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WO2024161042A1 - Dosages de colocalisation quantitative pour évaluer l'activité et l'efficacité de thérapies ciblant des troubles musculaires - Google Patents

Dosages de colocalisation quantitative pour évaluer l'activité et l'efficacité de thérapies ciblant des troubles musculaires Download PDF

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WO2024161042A1
WO2024161042A1 PCT/EP2024/052818 EP2024052818W WO2024161042A1 WO 2024161042 A1 WO2024161042 A1 WO 2024161042A1 EP 2024052818 W EP2024052818 W EP 2024052818W WO 2024161042 A1 WO2024161042 A1 WO 2024161042A1
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colocalization
interest
degree
nucleic acid
protein
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Oana LORINTIU
Tiphaine CHAMPETIER
Cécile GASTON
Mélanie FLAENDER
Erwann VENTRE
Luc Selig
Beatrice Darimont
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CYTOO
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells

Definitions

  • the present invention relates to methods of assessing the functionality of a cellular molecule in a muscle cell, assessing potency of a compound to modulate the functionality of a cellular molecule in a muscle cell, predicting the ability of a compound to treat a muscular disease, monitoring the response to a therapeutic compound of a patient affected with a muscular disease, selecting a patient affected with a muscular disease for a treatment with a therapeutic compound or determining whether a patient affected with a muscular disease is susceptible to benefit from a treatment with a therapeutic compound.
  • muscle fibers The unique structural and functional properties of muscles provide substantial challenges for the functional characterization of muscle disorders and discovery of therapies.
  • Particularly challenging are striated muscles, which are formed by the fusion of mononuclear progenitor cells, called myoblasts, to multinucleated myotubes. These myotubes then differentiate into myofibers that contain a contractile apparatus (sarcomere), which enables these fibers to contract upon stimulation from an attached motor neuron.
  • the ability of muscle fibers to differentiate and contract is dependent on their interactions with connective tissues that surround these fibers (basal lamina). These interactions are critical for the ability of muscle fibers to evade damage during contractions, to differentiate, and to respond to mechanical forces.
  • Each muscle fiber is innervated by a single motor neuron through a neuromuscular junction (NMJ).
  • NMJ neuromuscular junction
  • a process called excitation-contraction coupling converts the neuronal excitation mediated by the motor neuron into Ca 2+ signaling that induces the muscle contractions.
  • Skeletal muscle fibers are terminally differentiated. However, specific processes are in place to repair contraction-induced damages in the plasma membrane, or to replace damaged muscle fibers through activation of muscle stem cells.
  • Neuromuscular disorders are generally classified as dystrophies, myopathies and myasthenic syndromes dependent on the morphology of the diseased muscle fibers and on the function affected by the disease.
  • the underlying defects in these diseases are diverse and can affect either directly or indirectly the myofiber-matrix interactions, the excitationcontraction coupling, the contractile apparatus, the muscle metabolic activity, or the ability to regenerate (Dowling et al., Nat Rev Mol Cell Biol. 2021 Nov;22(ll):713-732).
  • neuromuscular disorders can also be caused by mutations in motor neurons (e.g., Spinal muscular dystrophy, Kennedy's disease), or in the basal lamina (e.g., Ullrich myopathy).
  • the structure and function of muscles can be impaired metabolically in response to malnutrition, immobility, advanced age, or acute and chronic diseases.
  • changes in the structure and function of muscles are often driven by changes in protein turnover mediated by the ubiquitin/proteasome system and autophagy-lysosomal pathways, by the inhibition of growth factor pathways, and/or activation of inflammatory pathways.
  • DMD Duchenne and Becker muscular dystrophies
  • DGC Dystrophin-Glycoprotein complex
  • This complex is involved in the regulation of muscle cell signaling and acts as a shock absorber that prevents the damaging of muscle cells during contractions.
  • Boys who do not express dystrophin usually lose the ability to walk around age 10 and have a shortened live span due to pulmonary and cardiac complications.
  • BMD patients express a partially functional dystrophin usually associated with less severe symptoms.
  • DM1 myotonic dystrophy
  • DM1 myotonic dystrophy
  • the disease is caused by the expansion of a CUG repeat in the DMPK pre-mRNA that forms a stem loop that is recognized by RNA splicing factors, including MBNL1. Interaction of these splicing factors with the mutant DMPK transcripts competes with the correct splicing of other RNAs, many of which are involved in critical muscle functions. It remains to be identified which of the miss-spliced RNAs are driving the DMl-associated phenotypes and whether other mechanisms are in play. The advances in understanding of the cellular processes involved in muscle function has opened the door for therapeutic discovery.
  • DMD a main challenge is the size of the DMD gene, the largest gene in the human genome which encodes a 420 kDa protein. Since current gene therapies rely on viral vectors that have limited load capacities, gene therapies targeting DMD can only deliver the information for a drastically truncated and functionally impaired dystrophin. Likewise, therapies altering the splicing of mutant DMD pre-mRNAs induces the expression of a partially functional dystrophin. Clinical data indicate that expression of the restored dystrophin alone does not correlate with the clinical efficacy of these therapies. Hence, functional assays are needed to guide the discover and clinical development of these therapies. The clinical success of these therapies has been further hampered by the lack of appropriate animal models that represent the diversity of the genetic backgrounds found in DMD patients. Similar challenges exist for many neuromuscular disorders.
  • the present invention relates to an in vitro method of assessing the functionality of a cellular molecule of interest in a muscle cell
  • the muscle cell is a myotube or cardiomyocyte.
  • the muscle cell is a cardiomyocyte and said least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of card io myocytes, and any combination thereof.
  • the muscle cell is a myotube and said least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof.
  • the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.
  • the present invention also relates to an in vitro method of assessing potency of a compound to modulate the functionality of a cellular molecule of interest in a muscle cell
  • the muscle cell is a myotube or cardiomyocyte.
  • the muscle cell is a cardiomyocyte and said least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of card io myocytes, and any combination thereof.
  • the muscle cell is a myotube and said least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof.
  • the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.
  • the present invention also relates to an in vitro method of predicting the ability of a compound to treat a muscular disease of interest comprising
  • the muscle cell is a myotube or cardiomyocyte.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a cardiomyocyte
  • said least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof
  • the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a myotube
  • said least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof
  • the muscular disease is a neuromuscular disease.
  • the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.
  • the present invention relates to an in vitro method for monitoring the response to a therapeutic compound of a patient affected with a muscular disease, wherein the method comprises
  • the muscle cell is a myotube or cardiomyocyte.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a cardiomyocyte, said least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof, and the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • ROI region of interest
  • the muscle cell is a myotube
  • said least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof
  • the muscular disease is a neuromuscular disease.
  • the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.
  • the present invention also relates to an in vitro method for selecting a patient affected with a muscular disease for a treatment with a therapeutic compound or for determining whether a patient affected with a muscular disease is susceptible to benefit from a treatment with a therapeutic compound, wherein the method comprises
  • the muscle cell is a myotube or cardiomyocyte.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a cardiomyocyte
  • said least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof
  • the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a myotube
  • said least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof
  • the muscular disease is a neuromuscular disease.
  • the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.
  • the muscle cells are derived from primary cells or are derived from immortalized cells.
  • the muscle cells have been cultured in constrained conditions allowing the production of homogeneous population of muscle cells.
  • the methods of the invention may further comprise comprises before step (i)
  • muscle cells in particular myoblasts or cardiomyocytes, in constrained conditions allowing the production of homogeneous population of muscle cells, in particular myotubes or cardiomyocytes, optionally in the presence of a compound to be tested;
  • the degree of colocalization is quantitatively determined by calculating one or several colocalization readouts selected from the group consisting of the Pearson's Colocalization Coefficient (PCC), the Manders' Colocalization Coefficient (MCC), the Rank-based intensity Weighting Coefficient (RWC), and any combinations thereof, and optionally applying a mathematical function on said coefficient(s).
  • PCC Pearson's Colocalization Coefficient
  • MCC Manders' Colocalization Coefficient
  • RWC Rank-based intensity Weighting Coefficient
  • said first molecule is a protein and said second molecule is a protein
  • the degree of colocalization is quantitatively determined by calculating the Pearson's Colocalization Coefficient (PCC) and the methods further comprise defining a threshold for high PCC values.
  • At least one of said first molecule and of said second molecule is a nucleic acid and in step (iii) the degree of colocalization is quantitatively determined by calculating the Manders' Colocalization Coefficient (MCC).
  • MCC Manders' Colocalization Coefficient
  • the muscular disease of interest is a neuromuscular disease selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes, motor neuron diseases and metabolic muscle disorders.
  • the muscular disease is Duchenne muscular dystrophy or myotonic dystrophy type 1 (DM1)
  • the first and second molecules are independently selected from the group consisting of proteins belonging to the Dystrophin Glycoprotein complex (DGC) and dysferlin, preferably selected from the group consisting of dystrophin, a-sarcoglycan, p-dystroglycan, a-dystroglycan and dysferlin, more preferably selected from the group consisting of dystrophin, a-sarcoglycan and
  • DGC Dystrophin Glycoprotein complex
  • the first molecule may be dystrophin and the second molecule may be a-sarcoglycan, or vice-versa, or the first molecule my be dystrophin and the second molecule may be p-dystroglycan, or vice-versa.
  • the muscular disease is myotonic dystrophy type 1 (DM1)
  • the first molecule is DMPK RNA
  • the second molecule is a RNA binding protein trapped by CTG repeats in the DMPK gene, preferably is MBNL1 protein, or vice-versa.
  • Figure 1 Characterization of DMD donor myotubes on MyoScreen micropatterned plates. Myotube differentiation and morphology of healthy and DMD donor cells under MyoScreen conditions was assessed using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes.
  • MHC myosin heavy chain
  • B Quantification of nuclei count, fusion index (ratio of the number of nuclei in myotubes over the total number of nuclei detected) and myotube mean area for HV and DMD donors.
  • DMD donors #5 and #6 show a decreased fusion index and mean area, indicating reduced differentiation compared to HV donors.
  • ANOVA with post-hoc multiple comparison test *, p ⁇ 0.05 ; **, p ⁇ 0.01; ***, p ⁇ 0.001 compared to HV #1 or #, p ⁇ 0.05 ; ##, p ⁇ 0.01 and ###, p ⁇ 0.001 compared to HV #2.
  • FIG. 2 Validation of the dystrophin antibodies used in the study.
  • A Dystrophin expression assessed using a N-terminal domain targeting antibody in two healthy (HV#1 and #2) and four DMD donors (DMD #1, #4, #5 and #6) (left panel). MHC and Hoechst staining show the presence and morphology of the myotubes (right panel). Scale bar, 100pm. As expected, no dystrophin expression can be detected in the four DMD donors.
  • B Dystrophin expression assessed using a C-terminal domain targeting antibody in healthy (HV #1 and #2) and DMD donors (DMD #5 and #6) (left panel). MHC and Hoechst staining show the presence and morphology of the myotubes (right panel).
  • the C-terminal domain targeting dystrophin antibody shows a detectable level of background staining in the DMD donors.
  • C Assessment of the dystrophin antibodies specificity using RNAi-mediated knockdown of dystrophin in two healthy donors (HV #1 and #2). Dystrophin staining decreases in a dose-response manner for both the N- terminal targeting antibody (upper panel) and the C-terminal targeting antibody (lower panel). Scale bar, 100pm.
  • D Quantitative assessment of dystrophin levels using N-terminal-targeting or C-terminal-targeting antibody in healthy and DMD donors.
  • both antibodies show similar staining than observed in DMD donors, suggesting that the residual staining shown by the antibody targeting the dystrophin C-terminal is due to a non-specific activity of this antibody.
  • the non-specific activity of this antibody is primarily cytosolic.
  • Figure 3 Characterizing the expression of selected dystrophin colocalization imaging targets in healthy and DMD donors in the absence and presence of dystrophin.
  • Three DGC proteins were selected to monitor colocalization with dystrophin: p-dystroglycan (b-DG), a transmembrane protein that interacts directly with dystrophin; a-sarcoglycan (a-SG), a transmembrane protein that interacts indirectly with dystrophin; a-dystroglycan (a-DG), an extracellular protein that interacts with the DGC through b-DG.
  • Dysferlin was included as a transmembrane protein that is not directly associated with the DGC.
  • Figure 4 General workflow to quantify colocalization between labelled image markers.
  • the masks of imaging marker 1 (IM1) positive areas and of imaging marker 2 (IM2) positive areas are obtained from their respective images, after thresholding staining from the respective secondary antibodies D.
  • the overlaid masks defined four areas: the area of the nuclei in myotubes (dotted line), the area of colocalization (hatched area), the area with IM1 signal above threshold (light grey) and the area with IM2 signal above threshold in the nuclei in myotubes (dark grey).
  • Formulas for three IM1/IM2 colocalization readouts 1. the Pearson's Colocalization Coefficient (PCC), 2. the Mander's Colocalization Coefficient (MCC) and 3. the Rank-based intensity Weighting Coefficient (RWC).
  • I IM2 the IM2 associated intensity for a given pixel
  • I IM1 the IM1 associated intensity for a given pixel
  • meanI IM2 ⁇ the mean IM2 intensity on the area indicated on the right of the formula
  • meanI IM1 the mean IM1 intensity on the area indicated on the right of the formula
  • W plxei a pixel weight that grows with the correlation of IM2 and IM1 intensity ranks for a given pixel.
  • the ranks of the intensities are calculated for each pixel
  • D pixei is the absolute difference between the ranks
  • Rn is the maximal rank of pixels (either in dystrophin channel or in IM2 channel) see Singan et al., 2011 (BMC Bioinformatics 12, 407).
  • the PCC is calculated on the area where at least one of the two proteins expression is above its specific threshold, that is to say, on the union of the two masks. 2-3.
