, none of the mutations tested abolishes the cleavage of this domain by calpains, as represented in Table 2.
These results suggest that calpains recognize and cleave a structural domain instead of a precise amino acids sequence. For instance, injured HEK cells transfected with dysferlin transcript 11 delN1 (deletion of exon 40a first seven aa) and transcript 11 delN2 (deletion of exon 40a second quarter) present a weaker mini-dysferlin band compared to injured HEK cells transfected with dysferlin transcript 11 (
In 2013, Lek et al (“Calpains, cleaved Mini-DysferlinC72 and L-type channels underpin calcium-dependent muscle membrane repair”, J. Neurosc., Mar. 20, 2013, 33(12):5085-94) demonstrated that during membrane repair process, dysferlin cleavage by calpains within exon 40a releasing a C-terminal fragment named mini-dysferlin which is enriched at membrane injury sites. To explore the fate of this mini-dysferlin, murine myoblasts were transfected with dysferlin transcript 1 (major transcript) or dysferlin transcript 11 (transcript containing exon 40a) to monitor the dysferlin localization after membrane injury (
To demonstrate the importance of the dysferlin transcript 11, a membrane repair assay was performed on wild-type human myoblasts (WT), dysferlin-null human myoblasts (DYSF-null) and transfected dysferlin-null human myoblasts (DYSF-null+mCherry-dysferlin1, DYSF-null+mCherry-dysferlin11 and DYSF-null+mCherry-dysferlin1+mCherry-dysferlin11) in presence of calcium and membrane-impermeable dye FM 1-43 (
2.4. Dysferlin Transcript 11 Participates in the Protection of Membrane Myoblasts from Mechanical Stress Induced by Osmotic Shock
The role of dysferlin transcript 11 in the protection of myoblast membrane was explored by an osmotic shock assay on wild-type human myoblasts (WT), dysferlin-null human myoblasts (DYSF-null) and transfected dysferlin-null human myoblasts (DYSF-null+GFP-dysferlin1, DYSF-null+GFP-dysferlin11 and DYSF-null+GFP-dysferlin1+GFP-dysferlin11) (
Several publications have involved dysferlin in protein vesicle trafficking. To explore the role of dysferlin transcript 11 in protein vesicle trafficking, a transferrin assay was performed on wild-type human myoblasts (WT), dysferlin-null human myoblasts (DYSF-null) and transfected dysferlin-null human myoblasts (DYSF-null+mCherry-dysferlin1, DYSF-null+mCherry-dysferlin11 and DYSF-null+mCherry-dysferlin1+mCherry-dysferlin11) (
This study demonstrates the importance of the dysferlin11 transcript which contains the alternative exon 40a. Indeed, membrane injuries of muscle cells induce the establishment of a repair mechanism that causes dysferlin cleavage by calpains within exon 40a, releasing a C-terminal fragment named mini-dysferlin.
Cell scrape injury and western blot analysis performed in this study show that cleavage in DYSF exon 40a is carried out by calpains 1 and/or 2. Several studies expose the predictions of calpains cleavage, but there seems to be no explicit rule for calpain specificity. Actually, amino acid sequences around cleavage sites by calpain are very diverse. It is therefore difficult to precisely determine the location of cleavage sites by calpain. Our results demonstrate that partial deletion of exon 40a decrease calpain cleavage (delC, delN1 and delN2) and deletion of exon 40a first half prevent calpain cleavage (delN). Therefore, the entire sequence of exon 40a is important for the cleavage of dysferlin by calpains but N-terminal part of this alternative exon is more important than others.
Results show that substitutions and small deletions in DYSF exon 40a do not prevent calpain cleavage. Thus, it has been observed that amino acid substitution or deletion of one to seven amino acids do not prevent dysferlin cleavage by calpains. Only the deletion of the twelve first amino acids of DYSF exon 40a avoids cleavage of dysferlin. These observations confirm that calpains recognize a large amino acid sequence to cleave dysferlin.
On the other hand, myoferlin, which is a member of ferlin protein family, is also cleaved by calpains within an alternative exon, releasing a C-terminal fragment that has 62.82% amino acid identity with the mini-dysferlin. There seems to be an evolutionary preservation of enzymatic cleavage of both dysferlin and myoferlin. Indeed, DYSF alternative exon 40a is conserved in the majority of mammals.
