Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2878—Muscular dystrophy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2878—Muscular dystrophy
  • G01N2800/2885—Duchenne dystrophy

Definitions

• the invention relates to methods for identifying individuals with, or who are likely to develop, muscular dystrophies, such as Duchenne Muscular Dystrophy (DMD) and limb girdle muscular dystrophy (LGMD), and to assay kits.
• muscular dystrophies such as Duchenne Muscular Dystrophy (DMD) and limb girdle muscular dystrophy (LGMD)
• DMD Duchenne Muscular Dystrophy
• LGMD limb girdle muscular dystrophy
• DMD Duchenne Muscular Dystrophy
• DGC dystrophin- glycoprotein complex
• Classic DMD which affects the entire skeletal musculature, arises from loss of an essential intracellular component of the DGC, dystrophin (Hoffman et al., 1987). The condition is characterised by early ( ⁇ 3 years age) and progressive disruption of skeletal muscle function leading to extensive muscle damage and causing early death (usually 20's). Death commonly results from complications arising from failure of respiratory muscles, however, 95% of patients with DMD develop a cardiomyopathy which, in 10-30% of cases is the primary cause of death (Cox and Kunkel, 1997).
• the mdx mouse a dystrophin deficient model of DMD, exhibits many of the post-natal features of DMD including cardiomyopathy; skeletal muscular dystrophy associated with bouts of fibre regeneration; fibrosis; hyperproliferation and apoptosis of skeletal muscle myoblasts (Harper et al., 2002; Smith et al., 1995).
• the phenotype is less severe than human DMD although mdx life span is curtailed with respect to WT (Chamberlain et al., 2007).
• Embryonic skeletal muscles originate from the dermamyotomal region of the somites (Cossu et al., 1996; Hollway and Currie, 2003). In the chick embryo all regions of the dermamyotome appear to contribute myogenic precursors to this process in a time dependent manner (Gros et al., 2004). Dystrophin is first expressed in embryonic somite from E9.5, and could thus have a function in early myogenesis (Huang et al., 2000; Ilsley et al., 2002).
• the myotome Under control of inducing factors secreted by surrounding tissues, the myotome produces cells which become the (myf-5 + ) stem cell populations which, respectively, generate (from E10.5) the epaxial (deep muscles of the back) and (from El 1.5) the hypaxial (limb, abdominal, diaphragm) muscles (reviewed in (Hollway and Currie, 2003)).
• Secondary myotubes are a morphologically distinct subset of myotubes, longer and thinner than primary myotubes which form in clusters around a single larger primary myotube (Cho et al., 1994).
• Myotube differentiation is dependent on the presence of functional muscle stem cell populations.
• Myf-5 is expressed in the somite, the earliest of the myogenic regulatory factors (MRF) at E8.5, it is the only MRF found prior to muscle differentiation and persists in all embryonic muscle groups throughout both primary and secondary myogenesis (Cossu et al., 1996). It is therefore a good marker for the embryonic myoblast population. Lineage analysis suggests that a majority of satellite cells derive from the somite (Armand et al., 1983).
• Pax-7 is a transcription factor, a repressor of myogenesis, and plays important roles in the maintenance and specification of the adult skeletal muscle stem cell population (the 'satellite' cell) and is expressed in undifferentiated muscle stem cells emerging from the somite from El 1.5 (Merrick et al., 2007; Relaix et al., 2006; Seale et al., 2000).
• the specificity of function of individual skeletal muscle groups depends on the appropriate localisation of fast myosin isoforms to different myotubes; a process initiated during the late stages of gestation (Merrick et al., 2007). In mammalian embryos developmental (embryonic and neonatal) myosin isoforms are co-expressed in newly formed secondary myotubes with adult fast myosin isoforms (Cho et al., 1994).
• myosin heavy chain (MyHC) isoforms in a muscle specific pattern, a process not completed until several weeks after birth (Agbulut et al., 2003; Merrick et al., 2007).
• MyHC myosin heavy chain
• the embryonic heart expresses two myosin isoforms (cardiac myosin ⁇ and slow/cardiac myosin ⁇ ) in a temporally regulated manner. Mutation of ⁇ -cardiac myosin is implicated in cardiomyopathy (Geisterfer-Lowrance et al., 1990).
• Dystrophin associates with the beta subunit of dystroglycan on the intracellular side of the sarcolemma and is an essential component of the DGC, a multifunctional protein complex, which links the extracellular matrix to the actin cytoskeleton in skeletal muscle myotubes (Ervasti and Campbell, 1993). In post-natal muscle, dystrophin deficiency results in complete breakdown of the DGC and the secondary down-regulation of a majority of the DGC proteins (Ohlendieck et al., 1993).
• Caveolin-3 localises both to skeletal muscle caveolae and to the DGC where, by means of a specific WW domain, it binds the same PPXY motif in the ⁇ - dystroglycan c-terminus that recognises and binds dystrophin, thus blocking the interaction between dystrophin and ⁇ -dystroglycan (Jung et al., 1995; Sotgia et al., 2000).
• the inventors have examined the impact of loss of caveolin-3 and dystrophin on skeletal muscle development in order to establish a functional role for these two proteins during myogenesis and have identified an assay for the early diagnosis of muscular dystrophy.