  • MCC and RWC the sums on pixels are realized in the zone highlighted next to the sum.
  • the area is the colocalization area, at the denominator it is the zone of IM2 expression.
  • the imaging marker called here I MCTL is used as a control protein that is not colocalizing with IM1. 1-3.
  • the Figures 4.D readouts are declined for I MCTL and IM1 proteins, with I CTL the I MCTL associated intensity for a given pixel, meanI CTL the mean I MCTL intensity on the area indicated on the right of the formula.
  • Figure 5 Defining a threshold for high PCC values.
  • One of the developed readouts is the percentage of ROI displaying a strong colocalization defined for the two imaging markers in the ROI.
  • a satisfying threshold is one for which 99% of the PCC values between Dystrophin and DGC proteins for DMD donors or MHC for all donor myotubes are under the threshold.
  • 0.6 was determined as a satisfying threshold for all the tested imaging markers and DMD donors as only outliers situated in the 99th percentile exceed this threshold value.
  • the High PCC% is the percentage of myotubes having a PCC value above the 0.6 threshold among the total number of . myotubes: 100.
  • Figure 6 Quantification methods for colocalization of dystrophin with a-sarcoglycan, P-dystroglycan, a-dystroglycan and dysferlin.
  • Dystrophin expression in two healthy donors was regulated by RNAi using DMD specific siRNAs in concentration ranges between 0.001 and 1 nM.
  • Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between a-sarcoglycan, p-dystroglycan, a-dystroglycan, dysferlin and dystrophin (N-terminal antibody). For HV#1 and HV#2, the colocalization readouts are then plotted against the dystrophin siRNA dose.
  • FIG. 7 Sensitivity of the colocalization between dystrophin and p-dystroglycan, a- sarcoglycan, a-dystroglycan, or dysferlin to changes in dystrophin levels.
  • Dystrophin expression in two healthy donors was regulated by RNAi using DMD siRNAs in concentration ranges between 0.001 and 1 nM. The resulting level of dystrophin determined by high content analysis.
  • Figure 8 Colocalization of restored dystrophin with p-dystroglycan and a-sarcoglycan in myotubes from DMD patients amenable to Exon 44 skipping. Exon 44 skipping was induced by treating myotubes with vivo PMOs targeting one of the exon 44 splice junction.
  • B Evaluation of morphological readouts (nuclei count, fusion index and myotube mean area) in untreated and vivo PMO treated healthy and DMD donors. Under the concentrations used, the vivo PMOs did not affect the growth and differentiation of the myotubes.
  • C Quantitative assessment of dystrophin, a-sarcoglycan and P-dystroglycan expression in vivo PMO-treated healthy and DMD donors. Statistical significance was assessed by ANOVA with Dunnett's test for multiple comparisons between HV and DMD-treated conditions.
  • Figure 9 Colocalization of restored dystrophin with p-dystroglycan and a-sarcoglycan in myotubes from DMD patients amenable to Exon 45 skipping. Exon 45 skipping was induced by treating myotubes with vivo PMOs targeting one of the exon 45 splice junction.
  • Myotubes were labeled for dystrophin (upper panel), p-dystroglycan (middle panel) and a-sarcoglycan (lower panel). Scale bar, 100pm.
  • B Evaluation of morphological readouts (nuclei count, fusion index and myotube mean area) in untreated and vivo PMO treated healthy and DMD donors. Under the concentrations used, the vivo PMOs did not affect the growth and differentiation of the myotubes.
  • C Quantitative assessment of dystrophin, a-sarcoglycan and p-dystroglycan expression in vivo PMO-treated healthy and DMD donors. ANOVA with Dunnett's multiple comparisons test for pairwise comparison. * - ** - ***, p ⁇ 0.05 - 0.01 - 0.001 for HV#1 and # - ## - ###, p ⁇ 0.05 - 0.01 - 0.001 for HV#2.
  • Figure 10 Colocalization of restored dystrophin with p-dystroglycan in myotubes from DMD immortalized cell line amenable to Exon 44 skipping. Exon 44 skipping was induced by treating myotubes with 4 PMOs targeting one of the exon 44 splice junction.
  • Myotube differentiation and morphology of healthy and DMD immortalized cell lines under MyoScreen conditions was assessed using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes.
  • MHC myosin heavy chain
  • D Quantitative assessment of dystrophin and p-dystroglycan expression in PMO-treated healthy and DMD immortalized cell lines. ANOVA with Dunnett's multiple comparisons test for pairwise comparison. * - ** - ***, p ⁇ 0.05 - 0.01 - 0.001 for HVimm. Treatment of DMDimm cell line with Exon 44 skipping PMOs partially restores dystrophin expression, in a distancedependent manner. The PMOs targeting closer to the splice junction (PMO1, 2 and 3) demonstrate higher dystrophin rescue than the PMO targeting further from the acceptor site (PMO4). Expression levels of
  • Figure 11 Characterization of DM1 donor myotubes on MyoScreen micropatterned plates. Myotube differentiation and morphology of healthy and DM1 donor cells under MyoScreen conditions was assessed using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes.
  • MHC myosin heavy chain
  • B Quantification of nuclei count, fusion index (ratio of the number of nuclei in myotubes over the total number of nuclei detected) and myotube mean area for HV and DM1 donors.
  • the MCC readout showed the largest dynamic range and distinguished itself as the preferred readout to monitor colocalization of DMPK foci and MBNL1.
  • Colocalization readout mean MCC for five DM1 donors and two healthy donors. We compare the response of the five DM1 donors to treatment with an ASO at 6 concentrations (2.7nM, 3.5nM, 4.6nM, 5.9nM, 7.7nM, lOnM). Statistical significance was assessed by ANOVA with Dunnett's test for multiple comparison between DM1 Mock and DM1 ASO-treated conditions. * - ** - ***, p ⁇ 0.05 - 0.01 - 0.001. For all tested donors, colocalization of nuclei foci and MBNL1 evaluated by the MCC readout provided a quantitative assessment of disease modulation by the DMPK ASO.
  • Figure 12 Colocalization of the C-terminal domain of dystrophin with p-dystroglycan and a-sarcoglycan in DM1 donors treated with DMPK ASO.
  • B Treatment of DM1 donors with DMPK ASO partially restores dystrophin C-terminal domain. The antibody targeting the C- terminal domain of dystrophin does not show any specific sarcolemmal staining in DM1 donors. Increasing DMPK ASO doses result in the restoration of dystrophin C-terminal signal in the DM1 donors (arrows show
  • Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between a-sarcoglycan or p-dystroglycan, and dystrophin (C-terminal antibody) in function of the ASO dose-response for DM1 donors (DM1 #1, #2, #3, #4 and #5). All donors show an ASO-dependent increase in the colocalization between dystrophin and
  • Colocalization between a-sarcoglycan or p-dystroglycan, and dystrophin (C- terminal) was analyzed using the High PCC % readout and is presented as a function of % of dystrophin normalized between the dystrophin expressed by the mock healthy donors (HV#1 and HV#2).
  • High PCC% is the highest for healthy donors ⁇ 90% and the lowest in DM1 mock conditions. All DM1 donors show an ASO-dose dependent restoration of dystrophin colocalization with a-sarcoglycan or p-dystroglycan. However, the degree of achieved colocalization was donor-dependent and much greater for donors #4 and #5 than for donors #1, #2, and #3.
  • Figure 13 Characterizing the expression of selected dystrophin and p-dystroglycan (b- DG) in human card io myocytes derived from induced pluripotent stem cells (hIPSC-CM) in the absence and presence of dystrophin.
  • hIPSC-CM untreated, mock and treated with a DMD siRNA (2nM) for five days, cardiomyocytes were labeled for p-dystroglycan (upper panel), dystrophin (middle panel) and MHC and Hoechst (lower panel). Scale bar, 20pm.
  • B Quantitative assessment of dystrophin and p-dystroglycan in card io myocytes shown in figure 13A.
  • Dystrophin expression in hIPSC-CM was regulated by RNAi using DMD specific siRNAs in concentration ranges between 0.0032 and 10 nM. Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between p-dystroglycan and dystrophin (N-terminal antibody). The colocalization readouts are then plotted against the dystrophin siRNA dose. The high PCC% readout displays the highest dynamic range.
  • Figure 14 Figure 2A of International patent application WO 2015/091593.
  • Example of a cell-adhesive pattern that can be used to pattern ECM proteins as substrates to culture myoblasts in the methods of the invention.
  • the inventors herein provide quantitative colocalization assays that monitor the interactions between cellular components that are critical for muscle function. These assays quantify the spatial overlap between two cellular components in situ labeled with specific labelling agents. Unlike coimmunoprecipitation assays or biochemical isolation of molecular complexes, these assays do not depend on physical interactions and can monitor direct, transient or indirect interactions between cellular components, as well as correlated changes in the subcellular localization of the labeled entities.
  • the quantitative colocalization assays of the invention can be used to quantitatively monitor the colocalization of dystrophin and p-dystroglycan (b-DG), a component of the dystrophin-glycoprotein complex (DGC) that directly interacts with dystrophin, and the colocalization of dystrophin and a-sarcoglycan (a-SG), a components of the DGC that does not directly interact with dystrophin, in in vitro cultured myotubes and cardiomyocytes.
  • b-DG dystrophin-glycan
  • DGC dystrophin-glycoprotein complex
  • a-SG colocalization of dystrophin and a-sarcoglycan
  • dystrophin/b-DG and dystrophin/a-SG colocalization assays demonstrate the ability of the dystrophin/b-DG and dystrophin/a-SG colocalization assays to quantitatively monitor the restoration of active dystrophin in myotubes from primary and immortalized cells from DMD patients treated with exon skipping and gene therapies, to distinguish the response of DMD patients to exon skipping therapies and to quantitatively monitor the reversal of disease phenotypes in myotubes from DM1 patients treated with antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the methods of the invention are based on quantitative colocalization analysis, i.e. on analysis quantifying in situ the spatial overlap between two cellular molecules labeled with specific labelling agents.
  • at least one image of at least one in vitro cultured muscle cell is used to quantify a degree of colocalization between two molecules by performing quantitative colocalization analysis.
  • the methods typically comprise
  • the type of cells, culture conditions (e.g. in the presence or absence of a specific compound) and molecules considered for the quantitative colocalization analysis may vary depending on the method and the disease of interest.
  • the degree of colocalization of the two molecules is defined by analyzing at least one image of at least one in vitro cultured muscle cell, i.e. at least one muscle cell produced by in vitro culture techniques.
  • the muscle cell is preferably a mammalian muscle cell, in particular a human muscle cell.
  • the muscle cell is a striated muscle cell, i.e. a skeletal muscle cell or a cardiac muscle cell. More preferably, the muscle cell is a myotube or a cardiomyocyte.
  • the muscle cell is a myotube, and in particular a human myotube.
  • the muscle cell is a cardiomyocyte, and in particular a human cardiomyocyte.
  • imaged muscle cells may be healthy muscle cells or diseased muscle cells.
  • Healthy muscle cells may be obtained by culturing muscle cells derived from at least one healthy subject, i.e. a subject who does not suffer from a disease of interest, preferably who does not suffer from any muscular disease affecting said muscle cells, in particular who does not suffer from any neuromuscular disease or cardiomyopathy as defined below.
  • Diseased muscle cells are muscle cells exhibiting at least one feature of a disease of interest.
  • Diseased muscle cells may be obtained by culturing muscle cells derived from at least one patient suffering from a disease of interest, in particular suffering from a neuromuscular disease or cardiomyopathy as defined below, or by culturing muscle cells which have been chemically or genetically modified to not express a molecule known as a disease driver or to express a mutated form thereof.
  • diseased muscle cells may be myotubes modified to not express dystrophin or to express a truncated form thereof.
  • Such modifications may be obtained by any method known by the skilled person such as RNA interference using siRNAs specifically targeting the gene of interest, or genetic alteration of the gene of interest.
  • diseased muscle cells are obtained by culturing muscle cells derived from at least one patient suffering from the disease of interest.
  • healthy myotubes may be obtained by culturing myoblasts derived from at least one healthy subject, preferably a subject who does not suffer from any muscular disease and in particular who does not suffer from any neuromuscular disease as defined below.
  • a myoblast is a mononucleate cell type that, by fusion with other myoblasts, gives rise to myotubes that maturate and later eventually develop into muscle fibers.
  • Diseased myotubes may be obtained by culturing myoblasts derived from at least one patient suffering from a neuromuscular disease of interest or by culturing myoblasts which have been chemically or genetically modified to not express a molecule known as a disease driver of a neuromuscular disease of interest or to express a mutated form thereof.
  • diseased myotubes are obtained by culturing myoblasts derived from at least one patient suffering from a neuromuscular disease of interest.
  • healthy cardiomyocytes may be obtained by culturing cardiomyocytes derived from at least one healthy subject, preferably a subject who does not suffer from any muscular disease and in particular who does not suffer from any neuromuscular disease or cardiomyopathy as defined below.
  • Diseased cardiomyocytes may be obtained by culturing card io myocytes derived from at least one patient suffering from a neuromuscular disease or cardiomyopathy of interest or by culturing cardiomyocytes which have been chemically or genetically modified to not express a molecule known as a disease driver of a neuromuscular disease or cardiomyopathy of interest or to express a mutated form thereof.
  • diseased card io myocytes are obtained by culturing cardiomyocytes derived from at least one patient suffering from a neuromuscular disease or cardiomyopathy of interest.
  • the terms "subject”, “individual” and “patient” are interchangeable and preferably refer to an animal, more preferably a mammal and even more preferably a human, including adult, child, newborns and human at the prenatal stage.
  • Muscle cells used in the present invention may be obtained by culturing muscle cells derived from primary cells or from immortalized cells.
  • muscle cells used in the present invention are obtained by culturing muscle cells derived from primary cells, in particular derived from primary cells obtained from a healthy subject or from a patient suffering from a neuromuscular disease or cardiomyopathy of interest.
