Quantitative colocalization assays to assess potency and efficacy of therapies targeting muscle disorders

Abstract

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, using quantitative colocalization assays.

Description

[0001] Quantitative colocalization assays to assess potency and efficacy of therapies targeting muscle disorders

[0002] FIELD OF THE INVENTION

[0003] 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.

[0004] BACKGROUND OF THE INVENTION

[0005] During the last decades genetic analysis identified more than 500 genes involved in the cause of neuromuscular disorders. The identification of these genes greatly advanced our understanding of muscle function and disease and opened many opportunities for therapeutic intervention. Despite those advances, for many neuromuscular diseases the disease mechanism is still unknown and effective therapies remain to be identified.

[0006] 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). A process called excitation-contraction coupling converts the neuronal excitation mediated by the motor neuron into Ca2+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.

[0007] 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). In addition to the muscle cells itself, 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). Furthermore, the structure and function of muscles can be impaired metabolically in response to malnutrition, immobility, advanced age, or acute and chronic diseases. In these cases, 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.

[0008] Among the best characterized dystrophies are Duchenne and Becker muscular dystrophies (DMD, BMD). In DMD patients, mutations in the DMD gene prevent the expression of a functional dystrophin and the assembly of the Dystrophin-Glycoprotein complex (DGC) at the plasma membrane (Gao & McNally, Compr Physiol. 2015 Jul 1;5(3):1223- 39). 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. In contrast to DMD, BMD patients express a partially functional dystrophin usually associated with less severe symptoms. Another example of a well-studied neuromuscular disease is myotonic dystrophy (DM1). In DM1 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. For an increasing number of disorders, various therapies have been developed and are currently passing through clinical development (Dowling et al., 2021 supra). The most promising are gene and RNA therapies that attempt to substitute a mutated disease driver or to correct disease-associated changes in post- transcriptional processing of a disease driver. However, even for well-understood neuromuscular disorders, discovery and development of these therapies have been challenging.

[0009] In case of 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.

[0010] Many muscle diseases are rare to ultra-rare diseases. With the increasing number of therapies under development, a small group of patients will be asked to support the clinical trials of these therapies and to choose between therapies. Functional assays performed on patient-derived cells could provide guidance to identify the therapy option that is most beneficial for each individuum.

[0011] The complex processes involved in the manufacturing of gene and RNA therapies and the high doses needed to obtain a therapeutic effect resulted in the need of potency assays at early stages in the clinical development to support batch releases. This requirement as led to several FDA-imposed holds during recent clinical trials targeting neuromuscular disorders.

[0012] Many neuromuscular diseases have cardiomuscular indications that often determine the life expectancy of the patient. Hence, evaluation of therapies targeting neuromuscular disorders in card io myocytes is of importance. Moreover, increasing understanding of the molecular mechanisms regulating cardiomuscular functions opened opportunities for targeted therapies for cardiomyopathies.

[0013] Together these examples highlight the strong need in the discovery and development of therapies for muscle disorders for quantitative, functional assays that can be conducted in cultured muscle cells, contribute to the mechanistic understanding of muscle disorders, support the clinical development of gene and RNA therapies, and enable evaluation of the benefit of novel therapies for individual disease genotypes.

[0014] SUMMARY OF THE INVENTION

[0015] In a first aspect, the present invention relates to an in vitro method of assessing the functionality of a cellular molecule of interest in a muscle cell

[0016] (i) providing at least one image of at least one in vitro cultured muscle cell, wherein said at least one muscle cell 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 has 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, cellular structures of muscle cells, and any combination thereof;

[0017] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0018] (iii) determining quantitatively a degree of colocalization of the cellular first molecule and the cellular second molecule in at least one ROI by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest in said at least one muscle cell.

[0019] Preferably, the muscle cell is a myotube or cardiomyocyte.

[0020] In some embodiments, 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. In some preferred embodiments, 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.

[0021] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0022] In a second aspect, 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

[0023] (i) providing at least one image of at least one in vitro cultured 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, 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 muscle cells, cellular structures of muscle cells, and any combination thereof; and

[0024] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0025] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0026] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0027] Preferably, the muscle cell is a myotube or cardiomyocyte.

[0028] In some embodiments, 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. In some preferred embodiments, 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.

[0029] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0030] In another aspect, 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

[0031] (i) providing at least one image comprising at least one in vitro cultured muscle cell exhibiting a feature of a muscular disease of interest ("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 muscle cells, cellular structures of muscle cells, and any combination thereof; and

[0032] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0033] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0034] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0035] Preferably, the muscle cell is a myotube or cardiomyocyte.

[0036] Preferably, the muscular disease is a neuromuscular disease or a cardiomyopathy.

[0037] In some embodiments, 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.

[0038] In some preferred embodiments, 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, and the muscular disease is a neuromuscular disease.

[0039] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0040] In a further aspect, 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

[0041] (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained from a sample of a patient affected with a muscular disease after administration of the therapeutic compound, wherein said at least one muscle cell 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 has 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, cellular structures of muscle cells, and any combination thereof; and

[0042] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0043] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0044] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured muscle cell obtained 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.

[0045] Preferably, the muscle cell is a myotube or cardiomyocyte.

[0046] Preferably, the muscular disease is a neuromuscular disease or a cardiomyopathy. In some embodiments, 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.

[0047] In some preferred embodiments, 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, and the muscular disease is a neuromuscular disease.

[0048] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0049] In another aspect, 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

[0050] (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained from a sample of said patient, wherein said at least one muscle cell has been contacted with a therapeutic compound, 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 has 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, cellular structures of muscle cells, and any combination thereof; and

[0051] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0052] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0053] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, said at least one reference muscle cell being at least one in vitro cultured muscle cell obtained 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 indicates that said patient is susceptible to benefit from a treatment with said therapeutic compound.

[0054] Preferably, the muscle cell is a myotube or cardiomyocyte.

[0055] Preferably, the muscular disease is a neuromuscular disease or a cardiomyopathy.

[0056] In some embodiments, 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.

[0057] In some preferred embodiments, 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, and the muscular disease is a neuromuscular disease.

[0058] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0059] Preferably, in the methods of the invention, quantitative colocalization analysis is carried out using pixel-based colocalization analysis.

[0060] Preferably, in the methods of the invention, the muscle cells are derived from primary cells or are derived from immortalized cells.

[0061] Preferably, in the methods of the invention, the muscle cells have been cultured in constrained conditions allowing the production of homogeneous population of muscle cells.

[0062] The methods of the invention may further comprise comprises before step (i)

[0063] - culturing 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;

[0064] - staining these muscle cells for said first molecule, for said second molecule and with said at least one labelling agent; and

[0065] - capturing at least one image of at least one stained muscle cell.

[0066] Preferably, in the methods of the invention, in step (iii) 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).

[0067] In some embodiments on the methods of the invention, said first molecule is a protein and said second molecule is a protein, and in step (iii) 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.

[0068] In some embodiments on the methods of the invention, 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).

[0069] Preferably, in the methods of the invention, 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.

[0070] In some embodiments on the methods of the invention, 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 |3- dystroglycan. In particular, 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.

[0071] In some embodiments on the methods of the invention, the muscular disease is myotonic dystrophy type 1 (DM1), 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.

[0072] BRIEF DESCRIPTION OF THE FIGURES

[0073] 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. A. Morphology of healthy (HV #1 and #2) and DMD (DMD #1, #4, #5 and #6) donors used in this study. Scale bar, 100pm. 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.

[0074] Figure 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). Scale bar, 100pm. Unlike the antibody targeting the N-terminal domain, 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. In healthy donors treated with high levels of DMD siRNAs, 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. ANOVA with post-hoc multiple comparison test, *, p <0.05 ; **, p<0.01; ***, p < 0.001 compared to Mock condition. E. Quantitative assessment of dystrophin signal as a function of siRNA dose. Corrected for the unspecific staining of the antibody targeting the dystrophin C-terminal domain, both dystrophin antibodies monitor changes in dystrophin expression with similar sensitivity. 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. A. Expression of a- sarcoglycan, p-dystroglycan, a-dystroglycan and dysferlin in myotubes of two healthy (HV#1 and #2) and four DMD donors (DMD #1, #4, #5 and #6). Scale bar, 100pm. B. Expression of |3- dystroglycan, a-sarcoglycan, a-dystroglycan and dysferlin after dystrophin (DMD) siRNA knock-down in healthy donors (HV#1 and #2). Scale bar, 100pm. C. Quantitative assessment of p-dystroglycan, a-sarcoglycan, a-dystroglycan and dysferlin in myotubes shown in figure 3B. ANOVA with post-hoc multiple comparison test, *, p <0.05 ; **, p < 0.01 compared to HV #1 or #, p<0.05 compared to HV #2.

[0075] Figure 4: General workflow to quantify colocalization between labelled image markers. A. Cytoplasmic or membrane-localized markers. Top. Myotube segmentation is realized on myosin heavy chain marker. Second and third rows. 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. B. Overlay of the masks defines four areas: the area of the myotube, the area of colocalization, the area with IM1 signal above threshold and the area with IM2 signal above threshold. C. Nuclear image markers. Top. The nuclei in myotube segmentation are realized on myosin heavy chain marker and HOECHST 33342 nuclei marker. Second and third rows. 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). E. 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). With IIM2the IM2 associated intensity for a given pixel, IIM1the IM1 associated intensity for a given pixel, meanIIM2■ the mean IM2 intensity on the area indicated on the right of the formula, meanIIM1the mean IM1 intensity on the area indicated on the right of the formula, Wplxeia pixel weight that grows with the correlation of IM2 and IM1 intensity ranks for a given pixel. For the calculus of RWC, the ranks of the intensities are calculated for each pixel, Dpixeiis 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). 1. 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. In MCC and RWC, the sums on pixels are realized in the zone highlighted next to the sum. In the numerator, the area is the colocalization area, at the denominator it is the zone of IM2 expression. F. 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 ICTLthe I MCTL associated intensity for a given pixel, meanICTLthe mean I MCTL intensity on the area indicated on the right of the formula.

[0076] 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. PCC between dystrophin and the four selected colocalization partners: |3- dystroglycan, a-sarcoglycan, a-dystroglycan, and dysferlin for all the myotubes in the DMD donors untreated and mock conditions. B. PCC between dystrophin and MHC for all myotubes in the Healthy and DMD donors untreated and mock conditions. In this example, MHC is used as a control protein that is not colocalizing with dystrophin. 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. In the shown example, 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.

[0077] 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.

[0078] Figure 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. 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 (HV#1 and HV#2).