• the inventors have found that examination of embryonic myogenesis of two distinct but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3 ';' ) establishes for the first time that key elements of the pathology of DMD and LGMD type 1C originate in disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx than cav-3 " ' ' consistent with the milder phenotype of LGMD-Ic and the earlier (E9.5) expression of dystrophin. Myogenesis is severely disrupted in mdx embryos with developmental delay, myotube morphology and displacement defects, and aberrant stem cell behaviour. Caveolin-3 protein is elevated in mdx embryos.
• cav-3 ⁇ ' from El 5.5
• mdx from El 1.5
• cav-3 'A there is a more restricted phenotype comprising hypaxial muscle defects, excess, malformed hypertrophic myotubes, 2- fold increase in myonuclei and reduced fast myosin heaving chain (FMyHC) content.
• FMyHC fast myosin heaving chain
• the test identified by the inventors is not based on polymorphisms in the dystrophin gene and potentially is applicable to all forms of muscular dystrophy. Moreover, the test is applicable to babies both before and after birth. More traditional forms of diagnosis are not carried out until the child reaches 3-5 years of age.
• the assay allows the early diagnosis of the disease which allows early treatment of that disease, for example, by replacement of pax-7 positive stem cells.
• Pax-7/Myf5 cells stem cells have been suggested to be used in therapeutic uses (WO 2007/059612). Such cells are suggested to be used as myogenic progenitor cells for the treatment of muscle diseases including muscular dystrophies. The cells are used as a source of new muscle cells for the treatment of diseases. However, there is no suggestion of using Pax-7 as a marker for a muscular dystrophy. The protein is simply used as one of two markers to identify a sub-population of stem cells to be used.
• the invention provides a method for identifying an individual exhibiting symptoms of, monitoring the treatment of or progression of, or having a propensity to develop symptoms of, a muscular dystrophy comprising determining the level of expression in a tissue sample from the patient, of one or more proteins selected from pax-7, caveolin-3 and/or fast-myosin.
• the method may be used to identify individuals exhibiting symptoms of or having a propensity to develop symptoms of the disease.
• the level in the sample may be compared to a level in a sample from the individual taken previously, such as days, weeks or months previously.
• the level of expression of the or each protein may be compared to a pre-determined level associated with, for example, an individual not having symptoms of, or having a propensity to develop, a muscular dystrophy, and/or compared to a pre-determined level that has previously been identified as being associated with individual exhibiting or having a propensity to develop, the muscular dystrophy.
• Pax-7 and caveolin-3 have been identified as having particular relevance in the early determination of whether, for example, a baby or pre-term baby is likely to develop a muscular dystrophy.
• Pax-7 may be measured.
• Caveolin-3 may be measured.
• Pax-7 and/or caveolin-3 may be measured they may be measured with or without measuring fast-myosin.
• the tissue sample may be obtained from an individual before or after the birth of the individual.
• Myogenesis typically occurs from 8-9 weeks of a pregnancy.
• samples could be taken from that stage onwards.
• the individual may be, for example, a child for example, less than 16, less than 12, less than 10, less than 6, less than 3, less than 2 or less than 1 year old, and may be still within the womb.
• the individual patient may be between birth and 3 or 6 months old.
• MD Muscular dystrophy
• DMD Duchenne
• LGMD limb girdle
• Emery-Dreyfuss oculopharyngeal
• distal myotonic, congenital, Becker, and/or facioscapulotumeral.
• the disease may be DMD and/or LGMD.
• a decrease in expression of pax-7 or altered expression of caveolin-3, compared to a normal individual is indicative of the individual exhibiting symptoms of, or having a propensity to develop symptoms of, a muscular dystrophy.
• Such animals Whilst the individual may be a human individual, there are a number of different animals which also develop muscular dystrophy-like symptoms. Such animals are typically mammals, and may include, mice, rats, sheep, dogs, horses, cats and non-human primates.
• Fast-myosin may be measured.
• the inventors have determined that fast-myosin has different expression levels in different diseases. For example, it is increased in the mouse model for Duchenne muscular dystrophy, but decreased in limb girdle muscular dystrophy. Hence, it is believed that assaying for the levels of expression of that protein gives an indication of the likely outcome of the disease.
• fast-myosin is assayed in combination with an assay for one or both of pax-7 and/or caveolin-3.
• Fast-myosin may also be used alone to diagnose, for example, particular types of muscular dystrophy such as DMD and LGMD.
• the assay may additionally comprise determining the level of expression of insulin-like growth factor-2 (Igf-2) in the tissue sample.
• Igf-2 is over expressed in muscular dystrophies such as Duchenne muscular dystrophy, and reduced in caveolin-3 deficient LGMD. Again this gives an indication of the prognosis of the disease.
• CDKNlc/P57kip2 the following gene products downstream of Igf-2 may be assayed: CDKNlc/P57kip2 and/or pAkt.
• Igf-2, CDKNl c/P57kip2 and/or pAkt may also be assayed separately to the other proteins. That is, for example, Igf-2 may be assayed separately without measuring pax-7 and caveolin- 3. It may be used with CDKNlc/P57kip2 and/or pAkt.