  • muscle cells may be obtained by culturing muscle cells derived from primary myoblasts, primary cardiomyocytes, primary stem cells, preferably non-embryonic stem cells, or induced pluripotent stem cells.
  • Methods to generate cardiomyocytes or myoblasts from induced pluripotent stem cells are known by the skilled person, see e.g. Ribeiro et al. 2015 (Proc Natl Acad Sci U S A.
  • muscle cells used in the present invention are obtained by culturing immortalized cells, in particular immortalized cells derived from a healthy subject or from a patient suffering from a neuromuscular disease or cardiomyopathy of interest.
  • immortalized cells in particular immortalized cells derived from a healthy subject or from a patient suffering from a neuromuscular disease or cardiomyopathy of interest.
  • two types of cells e.g. healthy cells and diseased cells, one type may be derived from primary cells and the other type may be derived from immortalized cells.
  • muscle cells of the same type may be obtained from a unique source, i.e. from the same subject or from a unique immortalized cell line, or may be obtained from several, at least two, sources, i.e. from several subjects and/or from several immortalized cell lines.
  • muscle cells of the same type e.g. healthy cells or diseased cells
  • muscle cells of the same type are obtained from a unique source, i.e. from the same subject or from a unique immortalized cell line.
  • muscle cells are in vitro cultured/produced on a suitable substrate and under suitable conditions known by the skilled person.
  • Methods for culturing muscle cells, and in particular for producing myotubes by culturing myoblasts or for producing cardiomyocytes are well known by the skilled person and include cultures on patterned or unpatterned substrates, on substrates with or without topological constraints, on soft substrates (e.g. synthetic hydrogels materials such as poly(hydroxyethyl methacrylate), polyacrylamide, polyethylene glycol, polyacrylic acid, poly(vinyl alcohol), polyvinylpyrrolidone, polyimide and polyurethane, natural hydrogel materials such as agarose, dextran, gelatin and matrigel, and silicone materials), or hard substrates (e.g.
  • in vitro cultured muscle cells are not organized in tissue.
  • in vitro cultured muscle cells are not comprised in an organized muscle tissue architecture comprising connective tissue such as endomysium, perimysium and epimysium, capillary vessels or adipocytes.
  • connective tissue such as endomysium, perimysium and epimysium, capillary vessels or adipocytes.
  • the image(s) provided in step a) is not the image of a tissue or biopsy sample.
  • muscle cells are cultured in constrained conditions, i.e. on a substrate allowing the production of homogeneous populations of muscle cells.
  • all muscle cells used in a method of the invention e.g. healthy, diseased or reference myotubes
  • are cultured in the same constrained conditions e.g. on a substrate with the same topological constraints or adhesive patterns.
  • a homogeneous population of muscle cells is a population of muscle cells exhibiting similar morphological parameters.
  • myotubes of a homogeneous population of myotubes may exhibit similar fusion index (ratio of nuclei within myotubes of the total number on nuclei), maturation index (number of nuclei per myotubes) and/or myotube area (or width/length ratio, width and/or length).
  • myotubes of a homogeneous population of myotubes exhibit a variation of fusion index of less than 30 %, preferably of less than 20 %, a variation of maturation index of less than 30 %, preferably of less than 20 %, and/or a variation of myotube area (or width/length ratio, width and/or length) of less than 30 %, preferably of less than 20 %.
  • myotubes of a homogeneous population of myotubes exhibit a variation of fusion index of less than 30 %, preferably of less than 20 %, a variation of maturation index of less than 30 %, preferably of less than 20 %, and a variation of myotube area (or width/length ratio, width and/or length) of less than 30 %, preferably of less than 20 %.
  • Cardiomyocytes of a homogeneous population of cardiomyocytes may exhibit similar cell area (or width/length ratio, width and/or length).
  • card io myocytes of a homogeneous population of card io myocytes exhibit a variation of cell area (or width/length ratio, width and/or length) of less than 30 %, preferably of less than 20 %.
  • Homogeneity is independently assessed for each population of muscle cells used in a method of the invention, i.e. muscle cells derived from a specific source (e.g. muscle cells derived from a healthy subject) and cultured in a specific condition (e.g. cultured in the presence of a compound to be tested).
  • a specific source e.g. muscle cells derived from a healthy subject
  • a specific condition e.g. cultured in the presence of a compound to be tested.
  • a culture in constrained conditions is a culture on a substrate with topological constraints or adhesive patterns.
  • substrates allowing the production of homogeneous population of muscle cells, and in particular the production of homogeneous population of myotubes and card io myocytes, are well known by the skilled person.
  • substrates include, but are not limited to, substrates with linear grooves formed in the surface of the substrate, e.g. by an etching technique, (Yamamoto et al., 2008, J. Histochem. Cytochem 56, 881-892; Rao et al. Biomaterials.
  • the myoblasts are cultured on a substrate containing adhesive patterns, preferably adhesive patterns as disclosed in WO 2015/091593, WO 2016/202850 and WO 2016/139312, and in particular as disclosed in Figure 2A of International patent application WO 2015/091593 and in Young et al. 2018 (Young et al. SLAS Discov. 2018 Sep;23(8):790-806).
  • Adhesive properties of patterns may be obtained by coating said patterns with one or several extracellular matrix proteins, preferably with fibronectin.
  • the myoblasts are culture on a substrate as disclosed in WO 2015/091593, in particular in Figure 2A of WO 2015/091593 or Figure 14 of the present application, i.e. a substrate containing at least one cell-adhesive pattern, wherein
  • said pattern (1) has an elongated surface comprising a central region (1C) and two lateral regions (IL) extending from said central region in both directions along a longitudinal axis of the pattern with a contour discontinuity between the central region (1C) and each lateral region (IL), the length (L) of the pattern being comprised between 100 and 1000 pm and the maximum width (Wc) of said pattern being comprised between 50 and 500 pm,
  • the pattern consists of the partial superposition of three elliptical surfaces: a first elliptical surface defining the central region of the pattern and second and third elliptical surfaces having a major axis coinciding with the major axis of the first ellipse defining the lateral regions of the pattern, and wherein the second and third elliptical surfaces intersect the first elliptical surface along their transversal axis.
  • the pattern is symmetrical according to its longitudinal axis (X) and to a transversal axis perpendicular to the longitudinal axis.
  • the ratio between the length (L) and the maximum width (Wc) of the pattern is of 2.5 and/or the area of said pattern is comprised between 5,000 and 500,000 pm 2 .
  • the method of culturing muscle cells is adaptable to high-throughput platforms and to perform high- throughput assays.
  • the muscle cells are stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first molecule of interest, i.e. the muscle cells are stained with a first labelling agent revealing the first molecule of interest and with a second labelling agent revealing the second molecule.
  • the muscle cells are also stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells, cellular structures of muscle cells, and any combination thereof.
  • ROI region of interest
  • cellular structures include, but are not limited to, nucleus, vacuole, mitochondrion, lysosome, cell membrane and cytoskeleton.
  • myotubes may be stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof.
  • Cardiomyocytes may be stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual card io myocytes, cellular structures of card io myocytes, and any combination thereof.
  • muscle cells are stained with at least one labelling agent revealing individual cells, in particular individual myotubes or cardiomyocytes.
  • the muscle cells, and in particular myotubes or cardiomyocytes may be further stained with at least one labelling agent revealing nuclei.
  • labelling agents that can be used to reveal individual muscle cell include, but are not limited to, antibodies directed against troponin-T or myosin heavy chain (MHC).
  • labelling agents that can be used to reveal nuclei include, but are not limited to, Hoechst and DAPI dyes.
  • labelling agents that can be used to reveal mitochondria include, but are not limited to, MitotrackerTM dyes.
  • ROI depends on the disease of interest and may be easily chosen by the skilled person based on his general knowledge. For diseases driven by a mutation in a gene related to the function of an organelle or inducing a change in the structure of said organelle, the skilled person may chose said organelle as ROI.
  • DM1 is known to induce RNA foci.
  • the disease of interest is DM1 and the myotubes are stained for two ROI, individual myotubes and nuclei in order to perform quantitative colocalization analysis in myotube nuclei.
  • the disease of interest is DMD and the myotubes are stained for one ROI, i.e. individual myotubes.
  • the myotubes may be stained for another ROI, i.e. nuclei, in order to perform colocalization analysis in myotube nuclei.
  • Staining may be performed during or after the culture of muscle cells.
  • the methods to stain/label cellular targets are well known by the skilled person.
  • the term "labelling agent” refers to any agent that is used to specifically detect and label a first or second molecule or a ROI. Said agent emits a signal that is visible on the captured images of stained muscle cells.
  • a labelling agent is able to specifically recognize the target (i.e. a first or second molecule or a ROI) and to emit a detectable signal, e.g. a fluorescent, luminescent, chemiluminescent or radioactive signal, preferably a fluorescent signal.
  • a labelling agent may comprise a moiety which is able to specifically recognize the target, i.e. an antibody or nucleic acid moiety, and a moiety emitting a detectable signal, i.e. a fluorochrome.
  • these moieties are covalently linked, e.g. an antibody or nucleic acid specifically recognizing the target and bearing a fluorochrome.
  • these moieties are born by two or more distinct molecules, e.g. a primary antibody or nucleic acid specifically recognizing the target and a secondary antibody or nucleic acid specifically recognizing the primary antibody or nucleic acid and emitting a detectable signal, e.g. bearing a fluorochrome.
  • the term "labelling agent" thus encompasses one or several molecules depending on the embodiment.
  • Each labelling agent emits a detectable and distinctive signal, i.e. a signal which can be distinguished from the signal of the other labelling agents used to stain the cells.
  • each labelling agent comprises a different fluorescence label having a different emission and/or excitation wavelength.
  • such labelling agent may be or comprise an antibody or nucleic acid probe conjugated to a fluorochrome (i.e. immunostaining).
  • a labelling agent may be a molecule emitting a detectable signal, e.g. a fluorescent dye, that naturally bind to the target, e.g. DAPI that naturally binds to DNA.
  • fluorescence labels can be selected such that their emission wavelengths are sufficiently separated and can be resolved by the imaging device used.
  • the imaging device used Depending on the spectral resolution of the detection device used, a person skilled in the art will be able to choose the appropriate labels that allow accurate colocalization determination. Conversely, if a particular set of labels is to be used, a person skilled in the art will be able to select the appropriate imaging device such that the labels can be separated and determined.
  • Labelling agents used to stain muscle cells may be easily chosen by the skilled person depending on the nature of molecules to be labelled.
  • the labelling agent may be an antibody directed against this protein, in its wildtype or mutated form, and comprising a moiety emitting a detectable signal, preferably a fluorescent signal, or may be a primary antibody directed against this protein, in its wild-type or mutated form, and a secondary antibody recognizing the primary antibody and emitting a detectable signal, preferably a fluorescent signal.
  • the labelling agent may be a nucleic acid probe which specifically hybridizes with this nucleic acid, in its wild-type or mutated form, and comprising a moiety emitting a detectable signal, preferably a fluorescent signal, or may be a first nucleic acid probe which specifically hybridizes with this nucleic acid, in its wild-type or mutated form, and a second nucleic acid probe which specifically hybridizes with the first nucleic acid probe and emits a detectable signal, preferably a fluorescent signal.
  • the molecules to be considered for the colocalization analysis depend on the disease of interest. Substantial advances in our molecular understanding of muscle function identified protein complexes and interactions between proteins or between proteins and nucleic acids that are critical for muscle function and are involved in the cause of muscular diseases, in particular in the cause of neuromuscular diseases or cardiomyopathies.
  • the first and second molecules are preferably selected from a group of molecules related to a cellular molecular complex or a cellular function affected by the disease of interest.
  • the first or the second molecule is a protein or a nucleic acid identified as a disease driver of the disease of interest, i.e. a known genetic or physiological cause of the disease, or is a protein or a nucleic acid known to be altered, in particular in its expression and/or activity, in the disease of interest.
  • the second molecule is a molecule interacting, i.e. known to directly or indirectly interact, with the first molecule in healthy or diseased muscle cells.
  • the first and second molecules are known to interact in healthy muscle cells and to have no, few or impaired interaction in diseased muscle cells, or vice-versa.
  • a direct interaction involves a physical contact between the two molecules.
  • dystrophin directly interacts with P-dystroglycan (b-DG).
  • An indirect interaction involves intermediate molecule(s) between the first and the second molecules.
  • the first and second molecules may belong to the same protein complex but without direct physical contact.
  • dystrophin and a- sarcoglycan (a-SG) indirectly interact; they are components of the dystrophin-glycoprotein complex (DGC) but do not directly interact together.
  • DGC dystrophin-glycoprotein complex
  • first and second molecules that can be selected depending on the disease of interest include, but are not limited to, molecules provided in Tables 1 and 2.
  • the first molecule may be chosen from the molecule(s) in the column "Examples of molecules useful as first molecules” and the second molecule may be chosen from the molecule(s) in the column "Examples of molecules useful as second molecules” or the first molecule may be chosen from the molecule(s) in the column “Examples of molecules useful as second molecules” and the second molecule may be chosen from the molecule(s) in the column "Examples of molecules useful as first molecules”.
  • the first molecule is chosen from the molecule(s) in the column "Examples of molecules useful as first molecules" and the second molecule is chosen from the molecule(s) in the column "Examples of molecules useful as second molecules”.
  • These tables also comprise examples of suitable ROI(s) depending on the disease and the molecules used for the colocalization analysis.
  • said at least one ROI comprises a ROI of Table 1 or 2 corresponding to the first and second molecules and the disease.