[0079] 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. A. Myotubes of healthy and DMD donors treated with a control vivo PMO (0.6 pM) and DMD donors treated with increasing doses of an exon 44 skipping vivo PMO (0.075-0.6 pM). 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. Statistical significance was assessed by ANOVA with Dunnett's test for multiple comparisons between HV and DMD-treated conditions. * - ** - ***, p<0.05 - 0.01 - 0.001 for HV#1 comparison and # - ## - ###, p<0.05 - 0.01 - 0.001 for HV#2 comparison. Treatment of DMD donors with Exon 44 skipping vivo PMOs partially restores dystrophin expression. Expression levels of |3- dystroglycan and a-sarcoglycan are not modified by the exon 44 vivo PMO treatments. D. High PCC % readout for colocalization between a-sarcoglycan, p-dystroglycan and dystrophin as a function of the exon 44 skipping vivo PMO dose (upper panel) or dystrophin level (% untreated healthy donors, lower panel).

[0080] 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. A. Myotubes of healthy and DMD donors treated with a control vivo PMO (2 pM) and DMD donors treated with increasing doses of an exon 45 skipping vivo PMO (0.25-2 pM). 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. Treatment of DMD donors with Exon 45 skipping vivo PMOs partially restores dystrophin expression. Expression levels of p-dystroglycan and a- sarcoglycan are not modified by the exon 45 vivo PMO treatments. D. High PCC % readout for colocalization between a-sarcoglycan, p-dystroglycan and dystrophin as a function of the exon 45 skipping vivo PMO dose (upper panel) or dystrophin level (% untreated healthy donors). As seen in Figure 9D the response to the PMO treatments is donor-dependent. However, both donors show similar dystrophin / oc-sarcoglycan colocalization as a function of dystrophin restoration, indicating that the restored dystrophin in these patients is similarly active with respect to this interaction. This was not the case when monitoring the dystrophin / P- dystroglycan colocalization, suggesting that the interaction between dystrophin and |3- dystroglycan is impaired in the restored dystrophin of DMD donor #1. This example demonstrates that the DGC colocalization assay is able to assess the activity of the restored dystrophin in a patient-dependent manner.

[0081] 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. A. Morphology of healthy (HVimm) and DMD (DMDimm) cell lines. Scale bar, 100pm. 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 immortalized cell lines. Statistical significance was assessed by Student test for comparison between HVimm and DMDimm cell lines. **, p<0.01. C. Myotubes of healthy and DMD cells treated with a control PMO (5 pM) and DMD cells treated with increasing doses of 4 exon 44 skipping PMO (0.3-10 pM) of different efficacy. PMOs are listed from 1 to 4 in order of predicted efficacy. Myotubes were labeled for dystrophin (upper panel) and p-dystroglycan (lower panel). Scale bar, 100pm. 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 |3 -dystroglycan is not modified by the exon 44 PMO treatments. E. High PCC % readout for colocalization between p-dystroglycan and dystrophin as a function of the exon 44 skipping PMO dose (left-hand side) or dystrophin level (% untreated healthy immortalized, right-hand side).

[0082] 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. A. Morphology of healthy (HV #1 and #2) and DM1 (DM1 #1, #2, #3, #4 and #5) donors used in this study. Scale bar, 100pm. 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. Statistical significance was assessed by ANOVA and Dunnett's test for multiple comparison between HV and DM1 donors. * - ** - ***, p<0.05 - 0.01 - 0.001 for HV#1 and # - ## - ###, p<0.05 - 0.01 - 0.001 for HV#2 (n>9 wells / condition). C. The presence of DMPK mRNA foci was assessed using a (CAG)5-Cy3 FISH probe and the immunofluorescent staining of MBNL1 was used to determine the presence of MBNL1 protein trapped in the DMPK mRNA foci. DMPK ASO is used to remove the aberrant DMPK mRNA in the DM1 patients. Quantification of the number of spots co-labelled with MBNL1 and DMPK mRNA foci signal relative to the nuclei area in healthy (HV #1 and HV#2) and DM1 donors (DM1 #1-5). ANOVA with Dunnett's multiple comparisons test for pairwise comparison. * - **, p<0.05 - 0.01 compared to the Mock condition for each donor. D. Colocalization of DMPK foci and MBNL1 was analyzed using mean PCC, MCC and RWC and high PCC% readouts as a function of the ASO dose-response for DM1 donors (DM1 #1, #2, #3, #4 and #5). In these examples, the MCC readout showed the largest dynamic range and distinguished itself as the preferred readout to monitor colocalization of DMPK foci and MBNL1. E. 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.

[0083] Figure 12: Colocalization of the C-terminal domain of dystrophin with p-dystroglycan and a-sarcoglycan in DM1 donors treated with DMPK ASO. A. 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 sarcolemma positive for dystrophin signal). Scale bar, 100pm. B. Expression of a-sarcoglycan (upper panel) and p-dystroglycan (lower panel) in healthy (HV#1 and #2) and DM1 donors (DM1 #1-5). Scale bar, 100 pm. C. Quantification of Dystrophin C-terminal, p-dystroglycan and a-sarcoglycan expression in healthy (HV#1 and #2) and DM1 donors (DM1 #1-5). ANOVA with Dunnett's multiple comparisons test for pairwise comparison. * - **, p<0.05 - 0.01 compared to the Mock condition for each donor. D. Colocalization method comparison. We compare the response of 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. 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 |3- dystroglycan or a-sarcoglycan. Among the tested readouts, high PCC% displayed the largest dynamic range. E. 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.

[0084] 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. A. 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. ANOVAwith post-hoc multiple comparison test, *, p <0.05 ; compared to mock condition. C. Selection of an optimal quantification method for colocalization of dystrophin with |3- dystroglycan. 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. D. High PCC% readouts calculated between p-dystroglycan and dystrophin (N-terminal antibody). *, p <0.05 ; compared to mock condition. Like in skeletal myotubes, in cardiomyocytes the colocalization of dystrophin and p-dystroglycan monitored by high PCC% responds to changes in dystrophin levels.

[0085] 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.

[0086] DETAILED DESCRIPTION OF THE INVENTION

[0087] Discovery and development of therapies targeting muscle disorders have been hampered by the lack of in vitro systems that enable the culturing of differentiated muscle cells, in particular myotubes, by difficulties to generate animal models that represent the diversity of the genetic backgrounds found in patients with neuromuscular disorders, and by the paucity of quantitative, cell-based, functional assays to assess the potency and efficacy of therapies. Many neuromuscular disorders require gene or RNA therapies. Due to the complex processes involved in the manufacturing of these drug products and the high doses needed to obtain a therapeutic effect, multiple batches of these drug products are needed to support even early-stage clinical trials. The release of these drug products requires potency assays that measure the activity of a disease driver as a function of the therapeutic compound. However, the lack of appropriate assays has led to several FDA-imposed holds during recent clinical trials targeting neuromuscular disorders. Furthermore, many muscle diseases are rare to ultra-rare diseases. With the increasing number of therapies under development, a small group of patients will be asked to support the clinical trials of these therapies and to choose between therapies. Functional assays performed on patient-derived cells could provide guidance to identify the therapy option that is most beneficial for individual patients.

[0088] In the present application, 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.

[0089] As a proof of concept, the inventors herein demonstrated that 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. They also showed that these assays can be used to quantitatively monitor the colocalization, and thus the interaction, between a protein and a nucleic acid, in particular between the splicing factor MBNL1 and mutated DMPK RNAs in DM1 patients. The inventors also demonstrated 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). Together these examples demonstrate that the colocalization assays developed by the inventors meet the need for quantitative, functional assays that contribute to the mechanistic understanding of muscle disorders, support the clinical development of therapies, in particular gene and RNA therapies, and enable evaluation of the benefit of novel therapies for individual disease genotypes.

[0090] Quantitative colocalization analysis

[0091] 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. In these methods, 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.

[0092] In particular, the methods typically comprise

[0093] (a) providing at least one image of at least one in vitro cultured muscle cell, wherein said at least one muscle cell has been stained for a first cellular molecule of interest and for a second cellular molecule interacting / known to interact with said first cellular molecule of interest; and

[0094] (b) determining quantitatively a degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing quantitative colocalization analysis.

[0095] 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.

[0096] 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.

[0097] The muscle cell is preferably a mammalian muscle cell, in particular a human muscle cell. Preferably, 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. In preferred embodiments, the muscle cell is a myotube, and in particular a human myotube. In some other embodiments, the muscle cell is a cardiomyocyte, and in particular a human cardiomyocyte. Depending on the method of the invention, imaged muscle cells may be healthy muscle cells or diseased muscle cells.

[0098] 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.

[0099] 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. For example, for DMD, 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. Preferably, diseased muscle cells are obtained by culturing muscle cells derived from at least one patient suffering from the disease of interest.

[0100] In particular, 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.

[0101] 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. Preferably, diseased myotubes are obtained by culturing myoblasts derived from at least one patient suffering from a neuromuscular disease of interest.

[0102] Similarly, 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. Preferably, diseased card io myocytes are obtained by culturing cardiomyocytes derived from at least one patient suffering from a neuromuscular disease or cardiomyopathy of interest.

[0103] As used herein, 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.

[0104] Muscle cells used in the present invention may be obtained by culturing muscle cells derived from primary cells or from immortalized cells.

[0105] In some particular embodiments, 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. In particular, 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. 2015;112(41):12705-12710), Funakoshi et al., 2021 (Nat Commun 12, 3155 (2021)), Lee et al. 2017 (Cell Stem Cell. 2017;21(2):179-194.e4) and Jerome Chai and Olivier Pourquie, 2017 (Development 15 June 2017; 144 (12): 2104-2122). Card io myocytes and myoblasts derived from induced pluripotent stem cells are also commercially available, see e.g. iCell Cardiomyocytes2 (Human iPSC-derived card io myocytes) from FUJIFILM Cellular Dynamics, and ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612, Abeam). In some other particular embodiments, 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. In embodiments wherein two types of cells are used, 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. In addition, muscle cells of the same type, e.g. healthy cells or diseased cells, 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. Preferably, muscle cells of the same type, e.g. healthy cells or diseased cells, are obtained from a unique source, i.e. from the same subject or from a unique immortalized cell line.

[0106] Before being imaged, muscle cells are in vitro cultured / produced on a suitable substrate and under suitable conditions known by the skilled person.

[0107] 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. glass, silicone or plastics such as polystyrene, polypropylene, polyethylene), on plates or in wells (see e.g. WO 2016 / 202850, WO 2016 / 139312, WO 2015 / 091593, EP 1 882 736 , EP 2 180 042, EP 1 664 266 and US 2008 / 299086 patent applications, herein incorporated by reference).

[0108] In preferred embodiments, in vitro cultured muscle cells are not organized in tissue. In particular, 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. Thus, the image(s) provided in step a) is not the image of a tissue or biopsy sample.

[0109] Preferably, muscle cells are cultured in constrained conditions, i.e. on a substrate allowing the production of homogeneous populations of muscle cells. Preferably, 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.

[0110] A homogeneous population of muscle cells is a population of muscle cells exhibiting similar morphological parameters. In particular, 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). Preferably, 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 %. More preferably, 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 %.

[0111] Cardiomyocytes of a homogeneous population of cardiomyocytes may exhibit similar cell area (or width / length ratio, width and / or length). Preferably, 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 %.