• Igf-2 insulin-like growth factor-2
• the tissue sample is typically a sample of muscle tissue, for example, in the form of a biopsy sample.
• fast-myosin and caveolin-3 may also be found within the blood of individuals with muscular dystrophy, due to degeneration of the muscle fibres within the individual.
• the protein expression may be determined by either measuring the level of protein expression directly, or indirectly by determining the level of mRNA in the sample.
• the expression level of the protein may be determined by immunoassay.
• Antibodies specific for the proteins described above are generally known in the art. Such antibodies may be labelled directly or indirectly by methods generally known in the art.
• the sample of tissue may be immunostained with a suitably labelled antibody.
• the sample may be fixed prior to staining.
• paraformaldehyde is particularly effective when used with antibodies specific for the proteins, and in particular for antibodies specific for pax-7.
• the fixative used is paraformaldehyde.
• the binding of antibodies to the proteins may be determined by methods generally known in the art. For example, avidin-biotin methods are generally known in the art.
• Such assays typically use horse-radish peroxidase in combination with a substrate such as 3, 3', 5, 5' tetramethyl-benzidine to form a coloured product, which may then be visualised under, for example, a microscope.
• a substrate such as 3, 3', 5, 5' tetramethyl-benzidine
• TSA tyramide signal amplification
• Alkaline phosphatase may also be used. This utilises a pre-formed cyclic enzyme anti- enzyme immunocomplex composed of three enzyme molecules (alkaline phosphatase) and two antibody molecules.
• the technique can be visualised by typically using a blue dye (Fast Blue BN) or a red dye (Fast Red TR).
• the immunoassays may be visualised using a directly labelled anti-protein antibody. Alternatively, they may be visualised indirectly by using an anti-immunoglobulin antibody to bind the anti protein antibody. That secondary antibody may itself be labelled.
• the or each antibody may be labelled with a fluorophore.
• fiuorphores are generally known in the art. They include, for example, fluoresceine, fiuoresceine isothiocyanate (FITC) and rhodamine. Such fiuorphores fluoresce at different colours.
• FITC fiuoresceine isothiocyanate
• rhodamine rhodamine.
• Such fiuorphores fluoresce at different colours.
• the antibodies used in the assays and indeed kits, defined below, have different fiuorophores and/or different methods of being visualised, for example with horse-radish peroxidase, to allow different proteins to be assayed from the same tissue sample.
• the proteins may also be visualised by adding the labelled antibody to the tissue sample, partially purified sample or purified proteins obtained from the sample, to identify the total antibody bound to the sample.
• the proteins in the sample may be purified and visualised by immunoblotting.
• the level of expression of the protein may be determined by identifying the level of mRNA expressed in this tissue sample.
• the level of mRNA expression may be determined by quantitative polymerase chain reaction (QPCR) or realtime PCR (RT-PCR). This technique utilises a pair of primers specific for the mRNA encoding the protein. Such techniques are generally well-known in the art.
• the invention also provides a kit for use in a method according to the invention, comprising two or more antibodies or a pair of PCR primers specific for two or more of pax-7, caveolin-3 and/or fast-myosin, or mRNA encoding such proteins.
• the protein may be pax-7 protein or mRNA encoding pax-7.
• the protein may be caveolin-3 or mRNA encoding caveolin-3.
• the protein may additionally be fast-myosin or mRNA encoding fast-myosin.
• the antibodies may be directly labelled with a suitable label, such as one of those described above.
• suitable label include fluorescent labels and enzyme-based labels such as alkaline phosphatase or horse radish peroxidase.
• the kit may comprise an antibody for one protein and a pair of primers for a second protein.
• the kit may comprise PCR primers for two or more other proteins.
• the kit may comprise two or more antibodies for different proteins. In the latter situation, the antibodies may be labelled with different labels to allow substantially simultaneous measurement of the amount of each protein.
• the kit may additionally comprise paraformaldehyde.
• Paraformaldehyde has been found to be particularly effective in allowing the fixation of muscle samples. This may be provided in combination with an antibody specific for pax-7 protein.
• the kit may additionally comprise an antibody or PCR primer pair specific for Igf-2. Where antibodies are provided in the kit, then the kit may comprise one or more reagents for tyramide signal amplification. This may include, for example, biotinylated tyramide.
• the kit according to the invention may comprise one or more fixatives for fixing the sample.
• the kit may comprise instructions for use with the kit.
• the instructions may comprise details of typical levels of protein expression associated with, for example, an individual exhibiting symptoms or, or having a propensity to develop, muscular dystrophy.
• the instructions may comprise details of expression levels in normal individuals. This allows a comparison of the data obtained from the sample from the individual to be compared to pre-determined values in order to allow a practitioner to assess the validity of the data obtained.
• One or more controls with known concentrations of the proteins may be provided.
• the kit may additionally comprise one or more labels or other reagents to allow quantitative PCR to be carried out. This may include, for example, SYBRTM green.
• the kit may also comprise one or more control samples of protein at a pre-determined concentration for use a standards within the assay.