Given the importance of the region coded by DYSF exon 40a (site of dysferlin cleavage by calpains during repair of the muscle cell membrane), dysferlin transcript 11 must have a particular importance in the functions of the muscle cell. Actually, our results indicate that this transcript is essential to the reparation of the muscle cell membrane. Main transcript of dysferlin (transcript 1) seems to be little involved in the repair mechanism of the muscle cell membrane when the latter suffer a laser-injury. In that case, only dysferlin transcript 11 which contains exon 40a makes it possible to repair the muscle cell membrane injured with a laser. These results are in line with the work of Lek and her team in 2013 that showed dysferlin cleavage at exon 40a by calpains and accumulation of mini-dysferlin at lesion site during repair of the muscle cell membrane.
Otherwise, dysferlin is involved in other muscle cell functions. Our results confirm this previous work: dysferlin-null myoblasts are more likely to die from hypo-osmotic shock unlike wild-type myoblasts. Our results also show that restoration of dysferlin transcript 1 and/or dysferlin transcript 11 seem to increase the cells resistance to mechanical stress induced by osmotic shock. This observation indicates that alternative exon 40a is not essential for muscle cell membrane protection, but dysferlin transcript 11 is able to accomplish it.
Moreover, dysferlin is involved in protein vesicle trafficking. It is known that dysferlin-null myoblasts accumulate transferrin and transferrin endocytic recycling is delayed in these cells. The present data show equally an abnormal vesicular trafficking in dysferlin-null myoblasts compared to wild-type myoblasts and dysferlin-null myoblasts with a dysferlin restoration. These data demonstrate that dysferlin-null myoblasts have a lower transferrin accumulation than wild-type myoblasts, and that they do not show a significant difference between endocytosis function (pulse) and recycling function (pulse-chase). When dysferlin transcript 1 and/or dysferlin transcript 11 is restored in dysferlin-null myoblasts, we show again a significant difference between endocytosis and recycling functions which demonstrates a restoration of vesicular trafficking. These two dysferlin transcripts seem to participate in transferrin trafficking in muscle cell. This observation indicates, as before, that alternative exon 40a is not essential for protein vesicle trafficking, but dysferlin transcript 11 is able to accomplish it.
All of these results indicate that dysferlin transcript 1 but also dysferlin transcript 11 have an important place in the functions of the muscle cell. In terms of therapy, these findings suggest that dysferlin transcript containing exon 40a should be restored in patients with dysferlinopathy. Currently, all therapeutic studies conducted so far restore only the main transcript of dysferlin (transcript 1). These studies show promising results: high levels of dysferlin expression, improvement of the histological aspect of the muscle, reduction of necrotic fibers and global improvement in locomotor activity.
Regarding the muscle membrane repair function, the improvements obtained do not restore the function to the same level as seen in the wild-type individuals. It would be interesting to restore dysferlin transcript 11 in models of dysferlinopathies to obtain better results in the repair function of the muscle cell membrane.
Among these SNPs, only two missense polymorphisms were predicted pathogenic by UMD-Predictor: c.4704C>T and c.4707G>T. Therefore, we carried out constructions in which we induced a substitution of the amino acids concerned by these two SNPs predicted pathogenic. Cell scrape injury and western blot analysis performed with these two constructions show that amino acid substitutions at these positions do not avoid the cleavage of dysferlin by calpains during the repair mechanism of muscle cell membrane. Moreover, our results indicate that substitutions and small deletions in DYSF exon 40a do not prevent calpain cleavage. These observations indicate that variants in exon 40a do not prevent the formation of mini-dysferlin, which has a major role in membrane repair mechanism. Knowing this, we can assume that variants in the alternative exon 40a do not lead to membrane repair defect. Nevertheless, as transcript containing exon 40a are strongly expressed in liver, lung, placenta and pancreas (between 40 and 60%), variants present in this exon could lead to non-muscular deleterious phenotype.
These data demonstrate the importance of the dysferlin transcript 11, which contains the alternative exon 40a, in muscle cell functions and more particularly in the reparation mechanism of the muscle cell membrane. During this mechanism, calpains 1 and/or 2 cleave dysferlin in the first part of DYSF exon 40a, releasing a C-terminal fragment named mini-dysferlin. Our results show that dysferlin transcript 11 is essential in muscle cell membrane repair, but it also participates in muscle cell membrane protection and protein vesicle trafficking. All these results indicate that dysferlin transcript 11 have an important place in the functions of the muscle cell. In terms of therapy, these findings suggest that dysferlin transcript containing exon 40a should be restore in patients with dysferlinopathy.