[0112] 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).

[0113] Typically, a culture in constrained conditions is a culture on a substrate with topological constraints or adhesive patterns. Such 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. Examples of such 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. 2013;34(10):2399-2411), and substrates with adhesive patterns forming lines, geometrical shapes such as circular, square and Y-shaped (Junkin et al. 2011, Journal of Cell Science 124, 4213-4220), substrates with adhesive "hybrid" patterns that consist of the combination of a linear element and an arcuate element centered on the linear element (Bajaj et al., 2011, Integrative Biology 3, 897-909) or with adhesive patterns disclosed in WO 2015 / 091593, WO 2016 / 202850 and WO 2016 / 139312, Young et al. 2018 (Young et al. SLAS Discov. 2018 Sep;23(8):790-806), in Bray et al., 2008 (Bray et al. 2008. Cell motility and the cytoskeleton 65(8): 641), or in Ribeiro et al., 2015 (Ribeiro et al. 2015. Proc Natl Acad Sci U S A. 2015;112(41):12705-12710. Preferably, 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.

[0114] In a particular embodiment, 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

[0115] - 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,

[0116] - the ratio between the maximum width (Wc) of the central region (1C) and the maximum width (WL) of the lateral regions (IL) is greater than or equal to 2,

[0117] - the ratio between the length (L) and the maximum width (Wc) of the pattern (1) is less than or equal to 4, and wherein

[0118] - 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.

[0119] Preferably, the pattern is symmetrical according to its longitudinal axis (X) and to a transversal axis perpendicular to the longitudinal axis.

[0120] Preferably, 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 pm2.

[0121] Preferably, the method of culturing muscle cells, and in particular myoblasts / myotubes or cardiomyocytes, is adaptable to high-throughput platforms and to perform high- throughput assays. Before being imaged, 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.

[0122] In preferred embodiments, 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. Examples of cellular structures include, but are not limited to, nucleus, vacuole, mitochondrion, lysosome, cell membrane and cytoskeleton.

[0123] In particular, 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. In preferred embodiments, muscle cells are stained with at least one labelling agent revealing individual cells, in particular individual myotubes or cardiomyocytes. Optionally, the muscle cells, and in particular myotubes or cardiomyocytes, may be further stained with at least one labelling agent revealing nuclei.

[0124] Examples of 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). Examples of labelling agents that can be used to reveal nuclei include, but are not limited to, Hoechst and DAPI dyes. Examples of labelling agents that can be used to reveal mitochondria include, but are not limited to, Mitotracker™ dyes.

[0125] The choice of 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.

[0126] DM1 is known to induce RNA foci. Thus, in a particular embodiment, 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. In another particular embodiment, the disease of interest is DMD and the myotubes are stained for one ROI, i.e. individual myotubes. Optionally, the myotubes may be stained for another ROI, i.e. nuclei, in order to perform colocalization analysis in myotube nuclei.

[0127] 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.

[0128] As used herein, 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.

[0129] 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. In some embodiments, these moieties are covalently linked, e.g. an antibody or nucleic acid specifically recognizing the target and bearing a fluorochrome. In some other preferred embodiments, 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.

[0130] 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. Preferably, each labelling agent comprises a different fluorescence label having a different emission and / or excitation wavelength. For example, such labelling agent may be or comprise an antibody or nucleic acid probe conjugated to a fluorochrome (i.e. immunostaining). Alternatively, 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.

[0131] Accurate colocalization determination in fluorescence microscopy can be achieved if emission spectra of the fluorochromes are sufficiently separated. To achieve this aim, fluorescence labels can be selected such that their emission wavelengths are sufficiently separated and can be resolved by 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.

[0132] Labelling agents used to stain muscle cells may be easily chosen by the skilled person depending on the nature of molecules to be labelled. Preferably, if the molecule to be labelled is a protein, 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. Preferably, if the molecule to be labelled is a nucleic acid, 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.

[0133] The first and second molecules are cellular molecules, i.e. molecules naturally present in healthy or diseased muscle cells. The first molecule of interest may be a protein, a nucleic acid, a lipid or a carbohydrate. Preferably, the first molecule of interest is a protein or a nucleic acid. The second molecule may be a protein, a nucleic acid, a lipid or a carbohydrate. Preferably, the second molecule is a protein or a nucleic acid. In preferred embodiments, the first molecule of interest is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid. Preferably, one of these molecules is a protein. The first and the second molecules are different, e.g. two different proteins, a protein and a nucleic acid, or two different nucleic acids.

[0134] 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. Preferably, 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.

[0135] 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. Preferably, 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. For example, dystrophin directly interacts with P-dystroglycan (b-DG). An indirect interaction involves intermediate molecule(s) between the first and the second molecules. In particular, the first and second molecules may belong to the same protein complex but without direct physical contact. For example, dystrophin and a- sarcoglycan (a-SG) indirectly interact; they are components of the dystrophin-glycoprotein complex (DGC) but do not directly interact together.

[0136] Examples of 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. For each disease in the column "neuromuscular disease" or "cardiomyopathy", 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". In preferred embodiments, 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. Preferably, in embodiments wherein the muscle cells are stained with at least one labelling agent revealing at least one region of interest (ROI), said at least one ROI comprises a ROI of Table 1 or 2 corresponding to the first and second molecules and the disease. As illustration, in embodiments wherein the disease of interest is Duchenne Muscular Dystrophy, 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.

[0137] 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 (*)

[0138] (1) LGMD: Limb-girdle muscular dystrophy. For 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. (2) Abbreviations: Dystrophin (DMD), Utrophin (UTRN), a-syntrophin (SNTA), b-syntrophin

[0139] (SNTB), 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), Filamin C (FLNC), Collagen VI alphal (COL6A1), Collagen VI alpha2 (COL6A2), Collagen VI alpha3 (COL6A3), Laminin alpha2 (LAMA2), integrin a7 (ITGA7), a-dystroglycan (DAG1), Desmin (DES), Fukutin (FKTN), Fukutin-related protein (FKRP), Protein O-mannosyl-transferase 1 / 2 (POMT1 / 2), Protein O-linked mannose betal,2-N-acetylglucosaminyltransferase 1 (POMGNT1), Xylosyl- and glucuronyltransferase LARGE1 (LARGE1), Myosin heavy chain 2 (MYH2), Desmin (DES), a-Actin (ACTA1), Nebulin (NEB), Myosin heavy chain 7 (MYH7), Myotilin (MYOT), Titin (TTN), Troponin T (TNNT3), muscle-RING-finger-1 (MURF1), Lamin A / C (LMNA), Emerin (EMD), Nesprin 1 / 2 (SYNE1 / 2), Transportin (TNPO3), Barrier-to-autointegration factor 1 (BAF1), Myc box-dependent-interacting protein 1 (BINI a.k.a amphiphysin 2), Dynamin 2 (DNM2), Striated Preferentially Expressed protein kinase (SPEG), Myotubularin 1 (MTM1), Ryanodine receptor 1 (RYR1), Voltage-dependent L-type calcium channel subunit alpha-lS (CACNA1S a.k.a DHPR), Caveolin 3 (CAV3), Calpain 3 (CAPN3), SH3 and cysteine rich domain 3 (STAC3), Calsequestrin-1 (CASQ1), Stromal interaction molecule 1 (STIM1), ORAI calcium release-activated calcium modulator 1 (Orail), Calcium voltage-gated channel auxiliary subunit beta 1 (CACNB1), Triadin (TRDN), Aspartyl / asparaginyl beta-hydroxylase (ASPH, a.k.a Junctin), ATPase sarcoplasmic / endoplasmic reticulum Ca2+ transporting 1 (SERCA1), Chloride channel protein 1 (CLCN1), Anoctamin-5 (ANO5), Dysferlin (DYSF), Myc box-dependent-interacting protein 1 (BINI a.k.a amphiphysin 2), Tripartite motif-containing protein 72 (TRIM72), EH domain-containing protein 1 (EHD1), EH domain-containing protein 2 (EHD2), Annexin Al (ANXA1), Annexin A2 (ANXA2), Annexin A5 (ANXA5), DnaJ homolog subfamily B member 6 (DNAJB6), Nucleotide exchange factor SIL1 (SIL1), BAG family molecular chaperone regulator 3 (BAG3), Heat shock protein beta-5 (HSPB5), E3 ubiquitin- protein ligase TRIM32 (TRIM32), Kelch-like protein 40 / 41 (KLHL40 / KLHL41), Lysosome-associated membrane glycoprotein 1 (LAMP1), Lysosome-associated membrane glycoprotein 2 (LAMP2), Sequestosome-l(SQSTMl), Lysosomal alpha-glucosidase (GAA), Muscle-RING-finger-1 (MURF1), Ectopic P granules protein 5 homolog (EPG5), Vacuolar ATPase assembly integral membrane protein VMA21 (VMA21), DMPK RNA, Muscleblind-like protein l(MBNLl), Muscleblind-like protein 2 (MBNL2), Muscleblind-like protein 3 (MBNL3), RNA binding protein fox-1 homolog 1 (RBFOX1), CCHC-type zinc finger nucleic acid binding protein (CNBP, a.k.a ZNF9) RNA, FXN (Frataxin), Citrate synthase (CS), Leucin rich pentatricopeptide repeat containing (LRPPRC), Solute carrier family 25 member 24 (SLC25A24), Translocase of inner mitochondrial membrane 44 (TIM M44), Glutaryl-CoA dehydrogenase (GCDH), TNF receptor associated protein 1 (TRAP1), Mitochondrially encoded cytochrome c oxidase I (MT-CO1), Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), Mitochondrially encoded cytochrome b (MT-CYB), ATP synthase Fl subunit beta (ATP5F1B), Heat shock protein family D member 1 (HSPD1), Coiled-coil-helix-coiled-coli-helix domain containing 2 (CHCHD2), Cytochrome C oxidase subunit 6B1 (COX6B1), Cytochrome c oxidase subunit 411 (COX4I1), ATP synthase membrane subunit e (ATP5ME), Solute carrier family 25 member 3 (SLC25A3), Translocase of outer mitochondrial membrane 20 (TOMM20), NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4). (*) Lapidos et al. Circulation Research. 2004; 94:1023-1031; Quan Gao and Elizabeth M.

[0140] McNally, Compr Physiol. 2015; 5(3): 1223-1239; Rahimov et al. J Cell Biol (2013) 201 (4): 499-510;

[0141] Dowling et al., Nature Reviews. 2021; Masayuki Nakamori et al Muscle Nerve. 2007 Aug;36(2):251-7;

[0142] Vihola et al. Neuropathol Appl Neurobiol. 2013 Jun; 39(4)

[0143] 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 (*)

[0144] Restrictive cardiomyopathy (RCM), Arrhythmogenic cardiomyopathy (ARVC).