  • the first molecule may be Dystrophin (DMD) or Utrophin (UTRN)
  • the second molecule may be selected from the group consisting of a-syntrophin, b-syntrophin, Ankyrin (ANK1, ANK2), a-dystroglycan (DAG1), b-dystroglycan (DAG1), sarcospan (SSPN), a-sarcoglycan (SGCA), b-sarcoglycan (SGCB), d-sarcoglycan (SGCD), g-sarcoglycan (SGCG), dystrobrevin (DTNA) and Filamin C (FLNC), and at least one ROI may be the myotube.
  • DMD Dystrophin
  • UTRN Utrophin
  • the second molecule may be selected from the group consisting of a-syntrophin, b-syntrophin, Ankyrin (ANK1, ANK2), a-dy
  • Table 1 Examples of molecules useful as first and second molecules in the methods of the invention in embodiments wherein the muscular disease is a neuromuscular disease (*)
  • LGMD Limb-girdle muscular dystrophy.
  • LGMD types Straub V, Murphy A, Udd B; LGMD workshop study group. 229th ENMC international workshop: Limb girdle muscular dystrophies - Nomenclature and reformed classification Naarden, the Netherlands, 17-19 March 2017. Neuromuscul Disord. 2018 Aug;28(8):702-710.
  • DMD Dystrophin
  • Utrophin Utrophin
  • SNTA a-syntrophin
  • SNTB Ankyrin
  • DAG1 a-dystroglycan
  • DAG1 b-dystroglycan
  • SSPN sarcospan
  • SGCA a- sarcoglycan
  • SGCB b-sarcoglycan
  • DTNA dystrobrevin
  • Filamin C FLNC
  • Collagen VI alphal COL6A1
  • Collagen VI alpha2 Collagen VI alpha2
  • COL6A3 Collagen VI alpha3
  • LAMA2 Laminin alpha2
  • IGA7 integrin a7
  • DAG1 Desmin (DES), Fukutin (FKTN), Fukutin-related protein (FKRP), Protein O-mannosyl-transferase 1/2 (POMT1/2), Protein O-linked mannose betal,2-N-acet
  • Table 2 Examples of molecules useful as first and second molecules in the methods of the invention in embodiments wherein the muscular disease is a cardiomyopathy (*)
  • RCM Restrictive cardiomyopathy
  • ARVC Arrhythmogenic cardiomyopathy
  • Actin Alpha Cardiac Muscle (ACTC1), Myosin Heavy Chain 7 (MYH7), Myosin Heavy Chain 6 (MYH6), Myosin binding protein C3 (MYBPC3), Troponin T2 cardiac type (TNNT2), Troponin 13 cardiac type (TNNI3), troponin Cl slow skeletal and cardiac type (TNNC1), tropomyosin 1 (TPM1), Myosin Light Chain 2 (MYL2), Myosin Light Chain 3 (MYL3), Titin (TTN), Titin-cap or telethonin (TCAP), Cysteine and glycine rich protein 3 (CSRP3), a-Actinin-2 (ACTN2), Myopalladin (MYPN), Ankyrin Repeat Domain-Containing Protein 1 (ANKRD1), Myozenin 2 (MYOZ2), muscle-RING-finger-1/2 (MURF1/2), Desmin (DES), LIM Domain Binding 3 (ACTC1), My
  • DCM Dilated cardiomyopathy
  • HCM Hypertrophic cardiomyopathy
  • RCM Restrictive cardiomyopathy
  • ARCV Arrhythmogenic cardiomyopathy
  • DCM dilated cardiomyopathy
  • ACTC1 Actin Alpha Cardiac Muscle
  • the first molecule may be ACTC1 and the second molecule may be selected from the group consisting of MYH7, MYH6, MYBPC3, TNNT2, TNNI3, TNNC1, TPM1, MYL2, MYL3, TTN, CSRP3, ACTN2.
  • MYPN, ANKRD1, MYOZ2 and MURF1/2 or vice versa.
  • the muscular disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy type 1 (DM1), LGMD R3, LGMD R4 ,LGMD R5, LGMD R6, Congenital muscular dystrophy (CMD), Muscle-eye-brain disease (MEB) and LGMD R16, and the first and second molecules are independently selected from the group consisting of proteins DMD, SNTA, SNTB, ANK1, ANK2, DAG1, SSPN, SGCA, SGCB, SGCD, SGCG, DTNA and FLNC.
  • DMD Duchenne Muscular Dystrophy
  • BMD Becker Muscular Dystrophy
  • DM1 Myotonic Dystrophy type 1
  • LGMD R3, LGMD R4 LGMD R5, LGMD R6, Congenital muscular dystrophy (CMD), Muscle-eye-brain disease (MEB) and LGMD R16
  • the first and second molecules are
  • the muscular disease is selected from the group consisting of LGMD D5, LGMD R22 and Ullrich myopathy (UM), and the first and second molecules are independently selected from the group consisting of proteins COL6A1, COL6A2, COL6A3, DAG1, LAMA2, ITGA7, DES, FKTN, FKPR, POMT1/2, POMGNT1 and LARGE1.
  • the muscular disease is selected from the group consisting of Walker-Warburg syndrome (WWS), LGMD R23, Congenital muscular dystrophy (CMD) and Myofibrillar myopathy (MFMP), and the first and second molecules are independently selected from the group consisting of proteins DAG1, POMT1/2, POMGNT1, LARGE1, FKTN, FKRP, LAMA2, ITGA7 and DES.
  • WWS Walker-Warburg syndrome
  • CMD Congenital muscular dystrophy
  • MFMP Myofibrillar myopathy
  • the muscular disease is selected from the group consisting of Centronuclear myopathy (CNM), Distal myopathy (DM), Inclusion body myopathy (IBM), LGMD RIO, Myofibrillar myopathy (MFMP), Nemaline myopathy (NEM2, NEM3), Protein aggregate myopathy (PAM), Rimmed vacuole myopathy (RVM) and Tibial muscular dystrophy (TMD), and the first and second molecules are independently selected from the group consisting of proteins TTN, MYH2, DES, ACTA1, NEB, MYH7, MYOT, TNNT3 and MURF1.
  • CCM Centronuclear myopathy
  • DM Distal myopathy
  • IBM Inclusion body myopathy
  • LGMD RIO Myofibrillar myopathy
  • MFMP Myofibrillar myopathy
  • NEM2 Nemaline myopathy
  • PAM Protein aggregate myopathy
  • RVM Rimmed vacuole myopathy
  • TMD Tibial muscular dystrophy
  • the muscular disease is selected from the group consisting of Congenital muscular dystrophy (CMD) and Emery-Dreifuss muscular dystrophy (EDMD), and the first and second molecules are independently selected from the group consisting of proteins LMNA, EMD, SYNE1/2, TNPO3 and BAF1.
  • the muscular disease is selected from the group consisting of Centronuclear myopathy (CNM) Congenital myopathy (CM) LGMD Rl, Malignant hyperthermia syndrome (MHS), Tubular aggregate myopathy (TAM), X-linked myotubular myopathy (XLMTM), Becker Muscular Dystrophy (BMD), Facioscapulohumeral Muscular Dystrophy (FSHD), Myotonic Dystrophy type 1 (DM1), and the first and second molecules are independently selected from the group consisting of proteins BINI, DNM2, MTM1, SPEG, RYR1, STAC3, CACNA1S, CACNB1, CAPN3, CASQ1, STIM1, ORAI1, TRDN, SERCA1 and CLCN1.
  • CCM Centronuclear myopathy
  • MHS Malignant hyperthermia syndrome
  • TAM Tubular aggregate myopathy
  • XLMTM X-linked myotubular myopathy
  • BMD Becker Muscular Dystrophy
  • FSHD Facioscapulohumer
  • the muscular disease is selected from the group consisting of LGMD R2, LGMD R12, Centronuclear myopathy (CNM), Miyoshi myopathy (MM), and the first and second molecules are independently selected from the group consisting of proteins DYSF, ANO5, BINI, TRIM72, EHD1, EHD2, ANXA1, ANXA2 and ANXA5.
  • the muscular disease is selected from the group consisting of LGMD DI, Marinesco-Sjbgren syndrome (MSS) and Myofibrillar myopathy (MFMP), and the first and second molecules are independently selected from the group consisting of proteins DNAJB6, SIL1, BAG3 and HSPB5.
  • the muscular disease is selected from the group consisting of LGMD R8, Nemaline Myopathy 8 (NEM8), Danon disease (DD), Myopathy, distal, with rimmed vacuoles (DMRV), Neurodegeneration with ataxia, dystonia, and gaze palsy, childhood-onset (NADGP), Pompe disease, Protein aggregate myopathy (PAM), Vici syndrome (VS) and X-linked myopathy with excessive autophagy (xMEA), and the first and second molecules are independently selected from the group consisting of proteins TRIM32, KLH40, KLH41, LAMP1, LAMP2, SQSTM1, GAA, MURF1, EPG5 and VMA21.
  • LGMD R8 Nemaline Myopathy 8
  • DD Danon disease
  • DMRV distal
  • DMRV rimmed vacuoles
  • PAM Protein aggregate myopathy
  • VS Vici syndrome
  • xMEA X-linked myopathy with excessive autophagy
  • the first and second molecules are independently selected from the group consisting of
  • the first and second molecules are independently selected from the group consisting of proteins belonging to the Dystrophin Glycoprotein complex (DGC) and dysferlin, preferably selected from the group consisting of dystrophin, a-sarcoglycan, -dystroglycan, a-dystroglycan and dysferlin, more preferably selected from the group consisting of dystrophin, a-sarcoglycan and -dystroglycan.
  • DGC Dystrophin Glycoprotein complex
  • the first molecule may be dystrophin and the second molecule may be selected from the group consisting of proteins belonging to the Dystrophin Glycoprotein complex (DGC) and dysferlin, preferably selected from the group consisting of a-sarcoglycan,
  • DGC Dystrophin Glycoprotein complex
  • the second molecule may be dystrophin and the first molecule may be selected from the group consisting of proteins belonging to the Dystrophin Glycoprotein complex (DGC) and dysferlin, preferably selected from the group consisting of a-sarcoglycan, p-dystroglycan, a-dystroglycan and dysferlin, more preferably selected from the group consisting of a-sarcoglycan and p-dystroglycan.
  • DGC Dystrophin Glycoprotein complex
  • the first molecule is DMPK RNA and the second molecule is a RNA binding protein trapped by CTG repeats in the DMPK gene, preferably is MBNL1 protein.
  • the second molecule may be DMPK RNA and the first molecule may be a RNA binding protein trapped by CTG repeats in the DMPK gene, preferably MBNL1 protein.
  • the skilled person can easily select a combination of a first molecule and a second molecule that can be used in the methods of the invention.
  • the skilled person can use a method comprising
  • Healthy and diseased cells are cultured in the same conditions to provide comparable degrees of colocalization.
  • the number of healthy and diseased cells is chosen in order to provide statistically significant results.
  • This step may be performed using any device suitable to capture microscopic images.
  • the microscopic images may for example be taken by bright-field imaging, dark-field imaging, cross-polarized light imaging, phase-contrast imaging, fluorescence imaging, confocal imaging and/or super-resolution imaging.
  • the imaging technique is chosen in order to provide images with a resolution in the range of 1pm to lOnm, preferably in the range of 800nm to lOOnm, more preferably in the range of 600nm to 200nm.
  • the choice of the imaging technique also depends on the nature of the signals emitting by the labelling agents.
  • the labelling agents emit fluorescence signals and the microscopic images are taken by fluorescence imaging and by acquiring each channels corresponding the labelling agents used to reveal the first molecule, the second molecule and optionally at least one ROL
  • an illumination function may be applied on the acquired images to correct uneven illumination.
  • each channel may be also referred to as a detection channel.
  • each channel corresponds to one label having a particular emission or excitation wavelength.
  • different channels are acquired as different images. Colocalization of two different la be Is/signa Is is also referred to as colocalization of two channels.
  • the imaged muscle cells have been stained for a first molecule of interest, for a second molecule interacting with said first molecule of interest, and with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual cells, a cellular structure, and any combination thereof.
  • ROI region of interest
  • an image segmentation is carried out with an algorithm on appropriate staining channel(s) in order to identify ROI(s).
  • Each image is thus segmented and may comprise one or a plurality of ROIs.
  • Appropriate staining channel(s) is(are) the channel(s) corresponding to the labelling agent(s) used to reveal ROI(s).
  • segmentation of individual myotubes and nuclei may be done using the channel of the labelling agent revealing Troponin T or Myosin heavy chain and the channel of Hoechst dye, respectively.
  • the threshold of segmentation is set-up in order to avoid detecting the background noise and eliminate aberrant small cellular structures, in particular aberrant small myotube or cardiomyocyte structures.
  • Segmentation of ROI(s) may be done by any method well known by the skilled person, for example using the open-source software Cell Profiler (Carpenter et al., 2006, Genome Biology, 7(10)) or appropriate software applications such as Matlab or Fiji or programming languages such as Python, Java, C++.
  • the degree of colocalization of the first molecule of interest and the second molecule in said muscle cell(s), or in said ROI(s), is determined by performing quantitative colocalization analysis.
  • each muscle cell or each ROI is individually analyzed.
  • the number of muscles cells and ROIs to be considered in the determination of the degree of colocalization can be easily adjusted by the skilled person in order to provide statistically significant results.
  • the degree of colocalization is preferably determined in at least 30 muscle cells, more preferably in at least 60 muscle cells.
  • the degree of colocalization is determined in at least 30 ROIs (preferably at least one ROI per muscle cell), and preferably in at least 60 ROIs (preferably at least one or two ROIs per muscle cell).
  • the quantitative colocalization analysis may be performed by using any method known by the skilled person such as quantitative pixel-based colocalization analysis or machine learning based analysis.
  • the methods of the invention do not comprise any step using a machine learning based analysis.
  • the quantitative colocalization analysis is performed using a quantitative pixel-based colocalization analysis.
  • colocalization refers to the presence of a signal from the first molecule label and a signal from the second molecule at the same pixel location.