• caveolin-3 may be used to alleviate symptoms of muscular dystrophy.
• the invention provides a method of treating a muscular dystrophy comprising administering caveolin-3 to a patient or increasing caveolin-3 expression in a patient.
• Methods of increasing the expression of protein, or presenting protein in the treatment of muscular dystrophy have previously been demonstrated for utrophin. It is expected that caveolin-3 may be used in a similar manner.
• Utrophin has been demonstrated to be useful for treatment of muscular dystrophies.
• US 2009/054327, US 2008/160108 and WO 97122696 disclose ways in which the protein has been used in such treatment.
• Pharmaceutical formulations comprising caveolin-3 in combination with a pharmaceutically acceptable carrier are also provided.
• the invention also provides caveolin-3 for use to treat muscular dystrophy.
• (F-H) Variable myotube diameters in (G) mdx and (H) cav-3 ' ' ' muscle fibres compared to (F) WT.
• (I) Displaced myotubes (combined tangential and misaligned myotubes) as a proportion of total counted. 0: no displaced fibres
• (J) Displaced myotube scoring strategy: tangential (T) > 25°, misaligned (M) ⁇ 25° from the median.
• El 4.5 atrial trabeculae (tb) are (I) short and stubby in cav-3 ' ' ' and (J) hookshaped in mdx.
• K- P E17.5 atrium;
• L-P attenuated N2.261 labelling and distension of cell layers in (L, O) cav-3 ' ' ' trabeculae (tb) and
• M, P mdx atrial wall, compared to (K, N) WT. Size bars 20 micrometers.
• Q low magnification image of MF20 labelled mdx heart to illustrate the orientation and matching of hearts sectioned sagitally through the most central portion of the heart.
• E- G transverse sections of E15.5 WT (E), mdx (F) and (G) cav-3 '1' lower proximal limb myotubes showing peripheral myonuclei (arrow) are associated with WT but not dystrophic embryo myotubes at this stage. The hypotrophy of mdx and hypertrophy of cav-3 -/- myotubes can also be seen; green line indicates WT myotube diameter.
• H WT,
• I mdx and
• J cav-3 ' ' ' lower magnification image of lower proximal limb region showing the reduced fibre density of mdx and increased density of cav-3 ' ' ' muscle fibres compared to WT in matched embryo sections.
• A-F, MN, QR E15.5,
• G-L E17.5.
• A-E; G-K low magnification;
• B-F; H-L high magnification; size bars 10 micrometers.
• Q-R whole region view of WT (Q) and mdxcav-3 +/ ⁇ lower proximal limb.
• Neomycin immunostaining confirms the presence of the cav-3KO transgene in cav-3 '1' and mdxcav-3 +l' .
• the reduced neomycin staining seen in mdxcav-3 +/ ⁇ embryos compared to cav-3 'A reflects their heterozygosity for the cav-3KO transgene.
• A-F FMyHC immunostaining in El 3.5, (A-C) diaphragm; (D-F) intercostals.
• A,D WT, (B,E) mdx and (C,F) cav-3 '1' showing (B,E) reduced FMyHC in mdx and (C,F) increased FMyHC in cav-3 '1' respiratory muscles.
• G-L FMyHC in El 5.5 respiratory muscles
• G-I diaphragm
• J-L intercostals
• G,J WT
• H,K mdx, (I,L) cav-3 '1' .
• K-P Higher magnification images of MF20 labelled intercostal muscle sections (matched 4 th intercostal for each embryo) showing the difference in myotube density between (K) WT, (L) mdx, (M) cav-3 "A and (N-P) three different mdxcav-3 +/ ⁇ E17.5 embryos.
• R fibre density assessed over a fixed grid area and (S) percent reduction in fibre density, in El 7.5 WT, mdx and mdxcav +/ ⁇ (het) embryonic intercostal muscle.
• T WT and (U) mdxcav+/- E17.5 intercostal labelled with My32 antibody showing that almost all fibres are FMyHC positive. Note that fibres are downregulating FMyHC in many WT but not mdxcav-3 +/ ⁇ intercostal fibres.
• Pax-7 human muscle biopsy
• FIG. 9 RT-PCR analysis (A) Densitometric analysis of pax-7 mRNA expression in six WT and six mdx mouse myoblast isolates establishes statistically significant suppression of Pax-7 expression in the dystrophic myoblasts. Data shown are mean and standard deviation of desitometry results individual myoblast isolates.
• A-D Wild type (WT) muscles show a characteristic striped pattern of Igf-2 where 50% of fibres are Igf-2 immunostain positive.
• E-H dystrophin-deficient (mdx) muscles over-express Igf-2 between E14.5 and El 7.5 and loose the 'stripy' immunostain pattern of WT.
• I-L Igf-2 is lost in caveolin-3 deficient ⁇ cav-3 '1' ) muscles.
• M Quantification of immunstain pattern confirms these conclusions.
• N RT-PCR establishes that Igf-2 message (mRNA) is elevated in mdx and cav-3 ⁇ ' embryos. The suppression of Igf-2 in caveolin deficient embryos is thus at the post-transcriptional level.
• Figure 12 shows depletion of Pax-7 myoblasts in human skeletal DMD.