[0145] (2) Abbreviations: 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 (LDB3), PDZ LIM domain protein 3 (PDLIM3), Metavinculin (VCL), Crystallin alpha B (CRYAB), Integrin-linked kinase (ILK), Laminin subunit alpha 4 (LAMA4), (Sarcoglycan Gamma (SGCG), Sarcoglycan beta (SGCB), Sarcoglycan alpha (SGCA), Sarcoglycan delta (SGCD), Dystrophin (DMD), Tafazzin (TAZ), Eyes-absent 4 (EYA4), ATP binding cassette subfamily C member 9 (SUR2), Desmoplakin (DSP), desmoglein 2 (DSG2), Desmocollin 2 (DSC2), Plakophilin 2 (PKP2), Junction plakoglobin (JUP), Lamin A / C (LMNA), Emerin (EMD), Lamina- associated polypeptide 2, isoform alpha (TMPO), Transmembrane protein 43 (TMEM43), Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A), ATP Binding Cassette Subfamily C Member 9 (ABCC9), Cardiac phospholamban (PLN), Ryanodine receptor 2 (RYR2), Calsequestrin 2 (CASQ2), presenilin 1 / 2 (PSEN1 / 2), Junctophilin 2 (JPH2), Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A), ATP Binding Cassette Subfamily C Member 9 (ABCC9), Cardiac phospholamban (PLN), Ryanodine receptor 2 (RYR2), CACNA1C (calcium voltage-gated channel subunit alphal C), Calsequestrin 2 (CASQ2), Junctophilin 2 (JPH2), BAG family molecular chaperone regulator 3 (BAG3), Tafazzin (TAZ), muscle- RING-finger-1 (MURF1), Titin (TTN), Mitochondrial import inner membrane translocase subunit Tim23 (TIM23).

[0146] (*) Hershberger et al. Genet. Med., 12 (2010), pp. 655-667; Cirino AL, Ho C. Hypertrophic Cardiomyopathy Overview. 2008 Aug 5 [Updated 2021 Jul 8], In: Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews® [Internet], Seattle (WA): University of Washington, Seattle; 1993-2023; McNally E, MacLeod H, Dellefave-Castillo L. Arrhythmogenic Right Ventricular Cardiomyopathy. 2005 Apr 18 [Updated 2017 May 25], In: Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews® [Internet], Seattle (WA): University of Washington, Seattle; 1993-2023; Ciarambino et al. Int J Mol Sci. 2021 Jul 19;22(14):7722; Linke et al. Circ Res. 2014 Mar 14;114(6):1052-68; Higashikuse et al. Dis Model Meeh. 2019 Nov 15;12(ll):dmm041103

[0147] Dilated cardiomyopathy (DCM), Hypertrophic cardiomyopathy (HCM), Restrictive cardiomyopathy (RCM) and Arrhythmogenic cardiomyopathy (ARCV) are large groups of cardiac pathologies that can be induced by a variety of genes mutations. Each cardiomyopathy of Table 2 may be further characterized by a mutation in the gene encoding the molecule indicated in the column "Examples of molecules useful as first molecules". As illustration, the cardiomyopathy may be a dilated cardiomyopathy (DCM) driven by a mutation in the gene encoding ACTC1 (Actin Alpha Cardiac Muscle). In this case, 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.

[0148] In some embodiments, 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.

[0149] In some embodiments, 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.

[0150] In some embodiments, 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.

[0151] In some embodiments, 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.

[0152] In some embodiments, 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.

[0153] In some embodiments, 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.

[0154] In some embodiments, 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.

[0155] In some embodiments, 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.

[0156] In some embodiments, 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.

[0157] In some preferred embodiments, in particular when the disease of interest is Duchenne muscular dystrophy or myotonic dystrophy type 1, 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. In particular, 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, |3- dystroglycan, a-dystroglycan and dysferlin, more preferably selected from the group consisting of a-sarcoglycan and p-dystroglycan. Alternatively, 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.

[0158] In some other preferred embodiments, in particular when the disease of interest is 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. Alternatively, 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.

[0159] 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. In particular, the skilled person can use a method comprising

[0160] (a) providing at least one image of at least one in vitro cultured healthy muscle cell, preferably at least one in vitro cultured healthy myotube or cardiomyocyte, and at least one image of at least one in vitro cultured muscle cell exhibiting at least one feature of a disease of interest ("diseased muscle cell"), preferably at least one in vitro cultured diseased myotube or cardiomyocyte, wherein said muscle cells have been stained for a first cellular molecule and for a second cellular molecule, preferably selected as described above; and

[0161] (b) determining quantitatively the degree of colocalization of the first cellular molecule and the second cellular molecule in said at least one healthy muscle cell and said at least one diseased muscle cell by performing quantitative colocalization analysis, wherein the combination of the first and second molecules can be used in the methods of the invention, in particular in methods relating to the disease of interest, when the degree of colocalization in said at least one healthy muscle cell is significantly different from the degree of colocalization in said at least one diseased muscle cell.

[0162] Healthy and diseased cells are cultured in the same conditions to provide comparable degrees of colocalization. Preferably, the number of healthy and diseased cells is chosen in order to provide statistically significant results.

[0163] As mentioned above, thanks to the possibility of using high-throughput platforms to culture, stain and capture images of muscle cells, this method may be easily reiterated without inducing an undue burden on the skilled person.

[0164] After muscle cells staining, images of these stained muscle cells are captured.

[0165] 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. Preferably, 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.

[0166] The choice of the imaging technique also depends on the nature of the signals emitting by the labelling agents. Preferably, 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

[0167] Optionally, an illumination function may be applied on the acquired images to correct uneven illumination.

[0168] As used herein, measurement of each different label may be also referred to as a detection channel. For example, in fluorescence detection, each channel corresponds to one label having a particular emission or excitation wavelength. In some embodiments, 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.

[0169] In preferred embodiments, 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. In these embodiments, before performing quantitative colocalization analysis, 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). For example, 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. Preferably, 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++.

[0170] After providing image(s) of stained in vitro cultured muscle cell(s), and optionally performing an image segmentation to identify ROI(s), 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.

[0171] To determine the degree of colocalization, 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. In particular, the degree of colocalization is preferably determined in at least 30 muscle cells, more preferably in at least 60 muscle cells. In preferred embodiments, 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).

[0172] 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. Preferably, the methods of the invention do not comprise any step using a machine learning based analysis. Preferably, the quantitative colocalization analysis is performed using a quantitative pixel-based colocalization analysis. In this case, 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.

[0173] Methods to determine quantitatively a degree of colocalization between two molecules by performing quantitative colocalization analysis, in particular using pixel intensity-based colocalization analysis, are well known by the skilled person (see e.g. Costes et al. Biophysical Journal Volume 86 June 2004 3993-4003; Zinchuk et al. Acta Histochem. Cytochem. 40 (4): 101-111, 2007 ; Wu et al. Biophysical Journal Volume 98 February 2010 493-504). Typically, the analysis is assisted by computer software. The software can estimate the degree of colocalization according to specialized algorithms within the image or the selected region(s) of interest (ROI).

[0174] When two molecules are spatially distributed over a region of the cell being analyzed, no specific colocalization exists between the two molecules when their spatial distributions are independent of each other, although some amount of random overlap will be present. Conversely, if the two molecules have some specific colocalization, then the overlay of the two spatial distributions will show a level of correlation that is in excess of the random overlap.

[0175] 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. Typically, the determination of threshold values is assisted / done by computer software. Typically, 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. In some preferred embodiments wherein the labelling agents of the first and second molecules comprise a primary and a secondary antibodies as disclosed above, 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. Optionally, before colocalization readout calculations, one or more filter steps may be applied on the image(s). In particular, 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.

[0176] 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. In particular, the degree of colocalization may be quantitatively determined by calculating a value representing an overlap coefficient of the two detection channels.

[0177] Preferably, 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). In particular, 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. Preferably, 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.

[0178] In some embodiments, determining the degree of colocalization comprises calculating the Pearson's Colocalization Coefficient (PCC) for each muscle cell or ROI, preferably each muscle cell.

[0179] 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. This later is more stringent as the region where none of the molecules is present positively affects the PCC. If the optional image filtration has been chosen, some ROI might have no pixel intensity above zero for one molecule related channel. In this case, the denominator value will equal zero, leading to an undefined PCC. Because the correlation between the other molecule related signal and this unvarying signal is null, the PCC may be set to 0.

[0180] In some embodiments, 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.

[0181] In some embodiments, 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 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 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.

[0182] In some embodiments, 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). In particular, 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. average of the overlap coefficients of the muscle cells or ROIs, e.g. mean PCC, mean MCC, mean RWC), a quantile value, e.g. a median value, or a ratio, e.g. ratio between colocalization value in first ROI, e.g. myotubes, and 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.

[0183] Optionally, in embodiments wherein determining the degree of colocalization comprises calculating the Pearson's Colocalization Coefficient (PCC), 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. To define this threshold, 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

[0184] Optionally, to strengthen the confidence in the colocalization readouts, 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. In this case, 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. In particular, the negative control molecule may be a molecule located all over the muscle cell in a homogeneous manner. For example, in embodiments wherein the muscle cell is a myotube, the negative control molecule may be the myosin heavy chain (MHC).

[0185] 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. In some embodiments, 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. In some other embodiments, 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).

[0186] Methods of the invention

[0187] The inventors herein demonstrated that the quantitative colocalization assays of the invention can be used to quantitatively monitor the colocalization of two cellular molecules known to directly or indirectly interact in healthy or diseased muscle cells, and thus to quantitatively monitor the functionality of these molecules.

[0188] In a first aspect, the present invention thus relates to an in vitro method of assessing the functionality of a cellular molecule of interest in a muscle cell.

[0189] The method comprises

[0190] (a) providing at least one image of at least one in vitro cultured muscle cell, wherein said at least one muscle cell 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

[0191] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest in said at least one muscle cell.

[0192] In preferred embodiments, 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.

[0193] Thus, in preferred embodiments, the method comprises

[0194] (i) providing at least one image of at least one in vitro cultured muscle cell, wherein said at least one muscle cell has been stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first molecule 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 muscle cells and cellular structures of muscle cells, and any combination thereof; and (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0195] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest in said at least one muscle cell.

[0196] 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.

[0197] The muscle cells used in this method may be healthy or diseased cells.

[0198] In an embodiment, the method comprises

[0199] (a) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube 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

[0200] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest.

[0201] In particular, the method may comprise

[0202] (i) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube has been stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first molecule 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 and cellular structures of myotubes, and any combination thereof; and

[0203] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0204] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest in said at least one myotube.

[0205] Preferably, 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.

[0206] In another embodiment, the method comprises

[0207] (a) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein said at least one cardiomyocyte has been stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first molecule of interest; and

[0208] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest.

[0209] In particular, the method may comprise

[0210] (i) providing at least one image of at least one in vitro cultured card io myocyte, wherein said at least one cardiomyocyte 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 has 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

[0211] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0212] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second molecule in at least one ROI by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular molecule of interest in said at least one card io myocyte.