  • the proper assessment of colocalization thus requires background correction, typically by thresholding.
  • Quantitative colocalization analysis using thresholding quantifies the colocalized fraction of each molecular species, but also requires a threshold value for each signal/channel, which is then used as a cutoff between specific staining versus nonspecific.
  • the threshold value is an intensity threshold above which it is considered that the labelled molecule is present at an image position (pixel).
  • Colocalized areas may thus be defined by regions where the signal from the first molecule is above a threshold Ti and the signal from the second molecule is above a threshold T2.
  • the determination of threshold values is assisted/done by computer software.
  • a threshold is determined on a control sample wherein the signal of interest is absent, e.g.
  • the threshold for the signal of the first molecule may be determined on an image of a muscle cell which is not stained for said molecule.
  • the thresholds may be determined on an image of a muscle cell which has been contacted with the secondary antibodies but not with the primary antibodies.
  • the threshold may also be determined by quantile statistics or by any other known method.
  • one or more filter steps may be applied on the image(s).
  • the respective threshold values of each labeled molecules may be subtracted from their corresponding images. The pixels values under zero may be clipped to zero.
  • the degree of colocalization of the first and the second molecules may be quantitatively determined by calculating a metric of colocalization between the two detection channels, i.e. the channel corresponding to the signal of the first molecule and the channel corresponding to the signal of the second molecule. This can be done by various approaches well known by the skilled person.
  • the degree of colocalization may be quantitatively determined by calculating a value representing an overlap coefficient of the two detection channels.
  • the degree of colocalization is quantitatively determined by calculating an overlap coefficient for each cell or ROI, and optionally applying a mathematical function on said coefficient(s).
  • the overlap coefficient may be selected from the group consisting of the Pearson's Colocalization Coefficient (PCC), the Manders' Colocalization Coefficient (MCC), the Rank-based intensity Weighting Coefficient (RWC), the Manders' Overlap Coefficient (MOC) and any combinations thereof.
  • the overlap coefficient is selected from the group consisting of the Pearson's Colocalization Coefficient (PCC), the Manders' Colocalization Coefficient (MCC), the Rank-based intensity Weighting Coefficient (RWC), and any combinations thereof.
  • Tools for quantifying PCC, MCC, and RWC are provided in nearly all image analysis software packages.
  • determining the degree of colocalization comprises calculating the Pearson's Colocalization Coefficient (PCC) for each muscle cell or ROI, preferably each muscle cell.
  • PCC Pearson's Colocalization Coefficient
  • PCC measures the pixel-by-pixel covariance in the signal levels of two images/channels. Typically, PCC values range from 1 for two images/channels whose signal intensities are perfectly, linearly related, to -1 for two images/channels whose signal intensities are perfectly, but inversely, related to one another. Values near zero reflect distributions of signals that are uncorrelated with one another.
  • the formula to calculate PCC exemplified with a first molecule IM1 (imaging marker 1) and a second molecule IM2 (imaging marker 2) colocalization case, is exemplified in Figure 4.E.I.
  • the PCC may be calculated on all the cell, all the ROI(s), or in the above threshold area.
  • the PCC may be set to 0.
  • determining the degree of colocalization comprises calculating the Manders' Colocalization Coefficient (MCC) for each muscle cell or ROI, preferably each muscle cell.
  • MCC is independent of pixel intensities correlation. Indeed, the MCC is a measure of co-occurrence. It measures the proportion of one molecule-related signal that overlaps with the other molecule signal.
  • the formula to calculate MCC exemplified with a first molecule IM1 (imaging marker 1) and a second molecule IM2 (imaging marker 2) colocalization case, is exemplified in Figure 4.E.2.
  • determining the degree of colocalization comprises calculating the Rank-based intensity Weighting Coefficient (RWC) for each muscle cell or ROI, preferably each muscle cell.
  • RWC Rank-based intensity Weighting Coefficient
  • the algorithm uses a non-parametric ranking of pixel intensities in each channel, and the difference in ranks of co-localizing pixel positions in the two channels is used to weight the pixel intensities in the numerator (see Figure 4.E.3). The closer the pixel rank of the two intensities, the higher the weight for the intensity in this pixel. This weighting is applied to co-occurring pixels thereby combining both co-occurrence and correlation.
  • the formula to calculate RWC exemplified with a first molecule IM1 (imaging marker 1) and a second molecule IM2 (imaging marker 2) colocalization case, is exemplified in Figure 4.E.3.
  • the degree of colocalization is quantitatively determined by calculating an overlap coefficient as defined above for each muscle cell or ROI, preferably each muscle cell, and applying a mathematical function on said coefficient(s).
  • the degree of colocalization may be quantitatively determined by calculating an overlap coefficient selected from the group consisting of the Pearson's Colocalization Coefficient (PCC), the Manders' Colocalization Coefficient (MCC), the Rank-based intensity Weighting Coefficient (RWC), and any combinations thereof, for each muscle cell or ROI, preferably each muscle cell, and applying a mathematical function on said coefficient(s).
  • the mathematical function may be chosen to calculate any significant value derived from the overlap coefficients obtained from individual cells or ROIs, such as a mean value (e.g.
  • a quantile value e.g. a median value
  • a ratio e.g. ratio between colocalization value in first ROI, e.g. myotubes
  • a second ROI e.g. nuclei
  • mean over max coefficient value e.g. mean over max PCC.
  • the mathematical function is chosen in order to reflect the degree of colocalization and to preserve the ability to compare different assays. The skilled person can easily choose such function.
  • the quantitative colocalization analysis may further comprise defining a threshold for high PCC values, i.e. a threshold above which the PCC is considered to reflect a strong correlation of the two signals in the area under consideration.
  • a threshold for high PCC values i.e. a threshold above which the PCC is considered to reflect a strong correlation of the two signals in the area under consideration.
  • the PCC and negative control PCC values of each muscle cell may be plotted.
  • a satisfying threshold may be, for example, one for which 99% of the PCC values. In this case, only outliers situated in the 99th percentile exceed this threshold value.
  • the High PCC% is the percentage of muscle cells having a PCC value above this threshold among the total number of muscle cells: 100
  • the method may further comprises repeating steps (a) and (b) while replacing i) said first molecule of interest with a negative control molecule that is known to not colocalize with said second molecule, and/or ii) said second molecule with a negative control molecule that is known to not colocalize with said first molecule.
  • the colocalization readouts only consider the areas wherein the signal of the first or second molecule is above the signal obtained with the negative control.
  • the negative control molecule may be a molecule located all over the muscle cell in a homogeneous manner.
  • the negative control molecule may be the myosin heavy chain (MHC).
  • the method to determine the degree of colocalization may be easily chosen depending on the couple first/second molecules and/or the muscle cell and/or the disease of interest.
  • the first and second molecules are proteins and the step of determining the degree of colocalization comprises calculating the Pearson's Colocalization Coefficient (PCC) and optionally defining a threshold for high PCC values.
  • PCC Pearson's Colocalization Coefficient
  • at least one of said first and second molecules is a nucleic acid and the step of determining the degree of colocalization comprises calculating the Manders' Colocalization Coefficient (MCC).
  • the present invention thus relates to an in vitro method of assessing the functionality of a cellular molecule of interest in a muscle cell.
  • the method comprises
  • said at least one muscle cell has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells and cellular structures of muscle cells, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the method comprises
  • Steps (a), (b), (i), (ii) and (iii) are detailed above in the section "Quantitative colocalization analysis”. All embodiments described above for these steps are also encompassed in this aspect.
  • the muscle cells used in this method may be healthy or diseased cells.
  • the method comprises
  • the method may comprise
  • said at least one in vitro cultured myotube is at least one human healthy or diseased myotube, in particular at least one myotube exhibiting a feature of a neuromuscular disease as defined below.
  • the method comprises
  • the method may comprise
  • said at least one in vitro cultured cardiomyocyte is at least one human healthy or diseased card io myocyte, in particular at least one cardiomyocyte exhibiting a feature of a neuromuscular disease or cardiomyopathy as defined below.
  • the degree of colocalization correlates with the functionality of the first cellular molecule, preferably protein or nucleic acid, of interest in the muscle cell(s) of interest.
  • the degree of colocalization may positively or negatively correlate with the functionality of the molecule of interest.
  • a positive correlation is a relationship between two variables that move in tandem, i.e. in the same direction.
  • a positive correlation exists when one variable decreases as the other variable decreases, or one variable increases while the other increase.
  • a negative correlation is a relationship between two variables in which one variable increases as the other decreases, and vice versa.
  • the degree of colocalization positively correlates with the functionality of the first cellular molecule of interest in the muscle cell(s) of interest. In this case, the higher the degree, the more functional the molecule is. In some other embodiments, the degree of colocalization negatively correlates with the functionality of the first cellular molecule of interest in the muscle cell(s) of interest. In this case, the lower the degree, the more functional the molecule is.
  • the skilled person knows if the functionality of the molecule of interest will positively or negatively correlate with the degree of colocalization. Indeed, if the two molecules are known to interact in healthy cells, the functionality of the molecule of interest will positively correlate with the degree of colocalization. For example, in embodiments wherein the first molecule is dystrophin and the second molecule is a protein of the dystrophin associated protein complex (DGC), the functionality of dystrophin positively correlates with the degree of colocalization because these proteins interact in healthy cells. On the other hand, if the two molecules are known to have no or few interaction in healthy cells but are known to interact in diseased muscle cells, the functionality of the molecule of interest will negatively correlate with the degree of colocalization.
  • DGC dystrophin associated protein complex
  • the first molecule is DMPK RNA and the second molecule is a RNA binding protein trapped by CTG repeats in the DMPK gene such as MBNL1 protein, or vice-versa
  • the functionality of molecule of interest, DMPK RNA or MBNL1 protein negatively correlates with the degree of colocalization because these molecules have no or few interaction in healthy cells by comparison to diseased cells.
  • a functionality of a nucleic acid may be its capacity of providing a functional encoded protein in a sufficient amount.
  • a functionality of DMPK RNA may be the capacity of providing a functional encoded DMPK protein in a sufficient amount.
  • mutant mRNA transcripts containing CUG expansions are retained in the nucleus and aggregate as nuclear foci negatively impacting the capacity of DMPK RNA to produce sufficient amount of DMPK protein.
  • These expansions form a stem loop that is recognized by RNA splicing factors, including MBNL1, that may be used as the second molecule.
  • a functionality of a protein may be its activity or one of its activities in muscle cells, in particular in relation with the interaction with the second molecule.
  • a functionality of dystrophin may be its activity as an essential component of the Dystrophin associated protein complex (DGC).
  • DGC Dystrophin associated protein complex
  • the inventors also demonstrated that the quantitative colocalization assays of the invention can be used to quantitatively monitor the effect of a compound on the functionality of the molecule of interest.
  • the present invention relates to an in vitro method of assessing potency of a compound to modulate the functionality of a molecule of interest in a muscle cell.
  • the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured muscle cell that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that said compound is able to modulate the functionality of the first cellular molecule of interest in said at least one muscle cell.
  • said at least one muscle cell has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells and cellular structures of muscle cells, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the method comprises
  • the muscle cells used in this method may be healthy or diseased cells.
  • the muscle cells used in the assay and as reference muscle cell are preferably healthy muscle cells.
  • the muscle cells used in the assay and as reference muscle cell are preferably diseased muscle cells, in particular muscle cells exhibiting a feature of a neuromuscular disease or cardiomyopathy of interest.
  • the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured myotube that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that said compound is able to modulate the functionality of the first cellular molecule of interest in said at least one myotube.
  • the method may comprises
  • the myotubes used in this method may be healthy or diseased myotubes, in particular myotubes exhibiting a feature of a neuromuscular disease of interest.
  • the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, said at least one reference cardiomyocyte being at least one in vitro cultured cardiomyocyte that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that said compound is able to modulate the functionality of the first cellular molecule of interest in said at least one card io myocyte.
  • the method may comprises
  • the cardiomyocytes used in this method may be healthy or diseased cardiomyocytes, in particular cardiomyocytes exhibiting a feature of a cardiomyopathy of interest.
  • the compound to be tested may be of any nature, e.g. a nucleic acid, a protein, a small molecule (i.e. an organic or inorganic compound, usually less than 1000 daltons), a lipid, a carbohydrate or a combination thereof.
  • this compound may be a drug authorized by a regulatory authority such as FDA or EMA.
  • the muscle cells may have been contacted with the compound to be tested before, during or after the culture of said cells, preferably during the culture.
  • the muscle cells Preferably, the muscle cells have been contacted with the compound to be tested prior to be stained with the labelling agents (for the first and second molecules and, optionally for the ROI(s)).
  • the reference degree of colocalization is obtained by performing steps (a) and (b) or (i) to (iii) on at least one reference muscle cell.
  • the reference muscle cells are cultured in the same conditions (same culture medium/substrate, same incubation parameters etc.) than the muscle cells contacted with the compound to be tested, i.e. the conditions differ only in the concentration or presence/absence of the compound to be tested.
  • the reference muscle cells may be healthy muscle cells or diseased muscle cells.
  • the muscle cells used in the assay and as reference muscle cells are of the same type, in particular are myotubes or cardiomyocytes, preferably of the same status, i.e. healthy or diseased cells, and more preferably are obtained from the same source, e.g. from the same donor.
  • the reference muscle cells can be easily chosen by the skilled person based on the nature of the muscle cells, in particular myotubes or card io myocytes, and the compound to be tested.
  • the method may further comprise determining a reference degree of colocalization.
  • the method may further comprise
  • the method may further comprise
  • the ROI(s) used to determine the reference degree of colocalization is(are) the same than the ROI(s) used in the assay, e.g. individual muscle cells and nuclei.