• Figure 13 shows a graph showing depletion of Pax-7 myoblasts in human DMD and BMD compared to controls.
• Figure 14 Reducing caveolin- 3 levels to wild-type in mdx, increases the severity and prolongs the progression of dystrophic phenotype in mdx mouse muscle.
• Figure 15 The affect of Igf-2 on dystrophic myoblasts; Pax-7 and Pax-3 are up regulated by Igf-2 treatement. Increased caveolin-3 levels in dystrophic myoblasts is enhanced by Igf-2 treatment.
• C57BL10 and isogenic mdx and cav-3 '1' mouse strains were used, cav-3 '1' dystrophic mice on C57B110 background were from Yoshito Hagiwara (Tokyo) (Hagiwara et al., 2000). Mdx and C57BL10 were generated in house (Merrick et al., 2007).
• Double mutant mice were null for dystrophin (dys-/-) and heterozygous for caveolin-3 (cav-3+/- ) and were generated by intercrossing mdx and cav-3 *A using a strategy described previously to generate dystrophin deficient mutants heterozygous for an Igf-2 transgene (mdxIgf-2+/-; (Smith et al., 2000)). Genotyping was achieved by PCR for neomycin (to detect cav-3KO transgene) and caveolin-3 (expressed by WT, mdx and cav-3 +/ ⁇ but not cav-3 "A ; Figure 7).
• CDNKl c/p57kip2 CDNKl c/p57kip2, pAkt, IGF-2 and FMyHC staining was quantified as previously described forFMyHC and IGF-2 (Merrick et al., 2007) bu counting the proportion of antibody positive myotubes over a fixed grid area. Data were analyzed using Student's t-test and ANOVA. . At least 3000 myotubes were counted for each data point.
• Pax-7 immunostaining was also carried out in archival juvenile human muscles biopsies (see Figure 8).
• Sagital cut MF20 stained embryos from WT, cav-3 " ' " and mdx were matched for stage, plane and angle of section. To avoid artefacts the inventors counted only splits and branches entirely in the plane of cut, this was carefully controlled between sections. This analysis may underestimate splitting/branching events.
• Sagital sections from E13.5, E15. 5 and E17.5 embryos were carefully matched for location of morphological features against a standard published mouse atlas (Kaufman, 1995) at the midline point of the embryo to facilitate matching. Sections were counted blind and by two separate observers for both misalignment and branching phenotypes and subject to statistical analysis.
• Branching was scored over a fixed area using a grid graticule in the same longitudinally presenting muscles in the intercostals, upper and lower limb and facial muscle regions, for each mutant and WT embryo scored. Scoring of misaligned fibres was achieved by orienting the direction of the muscle fibres in longitudinal sections to a grid graticule and scoring over a fixed area for fibres which deviated by more than a 25 degree angle. We are confident our data are a reliable indicator of splitting/branching: when two experimenters counted slides, the second being unaware of the strain, splitting/branching was only identified in mdx and was statistically significant.
• E11.5-E17.5 embryos were dissected to isolate areas rich in skeletal muscle cells. In all embryos head, spinal cord and internal organs were removed. In older embryos (E15.5-E17.5) skin and cartilage/bone were removed. Muscle-rich tissues were micro-dissected into micro- explants and cultured in microwells (Smith and Merrick, 2008; Smith and Schofield, 1994).
• CM explant conditioned medium
• Protein was extracted from embryos directly into a glass homogeniser (1-5 ml; VWR International, UK) containing RIPA buffer. Immunoblotting was carried out using standard protocols and detected by ECL (Pierce Endogen Hyclone). Antibodies: fast myosin (My32, 1:1000); ⁇ -tubulin (1:1000, Sigma); pax-7 (1/1000); caveolin-3 (l ⁇ OOO).Goat anti-mouse IgG-HRP (1:2000, Santa Cruz Biotechnology). Protein concentration was determined using an ELISA form of the Bradford assay (Merrick et al., 2007).
• RT-PCR Reverse transcribed (RT) PCR was carried out to quantitate the amount of pax-7 mRNA in myoblasts isolated from WT and mdx mouse skeletal muscle (Smith & Schofield, 1994). To obtain quantitative data linearisation and equalisation of the Pax-7 product were carried out as described for Igf-2 (Merrick et al, 2007). RT-PCR was carried out under these quantitative on six separate isolates of WT and six isolates of mdx myoblasts.
• PCR Polymerase Chain Reaction
• the Taq polymerase was added last in the PCR mix (as above), to prevent random transcription.
• the samples were loaded into a programmable thermal controller, which had been programmed specifically for the gene as follows: Pax7
• Insulin-like growth factor 2 (Igf-2) has been previously reported. Staining methods and PCR methods are discussed in Merrick D et al (2007). Typically muscle biopsies were assayed.
• WT, c ⁇ v 5 "A and mdx mice were immunostained with pan-myosin antibody (MF20) to reveal muscle fibre architecture (Fig. 1). Branching and fibre splitting is found in mdx epaxial (back), hypaxial (limb, respiratory muscles) and facial muscles from E13.5 to E17.5 and shows a dynamic temporal and spatial pattern consistent with it being an early event associated with myotube formation (Fig. IA- C and G). At matched stages and muscle groups mdx myotubes are hypotrophic, cav 3 ⁇ ' ⁇ myotubes hypertrophic and myotube width varies more in dystrophic embryos with respect to WT (Fig. IC-E, green bars).