The inventors designed a midi-dysferlin protein, comprising at least, from its Nt extremity to its Ct extremity:
Midi-dysferlin (SEQ ID No 21) was transfected into dysferlin-null myoblasts. Western blot performed with proteins from wild-type myoblasts (WT, C25), dysferlin-null myoblasts (DYSF-null, AB320) and transfected dysferlin-null myoblasts (DYSF-null+Midi-dysferlin), wherein Hamlet antibodies were used to detect dysferlin and midi-dysferlin, showed the expression of midi-dysferlin in transfected dysferlin-null myoblasts (
Experimental data with midi-dysferlin, which were performed in conditions similar to those described in example 1 with dysferlin, show that:
Experimental data show that a “midi-dysferlin” protein, which comprises exon 40a, can be cleaved by calpain and is able to repair muscle membrane lesion, to restore myoblasts membrane protection from mechanical stress induced by osmotic shock and to participate in protein vesicle trafficking. Said “midi-dysferlin” truncated protein is necessary and sufficient to trigger membrane repair pathway after injury.
AAV9 vector containing the nucleic acid sequence according to the invention were injected intramuscularly in the left quadriceps of mice at 2 months of age. For this purpose, the animals were positioned in lateral decubitus position to clear access to the posterior loge of the quadriceps. Injections were performed with 25G*5/8 needles: injection of 20 μL of 1×PBS for control mice and injection of 20 μL of vectors diluted in 1×PBS to reach a concentration of 5.1011 vg/mouse.
At 3 months mice injected and control were tested for their strength. The muscle strength of hind limbs of the mice was measured with the BIO-GS3 grip test (Bioseb). Mice were placed with hind limbs on the metal grid of the apparatus and slowly pulled backwards using their tails. The maximum tension was recorded, and the experiment was repeated 10 times for each mouse.
Following this test, mice were sacrificed, and muscle biopsy analyzed. Muscle sections were taken in a cryostat (ref CM1520, Leica, 7 μm) and fixed on superFrost Plus slides (ref 631-0108, VWR) through incubation with 4% paraformaldehyde for 10 minutes.
Hematoxylin and eosin (H&E) staining was performed by incubating for 2 minutes in hematoxylin (ref HHS32, Sigma-Aldrich), followed by washes with distilled water, and incubating for 10 minutes in eosin (ref HTAA0232, Sigma-Aldrich). The muscle sections were then incubated in 70% ethanol (once, 1 minute), 95% ethanol (once, 1 minute), 100% ethanol (twice, 1 minute), and in xylene (twice, 1 minute). Slides were stored at 4° C. until analysis under a microscope (Olympus BX51, 10× objective, Microvision). The percentage of centrally nucleated nuclei are monitored for each condition.
For detecting the presence of the midi-dysferlin we rely on immunofluorescence. The same muscle sections as above were permeabilized with a solution of 1×PBS and 0.5% Triton X-100 for 10 minutes. Blocking was performed for 1 hour at room temperature with the following solution: PBS 1×, BSA 5%, Donkey serum 5% and Triton X-100 0.1%. Then the dysferlin specific antibody was incubated according for one hour at 1/100 in the same buffer. Two washes with PBS 1× were performed, then the fluorescent antibody was incubated according to the manufacturer's recommendations.
Finally, two washes with 1×PBS were performed and the slides were mounted with Vectashield-Dapi (25 ng/mL, ref P36935, Life Technologies). The slides were stored at 4° C. until microscopic analysis (Zeiss Axio Imager Z2 microscope, 20× objective, ZEISS).
Experimental data clearly show that the administration in the Dysf mouse model of the midi-dysferlin construction according to the invention allows for: A significant increase in the strength developed by the AAV injected mice is detected compared to PBS-injected control mice (pvalue=0.0059) (
Immunofluorescence detection of dysferlin confirmed the presence of the midi-dysferlin protein in the muscle fibers of the injected mice. The amount of dysferlin detected is presented in
Histological analysis confirmed that midi-dysferlin presence in the injected muscles lowered the percentage of centronucleated cells in Dysf-treated fibers, compared to the PBS-injected control (
| Number | Date | Country | Kind |
|---|---|---|---|
| 21305569.2 | May 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/061409 | 4/28/2022 | WO |