  • the degree of colocalization and the reference degree of colocalization are determined by operating the same calculations, preferably by calculating the same overlap coefficient, e.g. PCC, for each cell or ROI, and optionally applying the same mathematical function on said coefficient.
  • the same overlap coefficient e.g. PCC
  • a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that the compound is able to modulate the functionality of the first cellular molecule of interest in the muscle cells used in the method.
  • No difference or a statistically non-significant difference between the degree of colocalization and the reference degree of colocalization indicates that said compound is not able to modulate the functionality of the first cellular molecule of interest in the muscle cells used in the method.
  • the compound may be able to positively or negatively alter the functionality of the first cellular molecule of interest.
  • the statistical significance of the difference may be assessed by any method known by the skilled person.
  • statistical significance of the difference may be assessed by carrying out a statistical test in order to determine a p-value between the degree of colocalization and the reference degree of colocalization.
  • a p-value below 0.05 indicates that the difference is significant.
  • the method of the invention of assessing potency of a compound may be performed several times with different concentrations of the compound to be tested, in particular to evaluate the dose-response effect of the compound to be tested.
  • the inventors also demonstrated that the quantitative colocalization assays of the invention can be used to quantitatively monitor the restoration of a cellular function, in particular of active dystrophin, in diseased muscle cells and in particular in myotubes from DMD patients treated with exon skipping therapies and from DM1 patients treated with antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the present invention also relates to an in vitro method of predicting the ability of a compound to treat a muscular disease of interest.
  • the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured diseased muscle cell that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a positive correlation between the concentration of the compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of said muscular disease.
  • said at least one diseased muscle cell has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells and cellular structures of muscle cells, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the method comprises (i) providing at least one image comprising at least one in vitro cultured diseased muscle cell, wherein said at least one muscle cell has been contacted with a compound to be tested, has been stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first cellular molecule of interest of interest, and has been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual myotubes, a structure of myotubes, and any combination thereof; and
  • ROI region of interest
  • Steps (a), (b), (i), (ii) and (iii) are detailed above in the section "Quantitative colocalization analysis”. All embodiments described above for these steps are also encompassed in this aspect. All embodiments described above for the method of assessing the functionality of a cellular molecule of interest or for the method of assessing potency of a compound to modulate the functionality of a cellular molecule of interest are also encompassed in this aspect.
  • the muscle cells used in this method are diseased muscle cells, i.e. muscle cells exhibiting a feature of a muscular disease.
  • muscle disease refers to a neuromuscular disease or a cardiomyopathy.
  • neuromuscular disorder and “neuromuscular disease” are used interchangeably and cover disorders that impair the functioning of the muscles, either directly, being pathologies of the voluntary muscle, or indirectly, being pathologies of nerves, neuromuscular junctions, or of the extracellular matrix.
  • muscular dystrophies such as selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy 1 (DM1), Myotonic Dystrophy 2 (DM2), Facioscapulohumeral Muscular Dystrophy (FSHD), Emery-Dreifuss muscular dystrophy, Limb-girdle muscular dystrophies, Walker-Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy such as Merosin-deficient congenital muscular dystrophy, Scapuloperoneal muscular dystrophy, Tibial muscular dystrophy and Autosomal Recessive Muscular Dystrophy; myopathies such as Ullrich myopathy, Myofibrillar myopathy, Distal myopathy, Rimmed vacuole myopathy, Myopathy, distal, with rimmed vacuoles (DMRV), Centronuclear
  • the neuromuscular disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy 1 (DM1), Myotonic Dystrophy 2 (DM2), Facioscapulohumeral Muscular Dystrophy (FSHD), Emery-Dreifuss muscular dystrophy, Limb-girdle muscular dystrophies (LGMD) (preferably LGMD Rl, R2, R3, R4, R5, R6, R8, RIO, R12, R16, R22, R23, DI and D5) Walker- Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy, Tibial muscular dystrophy, Ullrich myopathy, Myofibrillar myopathy, Distal myopathy, Rimmed vacuole myopathy, Myopathy, distal, with rimmed vacuoles (DMRV), Centronuclear myopathy (CNM), X-linked myotubular myopathy (XLM
  • the neuromuscular disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy 1 (DM1), Myotonic Dystrophy 2 (DM2), Facioscapulohumeral Muscular Dystrophy (FSHD), Emery- Dreifuss muscular dystrophy, Limb-girdle muscular dystrophies (LGMD) (preferably LGMD Rl, R2, R3, R4, R5, R6, R8, RIO, R12, R16, R22, R23, DI and D5) Walker-Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy, Tibial muscular dystrophy, Ullrich myopathy, Myofibrillar myopathy, Distal myopathy, Rimmed vacuole myopathy, Myopathy, distal, with rimmed vacuoles (DMRV), Centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM),
  • the neuromuscular disease of interest is selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes, motor neuron diseases and metabolic muscle disorders.
  • the neuromuscular disease of interest is selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes and motor neuron diseases.
  • the neuromuscular disease of interest is a muscular dystrophy selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy 1 (DM1), Myotonic Dystrophy 2 (DM2), Facioscapulohumeral Muscular Dystrophy (FSHD), Emery-Dreifuss muscular dystrophy, Limbgirdle muscular dystrophies, Walker-Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy, Scapuloperoneal muscular dystrophy, Tibial muscular dystrophy and Autosomal Recessive Muscular Dystrophy, preferably selected from the group consisting of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy 1 (DM1), Myotonic Dystrophy 2 (DM2), Facioscapulohumeral Muscular Dystrophy (FSHD), Em
  • DMD Du
  • the neuromuscular disease of interest is a myopathy selected from the group consisting of Ullrich myopathy, Myofibrillar myopathy, Distal myopathy, Rimmed vacuole myopathy, Centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), Tubular aggregate myopathy, Malignant hyperthermia syndrome, Inclusion body myopathy, Myofibrillar myopathy, Protein aggregate myopathy, Nemaline myopathy, Congenital myopathy (CM), Myoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjbgren syndrome, Neurodegeneration with ataxia, dystonia, and gaze palsy, childhood-onset (NADGP), Pompe disease and Primary mitochondrial myopathies, preferably selected from the group consisting of Ullrich myopathy, Myofibrillar myopathy, Distal myopathy, Rimmed vacuole myopathy, Centronuclear myopathy (CNM), X-linked myo
  • the neuromuscular disease of interest is a congenital myasthenic syndrome selected from the group consisting of Myasthenia gravis and other myasthenic syndromes driven by mutations in CHAT, COLQ, RAPSN, CHRNE, DOK7 and/or GFPT1 genes.
  • the neuromuscular disease of interest is a motor neuron disease selected from the group consisting of Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS) and Kennedy's disease.
  • SMA Spinal Muscular Atrophy
  • ALS Amyotrophic Lateral Sclerosis
  • Kennedy's disease a motor neuron disease selected from the group consisting of Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS) and Kennedy's disease.
  • the neuromuscular disease of interest is a metabolic muscle disorder selected from the group consisting of cachexia, sarcopenia and muscle atrophy.
  • the neuromuscular disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD) and Myotonic Dystrophy 1 (DM1).
  • DMD Duchenne Muscular Dystrophy
  • DM1 Myotonic Dystrophy 1
  • DCM dilated cardiomyopathy
  • HCM hypertrophic cardiomyopathy
  • RCM restrictive cardiomyopathy
  • ARCV arrhythmogenic cardiomyopathy
  • the cardiomyopathy is selected from the group consisting of dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and arrhythmogenic cardiomyopathy (ARCV), and said cardiomyopathy is driven by a mutation in a gene indicated in the column "Examples of molecules useful as first molecules" of Table 2.
  • the cardiomyopathy may be a dilated cardiomyopathy (DCM) driven by a mutation in the gene encoding ACTC1 (Actin Alpha Cardiac Muscle).
  • the muscle cells are preferably myotubes or cardiomyocytes, more preferably myotubes.
  • the muscle cells are preferably cardiomyocytes.
  • the muscular disease is a neuromuscular disease and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured diseased myotube that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a positive correlation between the concentration of the compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of said neuromuscular disease.
  • said at least one diseased myotube has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual myotubes and cellular structures of myotubes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease and the method comprises
  • the neuromuscular disease, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Table 1.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, said at least one reference cardiomyocyte being at least one in vitro cultured diseased cardiomyocyte that has not been contacted with said compound or that has been contacted with a higher or lower concentration of said compound, and wherein a positive correlation between the concentration of the compound a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of said neuromuscular disease or cardiomyopathy.
  • said at least one diseased cardiomyocyte has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual card io myocytes and cellular structures of cardiomyocytes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • the neuromuscular disease or cardiomyopathy, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Tables 1 and 2.
  • the compound to be tested may be as defined above.
  • the muscle cells may have been contacted with the compound to be tested before, during or after the culture of said cells, preferably during the culture.
  • the muscle cells Preferably, the muscle cells have been contacted with the compound to be tested prior to be stained with the labelling agents (for the first and second molecules and, optionally for the ROI(s)).
  • the reference degree of colocalization is obtained by performing steps (a) and (b) or (i) to (iii) on at least one reference muscle cell.
  • the reference muscle cells are cultured in the same conditions (same culture medium/substrate, same incubation parameters ect.) than the muscle cells used in the assay, i.e. the conditions differ only in the concentration or absence of the compound to be tested.
  • the reference muscle cells are diseased muscle cells.
  • the muscle cells used in the assay and as reference muscle cells are of the same type, in particular are myotubes or cardiomyocytes, preferably are obtained from the same source, e.g. from the same donor.
  • the muscle cells used in the assay and the reference muscle cells can be easily chosen by the skilled person based on the disease of interest and the compound to be tested.
  • the method may further comprise determining a reference degree of colocalization as described above.
  • a positive correlation between the concentration of the compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of said muscular disease.
  • a positive correlation exists when one variable decreases as the other variable decreases, or one variable increases while the other increase.
  • the first variable is the concentration of the compound and the second variable is the desired variation whether positive or negative, i.e. the absolute value of the desired variation.
  • a positive correlation thus implies that when the concentration of the compound used in the assay is higher than the concentration of the compound used to determine the reference degree of colocalization, the desired variation of the degree of colocalization increases by comparison to the reference degree of colocalization (i.e.
  • the absolute value of the desired variation increases) and when the concentration of the compound used in the assay is lower than the concentration of the compound used to determine the reference degree of colocalization, the desired variation of the degree of colocalization decreases by comparison to the reference degree of colocalization (i.e. the absolute value of the desired variation decreases).
  • the statistical significance of the variation of the degree of colocalization by comparison to the reference degree of colocalization may be assessed by any method known by the skilled person, in particular by carrying out a statistical test in order to determine a p- value between the degree of colocalization and the reference degree of colocalization. Typically, a p-value below 0.05 indicates that the variation is significant.
  • the desired variation is a variation in the direction of the healthy status.
  • This direction of variation may be easily determined by the skilled person depending on the concentration of the compound used to determine the reference degree of colocalization, i.e. a higher or lower concentration than used in the assay, and the chosen combination of first and second molecules.
  • the degree of colocalization may positively or negatively correlate with the health status of a muscle cell depending on the first and second molecules. If the two molecules are known to interact in healthy cells, the degree of colocalization positively correlates with the health status of the muscle cell. In this case, the higher the degree, the more healthy the cell is. It is the case for example when the first molecule is dystrophin and the second molecule is a protein of the dystrophin associated protein complex (DGC). If the two molecules are known to have no or few interaction in healthy cells but are known to interact in diseased muscle cells, the degree of colocalization negatively correlates with the health status of the muscle cell. In this case, the higher the degree, the less healthy the cell is. It is the case for example when the first molecule is DMPK RNA and the second molecule is a RNA binding protein trapped by CTG repeats in the DMPK gene such as MBNL1 protein, or vice-versa.
  • DGC dystrophin associated protein complex
  • the desired variation is a decrease of the degree of colocalization by comparison to the reference degree (the difference between the degree of colocalization and the reference degree is negative).
  • the desired variation is an increase of the degree of colocalization by comparison to the reference degree (the difference between the degree of colocalization and the reference degree is positive).
  • the desired variation is an increase of the degree of colocalization by comparison to the reference degree (the difference between the degree of colocalization and the reference degree is positive).
  • the desired variation is a decrease of the degree of colocalization (the difference between the degree of colocalization and the reference degree is negative).
  • a positive correlation between the concentration of the compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of the disease of interest.
  • the variation is in a direction opposite to the desired variation, then the assay does not indicate that the compound is useful in the treatment of the disease of interest.
  • the reference degree of colocalization is obtained with at least one reference muscle cell being at least one in vitro cultured diseased muscle cell that has not been contacted with the compound to be tested and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the compound is useful in the treatment of said neuromuscular disease.
  • the method of the invention of predicting the ability of a compound to treat a muscular disease is performed several times with different concentrations of the compound to be tested, in particular to evaluate the dose-response effect of the compound to be tested.
  • a positive correlation between the concentration of the compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization may be obtained in a specific range of concentrations of the compound and indicates that this compound is useful in the treatment of the muscular disease.
  • the inventors also herein demonstrated that the quantitative colocalization assays of the invention can be used to distinguish the response of patients, e.g. DMD patients, to a therapy, e.g. exon skipping therapy.
  • a therapy e.g. exon skipping therapy.
  • the present invention also relates to an in vitro method for monitoring the response to a therapeutic compound of a patient affected with a muscular disease.
  • the method comprises (a) providing at least one image of at least one in vitro cultured muscle cell obtained/derived from a sample of said patient after administration of the therapeutic compound, wherein said at least one muscle cell has been contacted with a compound to be tested, has been stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first cellular molecule of interest; and
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured muscle cell obtained/derived from a sample of said patient before administration of the therapeutic compound, and wherein a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the subject is responsive to the treatment.