• Cardiomyopathy is a significant clinical consequence of both LGMD (1C) and DMD.
• LGMD LGMD
• DMD DMD
• MF20 and cardiac ⁇ -myosin specific antibody N2.261 Fig. 2
• FMyHC skeletal fast myosin isoforms
• Cardiac ⁇ -myosin is present in WT and dystrophic ventricular and atrial myocytes between E13.5 and E17.5 (Fig. 2A-F, I-P). Both mutants exhibit ventricular wall thickening (Fig.
• myonuclei are evenly spaced along the length of the myotube and, except in newly formed myotubes, are arranged evenly and helically around the edge (Fig. 3A, white stars). In mdx, myonuclei are more frequently centrally located and slightly further apart than WT (Fig. 3B). In contrast, cav-3 'A myonuclei are closer together and exhibit severely disrupted myonuclei spacing and nuclei 'bunching' which is particularly evident at the ends of myotubes (Fig. 3C-D).
• the pax-7 skeletal muscle stem cell population is attenuated and disorganised in dystrophic embryos
• the WT pax-7 + stem cell population is more sparsely interspersed between skeletal muscle myotubes although pax-7 protein continues to increase with muscle size ((Merrick et al., 2007); Fig. 5A-B, G-H, M & O).
• pax-7 staining intensity is uniform and constant with gestational age (Fig. 5A-B, G-H & Q; lower proximal limb).
• cav-3 ' ' ' and mdx undergo attrition of their pax-7 + cell population throughout their musculature so that by El 7.5 there is significant reduction of pax-7 staining and pax-7 protein in both mutants (Fig.
• Pax-7 content increases in WT embryos with gestation but is substantially reduced in cav-3 ' ' ' and mdx at E15.5-E17.5 with the reduction being greater in cav-3 ' ' ' than mdx at both stages suggesting caveolin-3 may regulate pax-7 + myoblast survival in late gestation.
• pax-7 is slightly elevated in cav-3 ' ' ' and reduced in mdx , this may relate to the increase and decrease in myotube number found respectively in cav ⁇ " and mdx embryos at this stage (see Fig.3) and suggests that developmental timing may be disrupted in these embryos.
• FMyHC is present in small numbers of secondary myotubes as early as El 1.5 and by El 5.5 is strongly localised to around 45% of secondary myotubes across a wide range of muscle groups.
• This dynamic and muscle specific pattern of fast-myosin localisation is disrupted in cav-3 ' ' ' and mdx mutant embryos which show defects in developmental timing of FMyHC staining, in the total number of fast myosin positive myotubes present and in the intensity of fast myosin staining (Fig. 6, Fig. Sl).
• FMyHC is elevated above WT in some cav-3 '1' muscles (notably the respiratory muscles) but substantially attenuated in all mdx muscles and in cav-3 ' ' ' proximal muscles (Fig. 6A-F).
• RT-PCR was used to qualitise pax-7 mRNA levels. This showed lower pax-7 levels in DMD individuals than normal individuals. Igf-2 disrupted mice
• FIG. 9 shows Igf-2 in disrupted mice.
• Igf-2 message rnRNA
• rnRNA Igf-2 message
• the suppression of Igf-2 in caveolin deficient embryos is at the post-transcriptional level.
• pAkt phosphorylated Akt
• pAkt has a MD disease type specific response, for example in caveolin-3 deficiency/LGMD-lc pAkt is reduced compared to normal muscle and in mdx/DMD it is increased.
• the pattern of disturbance for pAkt is the same as that of Igf-2.
• myf-5 + mdx embryonic myoblasts The growth behaviour of myf-5 + mdx embryonic myoblasts is disrupted from El 1.5 and mdx myf-5+ myoblasts are hyperproliferative and apoptotic.
• Myf-5 marks the embryonic myoblast population; it is expressed in myotome (E8.5) before myotube differentiation is initiated and is found in all muscle groups throughout myogenesis (Hadchouel et al., 2003; Ott et al., 1991).
• Dystrophin is expressed at a crucial stage (E9.5) in myotome differentiation, one day later than Myf-5 expression at E8.5, and 24 hours prior (E10.5) to the appearance of the first fully differentiated myotomal (epaxial) myotubes and the first expression of myosin heavy chain (MyHC) (Houzelstein et al., 1992; Ott et al., 1991; Schofield et al., 1993).
• MyHC myosin heavy chain
• Early hypaxial and secondary myogenesis are severely disrupted and delayed in mdx embryos as shown by the late appearance of FMyHC, pax-7 and cav-3 proteins and by the incomplete formation and disorganisation of mdx musculature between E11.5-E13.5 (Fig. 1; Fig. 6 and Fig. Sl in supplementary material).
• myf-5+ myoblasts migrate from the myotome to initiate hypaxial muscle formation (Cusella-De Angelis et al., 1992).