[0213] Preferably, 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.

[0214] In this method, 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. Depending on the first and second molecules, 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. On the other hand, a negative correlation is a relationship between two variables in which one variable increases as the other decreases, and vice versa.

[0215] In some embodiments, 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.

[0216] Depending on the first and second molecules, 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. For example, in embodiments wherein 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.

[0217] By performing the method on different muscle cells or set of muscle cells, e.g. on healthy cells and diseased cells, it is thus possible to compare the functionality of a molecule in said cells or set of cells. In particular, when the degree of colocalization determined in the muscle cell(s) of interest is significantly different from the degree of colocalization determined in the same conditions in healthy muscle cells, this means that the functionality of the cellular molecule of interest is altered or impaired in the muscle cell(s) of interest.

[0218] As used herein, the term "functionality" refers to an activity of the molecule in a healthy muscle cell. In particular, a functionality of a nucleic acid may be its capacity of providing a functional encoded protein in a sufficient amount. As illustration, a functionality of DMPK RNA may be the capacity of providing a functional encoded DMPK protein in a sufficient amount. Indeed, 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. As illustration, a functionality of dystrophin may be its activity as an essential component of the Dystrophin associated protein complex (DGC).

[0219] 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.

[0220] Thus, in another aspect, 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.

[0221] The method comprises

[0222] (a) providing at least one image of at least one in vitro cultured 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 molecule of interest; and

[0223] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing quantitative colocalization analysis,

[0224] (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.

[0225] In preferred embodiments, 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.

[0226] Thus, in preferred embodiments, the method comprises

[0227] (i) providing at least one image of at least one in vitro cultured 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 cellular second molecule interacting with said first molecule 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 muscle cells and cellular structures of muscle cells, and any combination thereof; and

[0228] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0229] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis, and

[0230] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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. 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.

[0231] The muscle cells used in this method may be healthy or diseased cells.

[0232] In embodiments wherein the method is used to test the toxicity of a compound on muscle cells, the muscle cells used in the assay and as reference muscle cell are preferably healthy muscle cells. In embodiments wherein the method is used to test the capacity of a compound to treat a muscular disease or improve a feature of a muscular disease, 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.

[0233] In an embodiment, the method comprises

[0234] (a) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube 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

[0235] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing quantitative colocalization analysis,

[0236] (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.

[0237] In particular, the method may comprises

[0238] (i) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube 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 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 and cellular structures of myotubes, and any combination thereof; and

[0239] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0240] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis, and

[0241] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 molecule of interest in said at least one myotube.

[0242] The myotubes used in this method may be healthy or diseased myotubes, in particular myotubes exhibiting a feature of a neuromuscular disease of interest.

[0243] In another embodiment, the method comprises

[0244] (a) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein said at least one cardiomyocyte 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 molecule of interest; and

[0245] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing quantitative colocalization analysis,

[0246] (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.

[0247] In particular, the method may comprises

[0248] (i) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein said at least one cardiomyocyte 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 has 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 card io myocytes, and any combination thereof; and

[0249] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0250] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the cellular second molecule in at least one ROI by performing quantitative colocalization analysis, and

[0251] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0252] The cardiomyocytes used in this method may be healthy or diseased cardiomyocytes, in particular cardiomyocytes exhibiting a feature of a cardiomyopathy of interest.

[0253] 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. In particular, this compound may be a drug authorized by a regulatory authority such as FDA or EMA.

[0254] 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. 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)).

[0255] The reference degree of colocalization is obtained by performing steps (a) and (b) or (i) to (iii) on at least one reference muscle cell. Preferably, 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.

[0256] 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.

[0257] The method may further comprise determining a reference degree of colocalization. In particular, the method may further comprise

[0258] - providing at least one image of at least one in vitro cultured reference muscle cell, in particular at least one in vitro cultured reference myotube or cardiomyocyte, wherein said at least one reference muscle cell has not been contacted with the compound to be tested or has been contacted with a determined concentration of said compound, has been stained for the first cellular molecule of interest and for the second cellular molecule interacting with said first molecule of interest; and

[0259] - quantifying the reference degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one reference muscle cell by performing quantitative colocalization analysis.

[0260] In embodiments wherein the cells are stained for ROI(s), the method may further comprise

[0261] - providing at least one image of at least one in vitro cultured reference muscle cell, , in particular at least one in vitro cultured reference myotube or cardiomyocyte, wherein said at least one reference muscle cell has not been contacted with the compound to be tested or has been contacted with a determined concentration of said compound, has been stained for the first cellular molecule of interest and for the second cellular molecule interacting with said first molecule 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 muscle cells and cellular structures of muscle cells, and any combination thereof; and,

[0262] -performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI and

[0263] - determining quantitatively a reference degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis.

[0264] Preferably, 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.

[0265] To be comparable, 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.

[0266] 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. As explained above, the compound may be able to positively or negatively alter the functionality of the first cellular molecule of interest.

[0267] The statistical significance of the difference may be assessed by any method known by the skilled person. In particular, 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. Typically, a p-value below 0.05 indicates that the difference is significant.

[0268] Optionally, 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).

[0269] Thus, in another aspect, the present invention also relates to an in vitro method of predicting the ability of a compound to treat a muscular disease of interest.

[0270] The method comprises

[0271] (a) providing at least one image of at least one in vitro cultured diseased muscle cell, i.e. a muscle cell exhibiting a feature of a muscular disease of interest, 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

[0272] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased muscle cell by performing quantitative colocalization analysis,

[0273] (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.

[0274] In preferred embodiments, 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.

[0275] Thus, in preferred embodiments, 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

[0276] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0277] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0278] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 neuromuscular disorder.

[0279] 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.

[0280] The muscle cells used in this method are diseased muscle cells, i.e. muscle cells exhibiting a feature of a muscular disease.

[0281] As used herein, the term "muscular disease" refers to a neuromuscular disease or a cardiomyopathy. As used herein, the terms "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. This term encompasses a wide range of disorders including, but not limited to, 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 myopathy (CNM), X-linked myotubular myopathy (XLMTM), Tubular aggregate myopathy, Malignant hyperthermia syndrome, Inclusion body myopathy, Protein aggregate myopathy, Nemaline myopathies, Congenital myopathy (CM), Vacuolar aggregate myopathy, 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; congenital myasthenic syndromes such as myasthenia gravis and other myasthenic syndromes driven by mutations in CHAT, COLQ, RAPSN, CHRNE, DOK7 and / or GFPT1 genes; or motor neuron diseases such as Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS), Friedreich's Ataxia, Kennedy's disease, cachexia, sarcopenia and muscle atrophy.

[0282] Preferably, 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), Tubular aggregate myopathy, Malignant hyperthermia syndrome, Inclusion body myopathy, Protein aggregate myopathy, Nemaline myopathies (preferably Nemaline myopathy 2 or 8), Congenital myopathy (CM), Vacuolar aggregate myopathy, Myoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjbgren syndrome, Neurodegeneration with ataxia, Friedreich's Ataxia, dystonia, and gaze palsy, childhood-onset (NADGP) and Pompe disease. In particular, 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), Tubular aggregate myopathy, Malignant hyperthermia syndrome, Inclusion body myopathy, Protein aggregate myopathy, Nemaline myopathies (preferably Nemaline myopathy 2 or 8), Congenital myopathy (CM), Vacuolar aggregate myopathy, 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) and Pompe disease.

[0283] In an embodiment, 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. Preferably, the neuromuscular disease of interest is selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes and motor neuron diseases.

[0284] In a preferred embodiment, 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), Emery-Dreifuss muscular dystrophy, Limb-girdle muscular dystrophies, Walker-Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy and Tibial muscular dystrophy, more preferably selected from the group consisting of Duchenne Muscular Dystrophy (DMD) and Myotonic Dystrophy 1 (DM1).

[0285] In another particular embodiment, 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 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) and Pompe disease.

[0286] In another particular embodiment, 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.

[0287] In a further particular embodiment, 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.

[0288] In a further embodiment, the neuromuscular disease of interest is a metabolic muscle disorder selected from the group consisting of cachexia, sarcopenia and muscle atrophy.

[0289] In preferred embodiments, the neuromuscular disease is selected from the group consisting of Duchenne Muscular Dystrophy (DMD) and Myotonic Dystrophy 1 (DM1). As used herein, the term "cardiomyopathy" refers to a disease of the heart muscle where the walls of the heart chambers have become stretched, thickened or stiff. This affects the heart's ability to pump blood around the body and can lead to heart failure. The cardiomyopathy may be dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and arrhythmogenic cardiomyopathy (ARCV). Preferably, 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. As illustration, the cardiomyopathy may be a dilated cardiomyopathy (DCM) driven by a mutation in the gene encoding ACTC1 (Actin Alpha Cardiac Muscle).

[0290] In embodiments wherein the disease is a neuromuscular disease, the muscle cells are preferably myotubes or cardiomyocytes, more preferably myotubes. In embodiments wherein the disease is a cardiomyopathy, the muscle cells are preferably cardiomyocytes.

[0291] In an embodiment, the muscular disease is a neuromuscular disease and the method comprises

[0292] (a) providing at least one image of at least one in vitro cultured diseased myotube, i.e. a myotube exhibiting a feature of a neuromuscular disease of interest, wherein said at least one myotube 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

[0293] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased myotube by performing quantitative colocalization analysis,

[0294] (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.

[0295] In preferred embodiments, 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.

[0296] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease and the method comprises

[0297] (i) providing at least one image comprising at least one in vitro cultured diseased myotube, wherein said at least one myotube 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

[0298] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0299] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0300] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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. Preferably, 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.

[0301] In another embodiment, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0302] (a) providing at least one image of at least one in vitro cultured diseased cardiomyocyte, i.e. a cardiomyocyte exhibiting a feature of a neuromuscular disease or cardiomyopathy of interest, wherein said at least one cardiomyocyte 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

[0303] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased cardiomyocyte by performing quantitative colocalization analysis,

[0304] (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.

[0305] In preferred embodiments, 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.

[0306] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0307] (i) providing at least one image comprising at least one in vitro cultured diseased cardiomyocyte, wherein said at least one cardiomyocyte 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 card io myocytes, a structure of cardiomyocytes, and any combination thereof; and

[0308] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0309] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0310] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 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 or cardiomyopathy.

[0311] Preferably, 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.

[0312] The compound to be tested may be as defined above.

[0313] 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. 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)).

[0314] The reference degree of colocalization is obtained by performing steps (a) and (b) or (i) to (iii) on at least one reference muscle cell. Preferably, 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. Preferably, 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.

[0315] The method may further comprise determining a reference degree of colocalization as described above.

[0316] 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. As explained above, a positive correlation exists when one variable decreases as the other variable decreases, or one variable increases while the other increase. In this aspect, 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).

[0317] 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.

[0318] 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.

[0319] Indeed, as explained above, 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.