  • said at least one muscle cell has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells and cellular structures of muscle cells, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the method comprises
  • Steps (a), (b), (i), (ii) and (iii) are detailed above in the section "Quantitative colocalization analysis”. All embodiments described above for these steps are also encompassed in this aspect. All embodiments described above for the method of assessing the functionality of a cellular molecule of interest, for the method of assessing potency of a compound to modulate the functionality of a cellular molecule of interest are also encompassed in this aspect, and for the method of predicting the ability of a compound to treat a muscular disease.
  • the muscular disease is a neuromuscular disease and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured myotube obtained/derived from a sample of said patient before administration of the therapeutic compound, and wherein a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the subject is responsive to the treatment.
  • said at least one myotube has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual myotubes and cellular structures of myotubes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease and the method comprises
  • the neuromuscular disease, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Table 1.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, said at least one reference cardiomyocyte being at least one in vitro cultured cardiomyocyte obtained/derived from a sample of said patient before administration of the therapeutic compound, and wherein a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization indicates that the subject is responsive to the treatment.
  • said at least one cardiomyocyte has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes and cellular structures of cardiomyocytes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • the neuromuscular disease or cardiomyopathy, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Tables 1 and 2.
  • sample means any sample containing muscle cells derived from a subject, preferably a sample which contains myoblasts or cardiomyocytes.
  • samples include biopsies, tissues or cell samples.
  • the sample may be treated prior to its use, in particular to obtain isolated muscle cells, preferably isolated myoblasts or cardiomyocytes for cell cultures.
  • Muscle cells, in particular myotubes or card io myocytes may be obtained from the sample by isolating one or several myoblasts or card io myocytes and in vitro culturing said cells as described above to obtain in vitro cultured myotubes and cardiomyocytes. They also may be obtained from the sample by isolating cells such as fibroblasts from the sample, producing induced pluripotent stem cells from these cells and differentiating said iPSC into muscle cells. All these methods are well known by the skilled person.
  • the present invention also relates to an in vitro method for selecting a patient affected with a muscular disease for a treatment with a therapeutic compound or for determining whether a patient affected with a muscular disease is susceptible to benefit from a treatment with a therapeutic compound.
  • the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) to (b) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured muscle cell obtained/derived from a sample of said patient that has not been contacted with said therapeutic compound or that has been contacted with a higher or lower concentration of said therapeutic compound, and wherein a positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization is indicative that said patient is susceptible to benefit from a treatment with said therapeutic compound.
  • said at least one muscle cell has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual muscle cells and cellular structures of muscle cells, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the method comprises
  • Steps (a), (b), (i), (ii) and (iii) are detailed above in the section "Quantitative colocalization analysis”. All embodiments described above for these steps are also encompassed in this aspect. All embodiments described above for the method of assessing the functionality of a cellular molecule of interest, for the method of assessing potency of a compound to modulate the functionality of a cellular molecule of interest, for the method of predicting the ability of a compound to treat a muscular disease or for the method for monitoring the response to a therapeutic compound of a patient affected with a muscular disease are also encompassed in this aspect.
  • the muscular disease is a neuromuscular disease and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) to (b) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured myotube obtained/derived from a sample of said patient that has not been contacted with said therapeutic compound or that has been contacted with a higher or lower concentration of said therapeutic compound, and wherein a positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization is indicative that said patient is susceptible to benefit from a treatment with said therapeutic compound.
  • said at least one myotube has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual myotubes and cellular structures of myotubes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease and the method comprises
  • the neuromuscular disease, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Table 1.
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • step (c) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (a) to (b) on at least one reference card io myocyte, said at least one reference cardiomyocyte being at least one in vitro cultured myotube obtained/derived from a sample of said patient that has not been contacted with said therapeutic compound or that has been contacted with a higher or lower concentration of said therapeutic compound, and wherein a positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization is indicative that said patient is susceptible to benefit from a treatment with said therapeutic compound.
  • said at least one cardiomyocyte has also been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes and cellular structures of cardiomyocytes, and any combination thereof, and an image segmentation is performed with an algorithm on appropriate staining channel(s) in order to identify ROI before step (b) wherein the degree of colocalization is quantitatively determined in at least one ROI.
  • ROI region of interest
  • the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises
  • the neuromuscular disease or cardiomyopathy, the first molecule, the second molecule and optionally at least one ROI are selected according to the information provided in Tables 1 and 2.
  • the method may further comprise administering the therapeutic compound to the patient when said patient is susceptible to benefit from a treatment with said therapeutic compound.
  • the muscle cells may be contacted with the therapeutic compound to be tested before, during or after the culture of said cells, preferably before or during the culture.
  • the muscle cells have been contacted with the therapeutic compound to be tested prior to be stained with the labelling agents (for the first and second molecules and, optionally for the ROI(s)).
  • the compound to be tested may be as defined above.
  • the reference degree of colocalization may be obtained as described above.
  • the reference degree of colocalization is determined using muscle cells obtained/derived from the same sample than the muscle cells of the assay.
  • the method may further comprise determining a reference degree of colocalization as described above.
  • the definition of the desired variation is also as described above and depends on the concentration of the compound used to determine the reference degree of colocalization, i.e. a higher or lower concentration than used in the assay, and the chosen combination of first and second molecules.
  • the assay does not indicate that the patient is susceptible to benefit from a treatment with the therapeutic compound.
  • the method is performed several times with different concentrations of the therapeutic compound to be tested, in particular to evaluate the dose-response effect of this compound.
  • a positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation of the degree of colocalization by comparison to the reference degree of colocalization may be obtained in a specific range of concentrations of the compound and indicates that the patient is susceptible to benefit from a treatment with said therapeutic compound.
  • the methods of the invention may further comprise before step (a) or (i)
  • muscle cells in particular myoblasts or cardiomyocytes, preferably in constrained conditions allowing the production of homogeneous population of muscle cells, in particular myotubes or cardiomyocytes;
  • the muscular disease is Duchenne muscular dystrophy or myotonic dystrophy type 1 (DM1) and the first or second molecule is dystrophin and the first or second molecule is selected from the group consisting of proteins belonging to the Dystrophin Glycoprotein complex (DGC), and dysferlin, preferably selected from the group consisting of a-sarcoglycan, p-dystroglycan, a-dystroglycan and dysferlin, more preferably selected from the group consisting of a-sarcoglycan and
  • DGC Dystrophin Glycoprotein complex
  • the muscular disease is Duchenne muscular dystrophy and the first molecule is dystrophin and the second molecule is a-sarcoglycan or p-dystroglycan.
  • the muscular disease is myotonic dystrophy type 1 (DM1) and the first molecule is DMPK RNA and the second molecule is a RNA binding protein trapped by CUG repeats in the DMPK gene, preferably is MBNL1 protein.
  • DM1 myotonic dystrophy type 1
  • the present invention also relates to the use of a first molecule as an imaging marker for assessing the functionality of a second molecule in a muscle cell, assessing potency of a compound to modulate the functionality of a second molecule in a muscle cell, predicting the ability of a compound to treat a muscular disease, monitoring the response to a therapeutic compound of a patient affected with a muscular disease, selecting a patient affected with a muscular disease for a treatment with a therapeutic compound or determining whether a patient affected with a muscular disease is susceptible to benefit from a treatment with a therapeutic compound, using a quantitative colocalization assay, preferably using a method of the invention described above.
  • the first molecule is a protein belonging to the Dystrophin associated protein complex (DGC) and the second molecule is another molecule belonging to the Dystrophin associated protein complex (DGC) or dysferlin.
  • the first molecule is dystrophin and the second molecule is selected from the group consisting of a-sarcoglycan, P-dystroglycan, a-dystroglycan and dysferlin, preferably selected from the group consisting of dystrophin, a-sarcoglycan and p-dystroglycan, or vice-versa.
  • the muscle cell is a myotube or a cardiomyocyte and the muscular disease is a neuromuscular disease or a cardiomyopathy.
  • the muscle cell is a myotube or a cardiomyocyte and the muscular disease is a neuromuscular disease, more preferably a muscular dystrophy and even more preferably Duchenne muscular dystrophy (DMD) or myotonic dystrophy type 1 (DM1). More preferably, the muscle cell is a myotube and the muscular disease is a neuromuscular disease, more preferably a muscular dystrophy and even more preferably Duchenne muscular dystrophy (DMD) or myotonic dystrophy type 1 (DM1).
  • DMD Duchenne muscular dystrophy
  • DM1 myotonic dystrophy type 1
  • the first molecule is DMPK RNA and the second molecule is a RNA binding protein trapped by CTG repeats in the DMPK gene, preferably is MBNL1 protein, or vice-versa.
  • the muscle cell is a myotube and the muscular disease is a neuromuscular disease, more preferably a muscular dystrophy and even more preferably myotonic dystrophy type 1 (DM1). All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following examples which are given for purposes of illustration and not by way of limitation.
  • Cells expanded following patient biopsy collection were subsequently enriched for myoblasts using CD56+ cell sorting.
  • Primary vials were sourced, thawed, and the proportion of Desmin+ cells was determined.
  • Cells were expanded and cryopreserved into master banks (MB) at which point they were characterized using immunostaining (Desmin+ cells) and the Myoscreen platform (CYTOO, France, fusion index).
  • master bank vials were thawed, expanded, and finally cryopreserved into working cell banks (WB) at which point they were characterized using immunostaining (Desmin+ cells) and the Myoscreen platform (CYTOO, France, fusion index).
  • WB working cell banks
  • the growth medium was changed for a differentiation medium (DMEM/F12 (Invitrogen), 2% horse serum (GE Healthcare), 0.5% penicillinstreptomycin (Invitrogen)), in which myoblasts started differentiating and forming myotubes. Myotubes formation process was then continued for 8 or 9 days in differentiation medium without medium replacement.
  • DMEM/F12 Invitrogen
  • horse serum GE Healthcare
  • penicillinstreptomycin Invitrogen
  • the MyoScreen platform allows generation of myotubes from primary and immortalized cells under controlled conditions. Those myotubes are differentiated, striated and display the morphological features necessary to form neuromuscular junctions.
  • the standardized size and the controlled culture conditions facilitate quantitative, image-based analyses and are important for the robustness of the colocalization assays.
  • High-Throughput Cardiomyocytes culture iCell Cardiomyocytes2 (Human iPSC-derived cardiomyocytes) were purchased from FUJIFILM Cellular Dynamics. At day 0, cells were thawed according to manufacturer's instructions and seeded in micropatterned plates at 30 000 cells per well in lOOpL plating medium (Fujifilm) containing 1% of penicillin-streptomycin (Invitrogen). Micropattern design consist in rectangle of 132.3pm x 18.9pm as described in (Bray, Mark Anthony, Sean P. Sheehy, and Kevin Kit Parker. 2008. "Sarcomere Alignment Is Regulated by Myocyte Shape.” Cell motility and the cytoskeleton 65(8): 641).
  • DMD siRNA After four days of culture, differentiated healthy and DMD myotubes were transfected with DMD siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following manufacturers' instructions.
  • the DMD siRNA dose response was obtained through serial dilution for final concentration of 0.0016, 0.008, 0.04, 0.2, 1 and 5 nM.
  • DMD#2 siRNA (Table 4) After two days of culture in maintenance medium, medium was refreshed and cells were transfected with a DMD#2 siRNA (Table 4) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following manufacturers' instructions.
  • the DMD siRNA dose response was obtained through serial dilution for final concentration of 0.0032, 0.016, 0.08, 0.4, 2 and lOnM.
  • Morpholino oligonucleomers were used to perform exon-skipping of Exon 44 or 45 of the DMD transcript. PMOs targeting the exon 44 are listed from 1 to 4 according to the distance to the splicing acceptor site (e.g, PMO1 is closer to the acceptor site than PMO2). Vivo-phosphorodiamidate morpholino oligonucleomers (“vivoPMOs”) correspond to the same sequences, fused to an octa-guanidine dendrimer (vivo-group).
  • Exon 44-skipping vivoPMO was used at a final concentration of 0.075, 0.15, 0.3 and 0.6 pM.
  • Exon 45-skipping vivoPMO was used at final concentration of 0.25, 0.5, 1 and 2pM.
  • FISH Fluorescence In situ Hybridation
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • Triton X-100 Sigma-Aldrich
  • the cells are incubated with pre-hybridization buffer (phosphate buffer 0.5M + 40% formamide) for 20 min at room temperature.
  • pre-hybridization buffer phosphate buffer 0.5M + 40% formamide
  • the RNA probe (CAG)5-Cy3 is added to the hybridization buffer (7% dextran, 0.2% BSA in pre-hybridization buffer) and incubated overnight at 37°C.
  • the cells are grown on MyoScreen plates.
  • Four stainings are realized: HOECHST 33342 for the nuclei, DRAQ5 for the myosin heavy chain (MHC) or Troponin T staining and Cy3 and Alexa 488 channels that are related to identified disease biomarkers.
  • images of the plates were acquired on an Operetta HCS imaging system with a 20xN A objective in confocal mode.
  • images of cells were acquired with the Operetta HCS platform (Perkin Elmer) using a x40 objective.
  • Image processing and analysis were performed using dedicated algorithms developed on the Acapella High Content Imaging Software (Perkin Elmer) by the inventors and aims to determine the colocalization level between two proteins of interest.
  • Region of interest (ROI) identification is performed through segmentation algorithms on the appropriate staining channel.
  • the channel depends on the disease.
  • the myotubes are segmented using the Troponin T or MHC channel (see as example Figure 4.A first row).
  • Figure 4C represents another example of region of interest.
  • Figure 4A second row on the right presents a mask obtained by thresholding the imaging marker 1 channel in the myotube area.
  • the quantile was set to 0.99.
  • Figure 4A third row on the right presents a mask obtained by thresholding the imaging marker 2 channel in the myotube area.
  • the quantile was set to 0.99.