• Myoblasts migrate under control of hox genes (pax3 and lbx) and differentiate in response to growth factors (wntl and shh) secreted by adjacent tissues, (Gross et al., 2000; Hadchouel et al., 2003).
• the cues which trigger embryonic myoblast migration are not known, in other tissues stem cell migration is regulated by guidance cues from originating and target tissues, is essential for correct patterning of embryonic structures and can be easily disrupted (Lehmann, 2001).
• E10.5-11.5 epaxial myotubes provide signalling cues which trigger migration of myf-5+ myoblasts from the myotome and initiate hypaxial and secondary myogenesis. In the absence of dystrophin these cues are absent and both the migration and initiation processes are impaired.
• the aberrant behaviour of El 1.5 WT explants cultured in El 1.5 mdxCM (Fig. 41) supports the view that El 1.5 myotome releases secreted factors which modify the behaviour of the El 1.5 embryonic myoblast population. Disruption of early myogenesis in mdx suggests these early role(s) for dystrophin are distinct from those of utrophin.
• Caveolin-3 is regulated by muscle regulatory factors (MRF 's), activated during myotube differentiation and is expressed later than dystrophin in El 1.5 myotome (Biederer et al., 2000). This is consistent with our detection of caveolin-3 at El 1.5 in WT embryos. MRF's also regulate pax-7 which too emerges at El 1.5 (Merrick et al., 2007). In cav-3 ' ' ' embryos the early muscle patterning process appears intact, myf-5+ myoblast behaviour is not disrupted and the localisation and timing of hypaxial muscle formation is comparable to WT. Caveolin- 3 does not therefore seem to be required for these early stages of myogenesis. In mdx, the appearance of both caveolin-3 and pax-7 is delayed, this could be as a consequence of the developmental delay in myogenesis in these embryos or more specifically downstream of the failure to activate dystrophin.
• MRF 's muscle regulatory factors
• dystrophin in later embryonic events is complicated by the up- regulation of caveolin-3 in mdx embryos from El 3.5.
• over-expression of caveolin-3 causes dystrophin down-regulation and a DMD-like phenotype (Galbiati et al., 2000).
• Loss of dystrophin causes breakdown of the DGC and suppression of dystrophin- associated proteins (Ohlendieck et al., 1993; Vaghy et al., 1998).
• the muscles of DMD patients and adult mdx have 1.5 - 2.4 and 2-3 fold excess of caveolin-3 respectively (Vaghy et al., 1998).
• E 13.5 mdx embryos partially reproduce the over-expression phenotype having hypotrophic myotubes and reduced myotube numbers. Myonuclei number is not affected. In later stages (E15.5-E17.5) however, although caveolin-3 levels remain elevated, mdx myotube numbers are comparable to WT. This suggests caveolin- 3 may regulate myotube size but the E 13.5 myotube deficit is due to delayed hypaxial myogenesis in mdx (see section above) rather than excess caveolin-3.
• FMyHC+ myotubes appear first in WT epaxial muscles -El 1.5 and herald the start of secondary myogenesis (Merrick et al., 2007). Fibre-type switching begins at El 5.5 when some myotubes switch between slow and fast myosin expression and the process of establishing adult fibre-type ratios begins (Cho et al., 1994; Merrick et al., 2007). In mdx FMyHC+ myotube differentiation is significantly perturbed (see sections above) and fast fibre type specification is disrupted in both mdx and cav-3 'A (Fig. 6).
• FMyHC+ fibres are still over-represented at El 7.5 compared to WT suggesting that it is the balanced relationship between dystrophin and caveolin-3 which is crucial for correct fast fibre proportion rather than caveolin-3 levels alone.
• E15.5-E17.5 cav-3 '1' and mdx exhibit pax-7 + myoblast attrition and significant depletion of pax-7 protein.
• Loss of pax-7 occurs rapidly in cav-3 ⁇ ' ⁇ at El 5.5, a time-point when caveolin-3 is strongly up-regulated in WT embryos and the loss is greater than in El 5.5 mdx.
• Caveolin- 3 can elicit survival signalling in muscle as does pax-7 itself.
• PaX-T + myoblasts are crucial for normal post-natal satellite cell emergence (Relaix et al., 2006; Seale et al., 2000).
• pax-7 pax-7 "7" mice
• satellite cells are reduced in number and apoptosis is elevated.
• pax-3+ myoblasts satellite cells are not entirely lost and there appear to be sufficient satellite cells to establish and sustain (post-natal) juvenile muscle development in these mice, however regeneration in adults is impaired (Oustanina et al., 2004; Relaix et al., 2006; Seale et al., 2000).
• DMD and LGMD Ic are early onset, progressive skeletal muscle diseases of children affecting cardiac and skeletal muscle function and muscle stability (Hoffman et al., 1987; Minetti et al., 2002). In DMD there is widespread, progressive mal-function of the entire musculature, abnormal caveolin-3 expression, myoblast apoptosis, cardiomyopathy and regeneration defects (Cox and Kunkel, 1997; Smith et al., 1995; Vaghy et al., 1998).
• LGMD-IC exhibits similar but restricted myopathic changes particularly affecting the muscles of the limb, diaphragm and heart (Galbiati et al., 2001; Hagiwara et al., 2000; Smythe et al., 2003).