[0320] If the concentration of the compound used to determine the reference degree of colocalization is higher than the concentration used in the assay and the degree of colocalization positively correlates with the health status of the muscle cell, then 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).

[0321] If the concentration of the compound used to determine the reference degree of colocalization is lower than the concentration used in the assay (in particular when the reference muscle cell is not contacted with the compound) and the degree of colocalization positively correlates with the health status of the muscle cell, then 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).

[0322] If the concentration of the compound used to determine the reference degree of colocalization is higher than the concentration used in the assay and the degree of colocalization negatively correlates with the health status of the muscle cell, then 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).

[0323] If the concentration of the compound used to determine the reference degree of colocalization is lower than the concentration used in the assay and the degree of colocalization negatively correlates with the health status of the muscle cell, then the desired variation is a decrease of the degree of colocalization (the difference between the degree of colocalization and the reference degree is negative).

[0324] Thus, 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. When 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.

[0325] In preferred embodiments, 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.

[0326] Preferably, 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. In particular, 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.

[0327] 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.

[0328] Thus, in another aspect, 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.

[0329] 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

[0330] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing quantitative colocalization analysis,

[0331] (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.

[0332] In preferred embodiments, 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.

[0333] Thus, in preferred embodiments, the method comprises

[0334] (i) providing at least one image comprising 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 stained for a first cellular molecule of interest and for a second cellular molecule interacting with said first cellular molecule 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 muscle cells, cellular structures of muscle cells, and any combination thereof; and

[0335] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0336] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0337] 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.

[0338] In an embodiment, the muscular disease is a neuromuscular disease and the method comprises

[0339] (a) providing at least one image of at least one in vitro cultured myotube obtained / derived from a sample of a patient affected with a neuromuscular disease after administration of the therapeutic compound, wherein said at least one myotube 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

[0340] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing quantitative colocalization analysis,

[0341] (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.

[0342] In preferred embodiments, 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.

[0343] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease and the method comprises

[0344] (i) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from a sample of said patient after administration of the therapeutic compound, wherein said at least one myotube 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 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, cellular structures of myotubes, and any combination thereof; and

[0345] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0346] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0347] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0348] Preferably, 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. In another embodiment, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0349] (a) providing at least one image of at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient affected with a neuromuscular disease or cardiomyopathy after administration of the therapeutic compound, wherein said at least one cardiomyocyte 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

[0350] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing quantitative colocalization analysis,

[0351] (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.

[0352] In preferred embodiments, 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.

[0353] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0354] (i) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from a sample of said patient after administration of the therapeutic compound, wherein said at least one cardiomyocyte 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 has been stained with at least one labelling agent revealing at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes, cellular structures of card io myocytes, and any combination thereof; and

[0355] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0356] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0357] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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.

[0358] Preferably, 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.

[0359] The term "sample", as used herein, means any sample containing muscle cells derived from a subject, preferably a sample which contains myoblasts or cardiomyocytes. Examples of such 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.

[0360] In another aspect, 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.

[0361] The method comprises

[0362] (a) providing at least one image comprising at least one in vitro cultured muscle cell obtained / derived from a sample of a patient affected with a muscular disease, wherein said at least one muscle cell has been contacted with a therapeutic compound, 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

[0363] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing quantitative colocalization analysis, and

[0364] (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.

[0365] In preferred embodiments, 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.

[0366] Thus, in preferred embodiments, the method comprises

[0367] (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained / derived from a sample of a patient affected with a muscular disease, 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 muscle cells, cellular structures of muscle cells, and any combination thereof; and

[0368] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0369] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0370] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 indicates that said patient is susceptible to benefit from a treatment with said therapeutic compound.

[0371] 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.

[0372] In an embodiment, the muscular disease is a neuromuscular disease and the method comprises

[0373] (a) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from a sample of a patient affected with a neuromuscular disease, wherein said at least one myotube has been contacted with a therapeutic compound, 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 (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing quantitative colocalization analysis, and

[0374] (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.

[0375] In preferred embodiments, 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.

[0376] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease and the method comprises

[0377] (i) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from a sample of a patient affected with a neuromuscular disease, wherein said at least one myotube 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, cellular structures of myotubes, and any combination thereof; and

[0378] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0379] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 indicates that said patient is susceptible to benefit from a treatment with said therapeutic compound.

[0380] Preferably, 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.

[0381] In another embodiment, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0382] (a) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient affected with a neuromuscular disease or a cardiomyopathy, wherein said at least one cardiomyocyte has been contacted with a therapeutic compound, 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

[0383] (b) quantifying the degree of colocalization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing quantitative colocalization analysis, and

[0384] (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. In preferred embodiments, 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.

[0385] Thus, in preferred embodiments, the muscular disease is a neuromuscular disease or a cardiomyopathy and the method comprises

[0386] (i) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient affected with a neuromuscular disease or a cardiomyopathy, wherein said at least one cardiomyocyte 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 card io myocytes, cellular structures of cardiomyocytes, and any combination thereof; and

[0387] (ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and

[0388] (iii) determining quantitatively a degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing quantitative colocalization analysis; and

[0389] (iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 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 indicates that said patient is susceptible to benefit from a treatment with said therapeutic compound. Preferably, 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.

[0390] 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.

[0391] 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. Preferably, 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)).

[0392] The compound to be tested may be as defined above.

[0393] The reference degree of colocalization may be obtained as described above. Preferably, the reference degree of colocalization is determined using muscle cells obtained / derived from the same sample than the muscle cells of the assay.

[0394] The method may further comprise determining a reference degree of colocalization as described above.

[0395] 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.

[0396] When the variation is in a direction opposite to the desired variation, then the assay does not indicate that the patient is susceptible to benefit from a treatment with the therapeutic compound.

[0397] Preferably, 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. In particular, 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. Optionally, the methods of the invention may further comprise before step (a) or (i)

[0398] - culturing 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;

[0399] - staining these muscle cells, in particular myotubes or cardiomyocytes, for said first molecule, for said second molecule and optionally with said at least one labelling agent; and

[0400] - capturing at least one image of at least one stained muscle cell.

[0401] In some preferred embodiments of the methods of the invention, 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 |3- dystroglycan.

[0402] In particularly preferred embodiments of the methods of the invention, the muscular disease is Duchenne muscular dystrophy and the first molecule is dystrophin and the second molecule is a-sarcoglycan or p-dystroglycan.

[0403] In some other preferred embodiments of the methods of the invention, 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.

[0404] In the present application, a protein belonging to the Dystrophin Glycoprotein complex (DGC) is preferably selected from the group consisting of DMD, SNTA, SNTB, ANK1, ANK2, DAG1, SSPN, SGCA, SGCB, SGCD, SGCG, DTNA and FLNC.

[0405] In preferred embodiments, the methods of the invention are computer-implemented methods. As used herein, the term "computer-implemented method" refers to a method which involves a programmable apparatus, specifically a computer, a computer network, or a readable medium carrying a computer program, whereby at least one step of the method is performed by using at least one computer program. A computer-implemented method may further comprise at least one step that is not performed by using a computer program, e.g. a cell culture step. In preferred embodiments, the methods of the invention do not involve the use of a trained classifier. In another aspect, 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.

[0406] All embodiments disclosed above for the methods of the invention are also encompassed in this aspect.

[0407] In an embodiment, 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. Preferably, 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. Preferably, the muscle cell is a myotube or a cardiomyocyte and the muscular disease is a neuromuscular disease or a cardiomyopathy. Preferably, 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).

[0408] In another embodiment, 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. Preferably, 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.

[0409] EXAMPLES MATERIALS & METHODS

[0410] Cell source and maintenance

[0411] Primary human skeletal muscle myoblasts from Healthy and DMD donors were sourced from different donors (Table 3). Muscle cells were amplified to create master and working cell banks following suppliers' recommendations. Table 3: Donor Characteristics

[0412] 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). Finally, 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). Cells were selected based on consistency in their doubling time, proportion of Desmin+ cells and fusion index.

[0413] High-Throughput Myotube Formation

[0414] All steps were accomplished automatically using a Freedom EVO150 workstation (Tecan). Growth medium for Healthy and DMD cells myoblasts was Skeletal Muscle Cell Growth Medium provided by ZENBIO, while DM1 cells were cultured in DMEM / F10 (Thermo Fisher Scientific) supplemented with 20% Fetal Bovine Serum, 5pg / ml Bovine Insulin (Sigma), 0.4pg / ml Dexamethasone (Sigma) and lOng / ml FGF2 (Miltenyi Biotec).

[0415] For all experiments, at Day 0, MyoScreen™ plates (CYTOO, France, International patent application WO 2015 / 091593, Young et al. SLAS Discov. 2018 Sep;23(8):790-806) containing micropatterns coated with 10 pg / ml fibronectin (Invitrogen) were pre-filled with lOOpl / well of growth medium and stored in the incubator at 37°C. Human primary myoblasts were detached from the flasks, counted, and seeded into the plates with 15 000 cells per well in lOOpI of growth medium. At Day 1, 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.

[0416] 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.

[0417] 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). After 4h30, plating medium was replaced by lOOpL of maintenance medium (Fujifilm) containing 1% of penicillin-streptomycin (Invitrogen). On Day 2 and Day 6 maintenance medium was refreshed. siRNA treatment of myotubes and hIPSC-CM

[0418] 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.

[0419] Table 4: siRNA information for DMD siRNA treatment of hIPSC-CM

[0420] 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.

[0421] VivoPMO, PMO and ASO treatments

[0422] Morpholino oligonucleomers ("PMOs") 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).

[0423] After four days of culture, differentiated healthy and DMD myotubes were transfected following the manufacturer's instructions. 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.

[0424] Table 5: PMO and vivo PMO information After four days of culture, differentiated healthy and DM1 myotubes were transfected with a (CAG)7 ASO (antisense oligonucleotide) (Mulders et al., 2009, Cell, 106(33), 13915-13920) with doses ranging from 2.7 to lOnM final concentration using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to respective manufacturers' instructions.

[0425] Immunofluorescence staining

[0426] At Day 9, myotubes were fixed for 30 min in 3.3% formalin (Sigma-Aldrich) and for 20min in 3.3% formalin for hIPSC-CM. Subsequent immunofluorescence staining was according to Young et al., 2018 (Advancing Life Sciences R&D, 23(8), 790-806). Myotubes and hIPSC-CM were washed three times in Dulbecco's Phosphate-Buffered Saline (DPBS, Invitrogen) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich). After blocking in 1% bovine serum albumin for 20 min (BSA, Sigma-Aldrich), cells were incubated with primary antibodies listed in Table 6 prepared in BSA 1% for 2h at room temperature (or overnight for NCL-Dys2 antibody) and then washed three times in DPBS. Secondary antibodies (Thermo Fisher Scientific and Jackson ImmunoResearch) were added at room temperature for 2 h with Hoechst 33342 (Invitrogen). Cells were washed three times in DPBS before image acquisition.