  • the intersection of those two masks defines the colocalization zone, which is the area where both proteins are detected.
  • the union of the two masks is the above threshold area, where either one protein or the other, or both proteins are detected ( Figure 4B).
  • Figure 4C second row represents the mask obtained by thresholding the imaging marker 1 channel in the nuclei in myotubes area.
  • figure 4C third row represents the mask obtained by thresholding the imaging marker 2 channel in the nuclei in myotubes area.
  • one filter step may be applied on the proteins of interest images.
  • the respective threshold values of the labeled proteins are subtracted from their corresponding images.
  • the pixels values under zero are clipped to zero.
  • the Pearson's Colocalization Coefficient is one statistic calculation that can be used to quantify colocalization.
  • the formula exemplified with the imaging marker 1 (I M 1) and imaging marker 2 (I M2) colocalization case, is presented in the Figure 4E.1.
  • PCC measures the pixel-by-pixel covariance in the signal levels of two images. Plotting the pixel-intensity of one protein image against the pixel-intensity of the other protein, the more linear the relation will be, the higher the PCC.
  • the PCC may be calculated on all the ROI, or in the above threshold area. This later is more stringent as the region where none of the protein is present positively affects the PCC.
  • Manders colocalization measure is independent of pixel intensities correlation.
  • the MCC is a measure of co-occurrence. It measures the proportion of one protein-related signal that overlaps with the other protein signal.
  • the MCC for IM1 represents the proportion of IMl-related intensity that is colocalizing with IM2.
  • the numerator we find the sum of IM1 intensities for pixels in the colocalization zone, while in the denominator, we find the sum of the intensities of all pixels where IM1 is detected.
  • the RWC tries to add a notion of correlation in the MCC. Indeed, the algorithm uses a non-parametric ranking of pixel intensities in each channel, and the difference in ranks of colocalizing pixel positions in the two channels is used to weight the pixel intensities in the numerator (see Figure 4E.3). The closer the pixel rank of the 2 intensities, the higher the weight for the intensity in this pixel. This weighting is applied to co-occurring pixels thereby combining both co-occurrence and correlation.
  • the colocalization is also assessed between one of the two proteins and a protein that is known not colocalize.
  • IM2 is replaced by I MCTL.
  • the calculations of readouts are the same, except those intensities related to IM1 are replaced by intensities related to I MCTL, see in Figure 4F.
  • MHC is considered as the I MCTL .
  • the colocalization zone corresponds to the zone where the IM1 is above threshold.
  • Those readouts are considered negative controls for the colocalization readouts.
  • Another developed readout is the percentage of ROI displaying a strong colocalization.
  • the PCC and negative control PCC values of each myotube are plotted. The plots are realized for DMD donors untreated and mock, and for healthy donors untreated and mock for four pairs of dystrophin with a-dystroglycan, p-dystroglycan, a- sarcoglycan and dysferlin.
  • a satisfying threshold is one for which 99% of the PCC values between Dystrophin and DGC proteins for DMD donors or MHC for all donor myotubes are under the threshold.
  • 0.6 was determined as a satisfying threshold for all the tested imaging markers and DMD donors as only outliers situated in the 99th percentile exceed this threshold value.
  • the High PCC% is the percentage of myotubes having a PCC value above the 0.6 threshold among the total number of myotubes: 100.
  • the readouts can be corrected by subtracting the readout obtained with random colocalization. What we call the readouts with significance correction can be calculated this way:
  • the pixels of the considered zone are mixed multiple times.
  • the readout is calculated, and then these values are averaged.
  • this average we expect this average to be correlated to the number of molecules present in the zone. We use this measurement calculated on shuffled images by subtracting it from the measurement that is made on the original images.
  • DGC dystrophin-glycoprotein complex
  • the DGC links the intracellular actin cytoskeleton and the sarcomere to the extracellular matrix.
  • Dystrophin is an essential component of this complex and interacts physically with several proteins involved in this complex, such as p-dystroglycan (b-DG).
  • b-DG p-dystroglycan
  • the interaction of dystrophin with p-dystroglycan is critical for the formation of the DGC and has been demonstrated using a variety of biochemical and image-based assays (Cullen et al., J Histochem Cytochem. 1998 Aug;46(8):945-54 ; llsley et al., 2001, Cell Signal. 2001 Sep;13(9):625-32; Ervasti et al., Biochim Biophys Acta. 2007 Feb;1772(2):108-17).
  • the robustness of this interaction makes it an excellent candidate to monitor the restoration of a functional dystrophin in DMD patients by gene therapy or exon skipping RNA therapies.
  • the presented studies monitored the interactions of dystrophin with p-dystroglycan (b-DG), a- dystroglycan (a-DG), and a-sarcoglycan (a-SG). While a-dystroglycan (a-DG) and a-sarcoglycan (a-SG) are also components of the DGC, they do not directly interact with dystrophin. These interactions are difficult to monitor using physical isolation methods but have been monitored in situ using non-quantitative colocalization assays.
  • FIG. 1A shows that all donors form myotubes that display the standard MyoScreen morphology. Healthy donors have a larger mean myotube area than the DMD donors while their nuclei count is similar, resulting in a higher fusion index ( Figure 1A, B). This result indicates that healthy donors are more differentiated than the DMD donors, which is an expected DMD phenotype.
  • FIG. 2 shows the baseline expression of dystrophin in healthy and DMD donors using antibodies that either target the dystrophin N-terminal domain (Figure 2A) or the C-terminal domain ( Figure 2B).
  • Dystrophin detection is reduced to background noise in DMD donors when using the N-terminal targeting dystrophin antibody, and to approximately 40% of healthy donor level when using the C-terminal targeting dystrophin antibody ( Figure 2C, D).
  • dystrophin levels were modulated in healthy donors using RNAi ( Figure 2D and E).
  • the dystrophin signal decreased as the siRNA dose increased.
  • the observed dystrophin signal in the healthy donors mimicked the signal observed in DMD donors.
  • the 40% signal obtained in siRNA treated healthy donors and DMD donors upon monitoring dystrophin using the C-terminal targeting antibody suggests a lower specificity of this antibody.
  • both dystrophin antibodies are similar in their sensitivity to detect changes in dystrophin expression (Figure 2E).
  • the membrane-associated formation of the DGC is a biomarker for the recovery of dystrophin activity in DMD patients treated with therapies that restore dystrophin expression.
  • a-DG, b-DG and a- SG to monitor the recovery of dystrophin upon treating DMD patient-derived myotubes with either gene or exon skipping therapies.
  • Dysferlin was included in these studies as a transmembrane protein that is not directly associated with the DGC.
  • Figure 3 A and B shows the expression of a-DG, b-DG, a-SG and dysferlin in myotubes of healthy and DMD donors.
  • Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between a-sarcoglycan, p- dystroglycan, a-dystroglycan, dysferlin and dystrophin (N-terminal antibody).
  • HV#1 and HV#2 the colocalization readouts are then plotted against the dystrophin siRNA dose.
  • all four statistical methods yield similar results.
  • the High PCC% readout displays a larger dynamic range than PCC, MCC or RWC.
  • Dystrophin expression in two healthy donors was regulated by RNAi using DMD siRNA in concentration ranges between 0.001 and 1 nM. The resulting level of dystrophin determined by high content analysis. Colocalization between dystrophin and a-sarcoglycan, P-dystroglycan, a-dystroglycan, dysferlin was analyzed using the High PCC % readout and is presented as a function of % of dystrophin in the untreated healthy donors ( Figure 7). Changes in the colocalization between dystrophin and -dystroglycan or a-sarcoglycan can be detected when dystrophin levels are reduced by more than 40%.
  • the sensitivity of these assays is aligned with the level of dystrophin restoration achieved by therapies targeting DMD.
  • the High PCC % does not show a significant colocalization between dystrophin and a- dystroglycan. While dysferlin shows significant colocalization with dystrophin, the colocalization has a high variability between donors and was very sensitive to changes in the dystrophin concentration.
  • DMD patients have deletions of various lengths in the DMD gene which shift the reading frame and prevent the expression of a functional dystrophin.
  • skipping exons at the boundary of these deletions can restore the reading frame and enable the expression of shortened albeit partially functional dystrophin.
  • the skipping of particular exons can be achieved with the help of specific oligonucleotides that prevent the splicing of specific exons by masking sequences required for the assembly of the spliceosome at specific intron/exon junctions.
  • the observed difference in the level of dystrophin restoration likely reflects differences in the uptake of the vivoPMOs by these donors.
  • the expression levels of b-DG and a-SG were not significantly modified by the vivoPMO treatments ( Figure 8A, C).
  • Colocalization of dystrophin with b-DG or a-SG was monitored using the high PCC % readout. Both assays gave comparable results ( Figure 8D).
  • DMD donor #6 shows a lower level of colocalization between dystrophin and b-DG or a-SG than DMD donor #5.
  • both donors upon normalization of their responses, both donors showed the same relative response to the vivoPMO treatment and consequent restoration of dystrophin (Figure 8D).
  • the activity of the restored dystrophin in these donors differed when monitoring the colocalization of dystrophin and p-dystroglycan versus dystrophin and a-sarcoglycan ( Figure 9D).
  • the dystrophin restoration is similar between both donors, the High PCC % of donor DMD#1 only reaches 30% while it goes up to 70% for DMD#4, which indicates that the restored dystrophin of donor #1 is less capable to interact with p-dystroglycan than the restored dystrophin of donor #4 ( Figure 9D).
  • the restored dystrophin from both donors interacted to a similar extent with a- sarcoglycan ( Figure 9D). Since donor #1 and #4 have different deletions within the DMD gene (see Table 3), the restored dystrophin in these patients will have different structural properties. This example demonstrates the ability of the developed colocalization assays to detect differences in the functional properties of the restored dystrophin.
  • FIG. 10 exemplify the colocalization assay between the restored dystrophin and P-dystroglycan in myotubes from DMD immortalized cell line amenable to Exon 44 skipping, and demonstrate the ability of the method to detect differences in products with known potencies.
  • Myotube differentiation and morphology of healthy and DMD immortalized cell lines under MyoScreen conditions was assessed using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes.
  • MHC myosin heavy chain
  • DMPK ASO is used to remove the aberrant DMPK mRNA in the DM1 patients. All DM1 donors show the presence of co-labelled DMPK mRNA and MBNL1 protein, representative of the DM1 phenotype. For the congenital donors (DM1 #4 and #5) the results are also presented at a different scale ( Figure 11C) for visibility of the dose-response effect.
  • MCC gives the highest range among all compared methods and for all the DM1 donors and is the most suited method for estimating colocalization between DMPK foci and MBNL1 in DM1 .
  • This chosen colocalization method we further compare the response of the five DM1 donors to treatment with an ASO at 6 concentrations. Colocalization in healthy donors is close to 0 since the MBNL1 protein is not captured by the nuclear RNA foci. In contrast, colocalization is highest in DM1 donors.
  • the ASO treatment releases MBLN1 from the nuclear RNA foci and the colocalization decreases (Figure HE).
  • DMPK ASO DMPK ASO-mediated loss of function results in the exclusion of exons 71 and 78 from the DMD mRNA, leading to a different C-terminus for the dystrophin protein.
  • the antibody targeting the C-terminal domain of dystrophin does not show any specific sarcolemmal staining in DM1 donors.
  • Increasing DMPK ASO doses result in the restoration of dystrophin C- terminal signal in the DM1 donors ( Figure 12A white arrows show sarcolemma positive for dystrophin signal).
  • the response is donor-dependent with lower restoration in non-congenital donors and higher restoration in congenital donors ( Figure 12A).
  • High PCC% gives the highest range among all compared methods and for all the DM1 donors and is thus the most suited method for estimating colocalization between a-sarcoglycan, p-dystroglycan and dystrophin (C-terminal antibody) in DM1.
  • Colocalization of C-terminal dystrophin with p-dystroglycan or a- sarcoglycan was monitored using the high PCC % readout. Both assays gave comparable results (Figure 12D, E). Colocalization in healthy donors is close to 100. In contrast, colocalization is lowest in DM1 donors.
  • Congenital donors show a colocalization level between p-dystroglycan or a- sarcoglycan and dystrophin situated between 30% and 80% for a dystrophin expression level comprised between 20% and 60%.
  • Non-congenital donors present lower colocalization levels: for dystrophin levels between 20% and 50% they show colocalization percentages from 0 to 70%.
  • FIG. 13A shows the expression of b-DG and dystrophin in control cardiomyocytes and cardiomyocytes treated with siRNA.
  • DMD siRNA downregulated dystrophin levels up to less to 50%.
  • the decrease in dystrophin levels were associated with decreases in the levels of b-DG as previously observed in myotubes ( Figure 13B).
  • the colocalization readouts are then plotted against the dystrophin siRNA dose ( Figure 13 C, D).
  • the High PCC% readout displays a larger dynamic range than PCC, MCC or RWC, as it was also measured for myotubes.

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Abstract

La présente invention concerne des procédés d'évaluation de la fonctionnalité d'une molécule cellulaire dans une cellule musculaire, d'évaluation de l'activité d'un composé pour moduler la fonctionnalité d'une molécule cellulaire dans une cellule musculaire, de prédiction de la capacité d'un composé à traiter une maladie musculaire, de surveillance de la réponse à un composé thérapeutique d'un patient atteint d'une maladie musculaire, de sélection d'un patient atteint d'une maladie musculaire pour recevoir un traitement avec un composé thérapeutique ou de détermination du fait qu'un patient atteint d'une maladie musculaire est susceptible de bénéficier d'un traitement avec un composé thérapeutique, à l'aide de dosages de colocalisation quantitative.
PCT/EP2024/052818 2023-02-03 2024-02-05 Dosages de colocalisation quantitative pour évaluer l'activité et l'efficacité de thérapies ciblant des troubles musculaires Ceased WO2024161042A1 (fr)

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