• mdx and cav-5 '1' post-natal phenotypes are sufficiently similar to the clinical pathologies of DMD and LGMD in most respects, to be widely used as disease models (Chamberlain et al., 2007; Chan et al., 2007; Galbiati et al., 2001; Hagiwara et al., 2000; Roig et al., 2004; Vaghy et al., 1998).
• mice The respective embryonic phenotypes of mdx and cav-3 '1' strengthen the validity of these mice as models for LGMD and DMD, provide new insight into the mechanisms underlying MD and the mode of function of dystrophin and caveolin-3 and suggest new approaches to dissecting the differences between the human and murine forms of the disease.
• the inventors identify key developmental stages; myotome differentiation (El 1.5) and secondary myogenesis (E13.5) at which dystrophin and caveolin- 3, respectively, play key roles and reveal two novel pathologies in late gestation; fast fibre specification and attrition of pax-7 myoblasts which provide insight into the mechanism underlying MD pathology and which suggest novel routes for therapeutic intervention and earlier diagnosis of MD.
• Cardiomyopathy, particularly left ventricular failure is a significant clinical consequence of DMD and many other MD's and an established pathology of cav-3 ' ⁇ , mdx and caveolin-3 over-expressing mice (Aravamudan et al., 2003; Cox and Kunkel, 1997; Hayashi et al., 2004; Quinlan et al., 2004; Woodman et al., 2002; Yue Y et al., 2003).
• Dystrophin and Caveolin-3 express in mouse embryonic heart at E9.5 and E10.5 respectively (Biederer et al., 2000; Houzelstein et al., 1992).
• E13.5 mdx have moderate thickening of the ventricular apex and atrial trabecular defects.
• the mdx finding suggests a developmental origin for a recent report of hypertrabeculation in a 28 year old DMD (Finsterer et al., 2005). Although post-natal mdx hearts are reported to have WT levels of ⁇ -cardiac myosin and increased levels of utrophin which may compensate for dystrophin deficiency these mice have a progressive cardiomyopathy (Quinlan et al., 2004). Localisation of ⁇ -cardiac myosin has not yet been established postnatally, its disrupted atrial localisation could therefore persist into the adult or may be lost in the peri-natal or juvenile period (Wilding et al., 2005).
• Hyperproliferation and elevated muscle cell apoptosis are well-established, wide-spread features of post-natal MD muscle pathology which characterise both mouse and human forms of OMDI mdx and LGMD- lc/cav-3 '/' as well as most other MD types (Baghdiguian et al., 1999; Smith et al., 1995; Smith et al., 2000; Smythe et al., 2003).
• Dystrophin, dystroglycan and caveolin-3 have roles in survival signalling (Glass, 2005; Smythe et al., 2003).
• pax-7 depletion is an early marker of DMD and BMD which can be used to identify these diseases from other non- MD myopathies and from non-diseased muscle biopsies in children. This marker can be used alone or in combination with a marker for muscle degradation (eg creatine Kinase), and/or for muscle regeneration (eg.
• Caveolin-3 is elevated above normal (wild-type) levels in mdx (mouse) embryonic and post natal tissues and in DMD (human) muscles. Double mutant mouse embryos which are deficient in dystrophin (mdx) and heterozygous for caveolin-3 have reduced levels of caveolin-3 and a more severe muscle pathology than that found in dystrophin deficiency (mdx) alone suggesting that elevated levels of Caveolin-3 may compensate for the loss of dystrophin.
• Figure 14 shows that when levels of caveolin-3 are reduced back to non-disease (wild-type) levels in mdx mouse that the post natal dystrophic phenotype of mdx also increases in severity confirming the therapeutic effect of increased levels of caveolin-3. In these mice ( Figure 14).
• Igf-2 has an ameliorative effect on the dystrophic (mdx) phenotype (Smith et al, 2000) but the mechanism for this is unknown.
• Figure 15 demonstrates that Igf-2 can upregulate the expression of Pax-7, Pax-3 and caveolin-3 in dystrophic myoblasts suggesting a direct mechanism for its therapeutic effect and providing strong support for the use of this protein as a therapeutic agent separately or in conjunction with other treatments such as caveolin-3 ( Figure 15).
• Igf-2 induces increased expression (mRNA) of Pax-7 in WT and dystrophic myoblasts within 15 minutes of treatment of 10-20ug/ml Igf-2.
• Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. Development 130, 5851-60.
• Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. Faseb J 21, 2195-204.
• dystrophin-glycoprotein complex As a transmembrane linker between laminin and actin. Journal of Cell Biology 122, 809-823.
• Lbxl is required for muscle precursor migration along a lateral pathway into the limb. Development 127, 413-24.
• Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. JCe// Biol 172, 91-102.
• dystrophin-related protein utrophin
• Dystroglycan mRNA expression during normal and mdx mouse embryogenesis a comparison with utrophin and the apo-dystrophins. Dev Dyn 204, 178-85.
• IGF-II ameliorates the dystrophic phenotype and coordinately down-regulates programmed cell death.
• Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag 1 -null mice.

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