[0427] Table 6: Antibodies

[0428] Fluorescence In Situ Hybridation (FISH)

[0429] At day 9, after fixation with 4% paraformaldehyde (Sigma-Aldrich) for 10 min the cells are washed three times in Dulbecco's Phosphate-Buffered Saline (DPBS, Invitrogen) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich). After three washes in DPBS, the cells are incubated with pre-hybridization buffer (phosphate buffer 0.5M + 40% formamide) for 20 min at room temperature. 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 next day, cells are washed with wash buffer (0.2% BSA in pre-hybridization buffer) warmed at 37°C followed by three washes in DPBS. Immunofluorescence staining of MBNL1 is performed on the same wells after the FISH procedure.

[0430] Myotubes morphology and DMD associated biomarkers high content analysis

[0431] After fixation and immunostaining, quantitative microscopy was performed using the Operetta HCS imaging system with a 10x / 0.3 NA objective (PerkinElmer). Images were analyzed using scripts developed in Acapella software (PerkinElmer). Eleven fields of view per well were acquired. First, segmentation of myotubes and nuclei were done using respectively the Troponin T or Myosin heavy chain staining and the Hoechst staining. One to two myotubes per micropattern were usually identified. The threshold of segmentation was set-up to avoid detecting the background noise and eliminate aberrant small myotube structures. At the end of this first step, specific morphological readouts were calculated in the whole well, like the nuclei count and the fusion index (percentage of nuclei included in Troponin T or MHC staining) and mean myotube area, to control the quality of the myotube viability and differentiation. Usually around 50 to 60 myotubes were detected per well in a control condition. Then, an image clean-up step was performed on the myotubes images to remove myotubes that touch the border of the image. The resulted myotubes were used to extract specific marker expression (e.g. dystrophin, a-sarcoglycan staining intensity...).

[0432] Dystrophin high content analysis in cardiomyocytes

[0433] After fixation and immunostaining, quantitative microscopy was performed using the Operetta-CLS HCS imaging system with a 40x / l.l NA confocal water objective (PerkinElmer). Images were analyzed using scripts developed in Acapella software (PerkinElmer). Sixty-four fields of view per well were acquired. Segmentation of myocytes spread on patterns was done using Myosin heavy chain staining. An average of four cardiomyocytes per micropattern were usually identified. The threshold of segmentation was set-up to avoid detecting the background noise and eliminate aberrant small structures. Usually around 100 to 150 micropatterns were detected and analyzed per well in a control condition. Then, an image clean-up step was performed on the myotubes images to remove patterns that touch the border of the image. The resulted patterns were used to extract specific marker expression (e.g. dystrophin staining intensity...) in the cardiomyocytes. Colocalization analysis

[0434] 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. In the DMD example, images of the plates were acquired on an Operetta HCS imaging system with a 20xN A objective in confocal mode. In the DM1 and cardiomyocyte examples, images of cells were acquired with the Operetta HCS platform (Perkin Elmer) using a x40 objective. For each condition, 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.

[0435] Region of interest (ROI) identification is performed through segmentation algorithms on the appropriate staining channel. The channel depends on the disease. In the case of DMD, 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. In this example, we segmented the nuclei inside the myotubes using Troponin T or MHC channel for the myotube and the HOECHST channel for nuclei.

[0436] 3. Protein presence threshold:

[0437] 3.1. Calculus

[0438] To study the colocalization of two proteins, we first look for an intensity threshold above which we will consider that the fluorescently marked protein is present at an image position (pixel). The threshold is determined on wells where, either the protein is not expressed, or the primary antibody is not added to. The threshold is determined by quantile statistics. At ROI level, a high quantile of pixel intensity distribution is taken. ROI values are aggregated at well and then plate level. For this plate, the final value determines the threshold specific to the protein. 3.2. Masks

[0439] 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.

[0440] 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.

[0441] 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).

[0442] Figure 4C second row represents the mask obtained by thresholding the imaging marker 1 channel in the nuclei in myotubes area. And figure 4C third row represents the mask obtained by thresholding the imaging marker 2 channel in the nuclei in myotubes area.

[0443] 3.3. Optional image filtration

[0444] Before colocalization readout calculations, 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.

[0445] 4. Colocalization readouts calculation

[0446] 4.1. Pearson's Correlation Coefficient (PCC)

[0447] The Pearson's Colocalization Coefficient (PCC) 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.

[0448] 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.

[0449] If the optional image filtration has been chosen, some ROI might have no pixel intensity above zero for one protein related channel. In this case, the denominator value will equal zero, leading to an undefined PCC. Because the correlation between the other protein related signal and this unvarying signal is null, the PCC is set to 0. 4.2. Manders' Colocalization Coefficient (MCC)

[0450] Manders colocalization measure is independent of pixel intensities correlation. Indeed, the MCC is a measure of co-occurrence. It measures the proportion of one protein-related signal that overlaps with the other protein signal. As exemplified in the Figure 4E.2, the MCC for IM1 represents the proportion of IMl-related intensity that is colocalizing with IM2. In 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.

[0451] 4.3. Rank-based intensity Weighting Coefficient (RWC)

[0452] 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.

[0453] 4.4. A negative control protein

[0454] To strengthen the confidence in the readouts, the colocalization is also assessed between one of the two proteins and a protein that is known not colocalize. In the example case, 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. In Figure 5B, MHC is considered as the I MCTL . As the MHC is located all over the myotube in a homogeneous manner, the colocalization zone corresponds to the zone where the IM1 is above threshold. Those readouts are considered negative controls for the colocalization readouts.

[0455] 4.5. Defining a threshold for high PCC values

[0456] Another developed readout is the percentage of ROI displaying a strong colocalization. To define what is a strong colocalization, we need to set up threshold above which the PCC is considered to reflect a strong correlation of the two markers in the ROI. In the DGC and dystrophin case (Figure 5), 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. In the shown example, 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.

[0457] 4.6. Readouts with significance correction

[0458] 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:

[0459] For each of the two images, the pixels of the considered zone are mixed multiple times. For each of the shuffles, the readout is calculated, and then these values are averaged. For the MCC, 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.

[0460] RESULTS

[0461] Selection of cellular components to distinguish healthy and diseased cells using immunofluorescence (IF)-based colocalization

[0462] The following examples of colocalization assays monitored the assembly of the dystrophin-glycoprotein complex (DGC) and interaction of nuclear RNA foci with RNA splicing factors. These assays were then used to assess the potency and efficacy of therapies targeting DMD and DM1.

[0463] 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). 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.

[0464] Like the dystrophin / b-DG interaction, the interaction between the splicing factor MBNL1 and mutated DMPK RNAs in DM1 patients is well studied and documented (Mankodi et al., 2001; Fardaej et al., 2001; Fardaej et al., 2002). No reports have been published characterizing the interactions of miss-spliced dystrophin as found in DM1 patients with components of the DGC.

[0465] Characterization of DMD donors and imaging targets

[0466] To characterize the growth behavior and differentiation of healthy and DMD donors under MyoScreen conditions, an evaluation of the myotube morphology in untreated condition was performed. Two healthy and four DMD donors were labelled using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes. Figure 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.

[0467] A functional assay to assess the structural integrity of the muscle cells should demonstrate a good correlation between the level of rescue induced by the therapeutic agent and the assay readout. In DMD pathology, it would translate as a correlation between dystrophin expression and the colocalization of dystrophin with other components of the DGC. Figure 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). To assess the correlation between dystrophin expression levels and the colocalization assay readouts, dystrophin levels were modulated in healthy donors using RNAi (Figure 2D and E). As expected, the dystrophin signal decreased as the siRNA dose increased. At the highest siRNA concentration, the observed dystrophin signal in the healthy donors mimicked the signal observed in DMD donors. Hence 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. Despite the higher background when using the C- terminal targeting dystrophin antibody, both dystrophin antibodies are similar in their sensitivity to detect changes in dystrophin expression (Figure 2E).

[0468] 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. In the examples below we evaluated the colocalization of dystrophin with 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. Besides variability in the expression levels of a-DG, b-DG, and a-SG between the healthy donors, there was no significant difference in the expression levels of these proteins between healthy and diseased donors. Treatment of the healthy donors with DMD siRNAs downregulated dystrophin levels up to less than 20%. The decrease in dystrophin levels was associated with decreases in the levels of a-DG, b-DG, and a-SG, but had no significant effect on the expression level of dysferlin (Figure 3C).

[0469] Quantification methods for colocalization of dystrophin with a-sarcoglycan, P- dystroglycan, a-dystroglycan and dysferlin

[0470] Four quantification method for colocalization of dystrophin with a-sarcoglycan, p- dystroglycan, a-dystroglycan and dysferlin in myotubes were used and the dynamic range, discrimination, and selectivity of the MCC, RCW, PCC, and High PCC % readouts were compared in healthy donors upon modulation of dystrophin levels by RNAi (Figure 6). Dystrophin expression in two healthy donors was regulated by RNAi using DMD specific siRNA 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. In case of the colocalization between dystrophin and -dystroglycan all four statistical methods yield similar results. For the colocalization between dystrophin and a-sarcoglycan or dysferlin, all four statistical methods yield satisfying results but the High PCC% readout displays a larger dynamic range than PCC, MCC or RWC.

[0471] Sensitivity of the colocalization between dystrophin and p-dystroglycan, a-sarcoglycan, a- dystroglycan, or dysferlin to change in dystrophin levels

[0472] 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. These results revealed that among the four tested colocalization pairs and for the tested donors, the colocalization between dystrophin and p-dystroglycan or a-sarcoglycan analyzed by High PCC% provided optimal conditions to monitor the dystrophindependent assembly of the DGC.

[0473] These experiments show that quantitative colocalization assays monitoring dystrophin and b-DG or dystrophin and a-SG, preferably using the high PCC % readout, are both equally suited as functional assays to assess the potency and efficacy of DMD therapies that restore the expression of dystrophin.

[0474] Assessing the potency and efficacy of DMD therapies by monitoring the colocalization of dystrophin with p-dystroglycan or a-sarcoglycan

[0475] The examples presented in Figures 8 and 9 demonstrate that dystrophin-dependent assembly of the DGC can be quantitatively monitored using the colocalization assays of the invention. The following examples provide evidence that these assays can be used as a cellbased functional assays to monitor the activity of dystrophin restored in primary and immortalized patient-derived DMD myotubes by gene or RNA therapies, to predict the ability of a therapeutic compound to treat DMD, and to evaluate the susceptibility of various DMD patient genotypes for particular therapies.

[0476] Exon skipping therapy in primary DMD patient-derived myotubes

[0477] Most DMD patients have deletions of various lengths in the DMD gene which shift the reading frame and prevent the expression of a functional dystrophin. In more than 50% of these patients 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.

[0478] To skip exon 44 in DMD patients amenable to exon 44 skipping, we used a PMO targeting the exon 44, fused to an octa-guanidine dendrimer (vivo group) for better cellular delivery. As shown in Figure 8B, treatment of myotubes from two DMD donors with this vivoPMO had no impact on nuclei count, fusion index and myotube mean area. The level of dystrophin restoration was assessed using the dystrophin antibody that targets the dystrophin N-terminal domain and reached 39% (DMD #5 donor) and 22% (DMD #6 donor) of the mean dystrophin signal from healthy donors treated with a control PMO (Figure 8C). The two DMD donors have the same genotype (deletion of exon 45). Hence, 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). In line with the lower level of dystrophin restoration, DMD donor #6 shows a lower level of colocalization between dystrophin and b-DG or a-SG than DMD donor #5. However, upon normalization of their responses, both donors showed the same relative response to the vivoPMO treatment and consequent restoration of dystrophin (Figure 8D). These results confirm the utility of the dystrophin / b-DG and dystrophin / a-SG colocalization assays to monitor the restoration and function of dystrophin in patient-derived myotubes upon exonskipping therapy. To skip exon 45 in DMD patients amenable to exon 45 skipping, we used a vivo PMO targeting one of the exon 45 splice junction. As shown in Figure 9A-B, treatment of myotubes from two DMD donors with this vivoPMO had no impact on nuclei count, fusion index and myotube mean area. Both donors responded to the Exon 45 vivo PMO treatment and restored dystrophin to similar extent (Figure 9C). However, 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). Although 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). In contrast, 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.

[0479] Exon in immortalized DMD

[0480] Potency assays necessitate a steady supply of its critical reagents, and cannot be easily executed in primary cells that are in limited supply and at risk to derive and lose their features over time. Figure 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. Morphology of healthy (HVimm) and DMD (DMDimm) cell lines do not show any significant differences (Figure 10A, B). Myotubes of healthy and DMD cells were treated with a control PMO (5 pM) and DMD cells were treated with increasing doses of 4 exon 44 skipping PMOs (0.3-10 pM) listed by order of predicted efficacy (Figure 10C, D). Treatment of DMD cell line with Exon 44 skipping PMOs partially restores dystrophin expression, in a distance-dependent 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 p -dystroglycan is not modified by the exon 44 PMO treatments (Figure 10D). Colocalization of dystrophin with P-dystroglycan was monitored using the high PCC % readout as a function of the exon 44 skipping PMO dose or dystrophin level % (Figure 10E). We observe that PMO4 displays the lowest activity followed by the PMO3. PMO1 and 2 show comparable colocalization levels. This result indicates that the colocalization assay is able to distinguish between products of different potencies.

[0481] Establishing quantitative colocalization assays for DM1

[0482] Characterization of DM1 donors and assessment of quantitative colocalization of nuclear DMPK RNA foci and the splicing factor MBNL1

[0483] To characterize the growth behavior and differentiation of healthy and DM1 donors under MyoScreen conditions, an evaluation of the myotube morphology in untreated condition was performed. Two healthy and 5 DM1 donors were labelled using Hoechst as a nuclei dye and myosin heavy chain (MHC) as a marker of differentiation to separate myoblasts and myotubes. Figure 11A shows that all donors form myotubes that display the standard MyoScreen morphology. DM1 #2 donor has a lower fusion index than the other HV and DM1 donors which might indicate changes in differentiation compared to the other donors. (Figure 11A, B). The presence of DMPK mRNA foci was assessed using a (CAG)5-Cy3 FISH probe and the immunofluorescent staining of MBNL1 was used to determine the presence of MBNL1 protein trapped in the DMPK mRNA foci. 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. Increasing DMPK ASO dose leads to a decrease of MBNL1 / DMPK foci co-labelled spots indicating the disappearance of RNA foci and the liberation of MBNL1 protein. The same effect is observed in all donors, although statistical significance is reached only for DM1 #3 and #5 due to the important variability in the other donors. (Figure 11C). Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between DMPK foci and MBNL1 in function of the ASO dose-response for DM1 donors (Figure 11D). 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 . With 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).

[0484] Monitoring the colocalization of miss-spliced dystrophin with p-dystroglycan or a- sarcoglycan

[0485] In DM1 patients, MBNL1 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. 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 (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). Treatment of the DM1 donors with DMPK ASO led to a partial rescue of the C-terminal domain of dystrophin expression levels while the expression levels of a-sarcoglycan and p-dystroglycan are not modified by the DMPK ASO treatment (Figure 12B, C). Colocalization was analyzed using mean PCC, MCC and RWC and high PCC% readouts calculated between a-sarcoglycan or |3- dystroglycan, and dystrophin (C-terminal antibody) in function of the ASO dose-response for DM1 donors (Figure 12D). 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. We further compared the response of the five DM1 donors to treatment with an ASO at 6 concentrations. 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%. Colocalization of dystrophin with p-dystroglycan in cardiomyocytes

[0486] To assess the feasibility to apply colocalization assay to cardiomyocytes, expression of b-DG and dystrophin was determined in human cardiomyocytes derived from induced pluripotent stem cells (hlPSC-CM) knocked-down with DMD siRNA. Figure 13A shows the expression of b-DG and dystrophin in control cardiomyocytes and cardiomyocytes treated with siRNA. Treatment of the cells with 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).

[0487] To determine the optimal quantification method for colocalization of dystrophin with P-dystroglycan in hlPSC-CM, we compared the dynamic range, discrimination, and selectivity of the MCC, RCW, PCC, and High PCC % readouts in healthy cells upon modulation of dystrophin levels by RNAi (Figure 13). Dystrophin expression 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 |3- dystroglycan and dystrophin (N-terminal antibody). For hlPSC-CM, the colocalization readouts are then plotted against the dystrophin siRNA dose (Figure 13 C, D). For hlPSC-CM the colocalization between dystrophin and p-dystroglycan, the High PCC% readout displays a larger dynamic range than PCC, MCC or RWC, as it was also measured for myotubes.

Claims

CLAIMS1. An in vitro computer-implemented method of assessing the functionality of a cellular protein or nucleic acid of interest in a myotube(i) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube has been stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid interacting with said first cellular protein or nucleic acid 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, cellular structures of myotubes, and any combination thereof;(ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and(iii) determining quantitatively a degree of colocalization of the cellular first protein or nucleic acid and the cellular second protein or nucleic acid in at least one ROI by performing quantitative colocalization analysis, wherein the degree of colocalization correlates with the functionality of the cellular protein or nucleic acid of interest in said at least one myotube.

2. An in vitro computer-implemented method of assessing potency of a compound to modulate the functionality of a cellular protein or nucleic acid of interest in a myotube(i) providing at least one image of at least one in vitro cultured myotube, wherein said at least one myotube has been contacted with a compound to be tested, has been stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid interacting with said first cellular protein or nucleic acid 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, cellular structures of myotubes, and any combination thereof; and(ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and(iii) determining quantitatively a degree of colocalization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing quantitative colocalization analysis; and(iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 protein or nucleic acid of interest in said at least one myotube.

3. An in vitro computer-implemented method of predicting the ability of a compound to treat a neuromuscular disease of interest comprising(i) providing at least one image comprising at least one in vitro cultured myotube exhibiting a feature of a neuromuscular disease of interest ("diseased myotube"), wherein said at least one myotube has been contacted with a compound to be tested, has been stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid interacting with said first cellular protein or nucleic acid 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, cellular structures of myotubes, and any combination thereof; and(ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and(iii) determining quantitatively a degree of colocalization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing quantitative colocalization analysis; and(iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) 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 thereference degree of colocalization indicates that the compound is useful in the treatment of said neuromuscular disease.

4. An in vitro computer-implemented method for monitoring the response to a therapeutic compound of a patient affected with a neuromuscular disease, wherein the method comprises(i) providing at least one image comprising at least one in vitro cultured myotube obtained from a sample of a patient affected with a neuromuscular disease after administration of the therapeutic compound, wherein said at least one myotube has been stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid interacting with said first cellular protein or nucleic acid 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, cellular structures of myotubes, and any combination thereof; and(ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and(iii) determining quantitatively a degree of colocalization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing quantitative colocalization analysis; and(iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured myotube obtained 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.

5. An in vitro computer-implemented method for selecting a patient affected with a neuromuscular disease for a treatment with a therapeutic compound or for determining whether a patient affected with a neuromuscular disease is susceptible to benefit from a treatment with a therapeutic compound, wherein the method comprises(i) providing at least one image comprising at least one in vitro cultured myotube obtained from a sample of said patient, wherein said at least one myotube has been contacted with a therapeutic compound, has been stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid interacting with said first cellular protein or nucleic acid 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, cellular structures of myotubes, and any combination thereof; and(ii) performing an image segmentation with an algorithm on appropriate staining channel(s) in order to identify ROI; and(iii) determining quantitatively a degree of colocalization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing quantitative colocalization analysis; and(iv) comparing said degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, said at least one reference myotube being at least one in vitro cultured myotube obtained 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 indicates that said patient is susceptible to benefit from a treatment with said therapeutic compound.

6. The method of any one of claims 1 to 5, wherein quantitative colocalization analysis is carried out using quantitative pixel-based colocalization analysis.

7. The method of any one of claims 1 to 6, wherein the myotubes are derived from primary cells or are derived from immortalized cells.

8. The method of any one of claims 1 to 7, wherein the myotubes have been cultured in constrained conditions allowing the production of homogeneous population of myotubes.

9. The method of any one of claims 1 to 7, wherein the method further comprises before step (i)- culturing myoblasts, preferably in constrained conditions allowing the production of homogeneous population of myotubes, optionally in the presence of a compound to be tested;- staining these myotubes for said first protein or nucleic acid, for said second protein or nucleic acid and with said at least one labelling agent; and- capturing at least one image of at least one stained myotube.

10. The method of any one of claims 1 to 9, wherein in step (iii) 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).

11. The method of any one of claims 1 to 10, wherein said first protein or nucleic acid is a protein and said second protein or nucleic acid is a protein, wherein in step (iii) the degree of colocalization is quantitatively determined by calculating the Pearson's Colocalization Coefficient (PCC) and optionally applying a mathematical function on said coefficient and / or defining a threshold for high PCC values.

12. The method of any one of claims 1 to 10, wherein at least one of said first protein or nucleic acid and of said second protein or nucleic acid is a nucleic acid and wherein in step (iii) the degree of colocalization is quantitatively determined by calculating the Manders' Colocalization Coefficient (MCC) and optionally applying a mathematical function on said coefficient.

13. The method of any of claims 3 to 12, wherein 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.

14. The method of any of claims 3 to 12, wherein the neuromuscular disease of interest 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, Limbgirdle muscular dystrophies (LGMD), 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), Tubular aggregate myopathy, Malignant hyperthermia syndrome, Inclusion body myopathy, Protein aggregate myopathy, Nemaline myopathies, Congenital myopathy (CM), Vacuolar aggregate myopathy, Myoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjbgren syndrome, Neurodegeneration with ataxia, Friedreich's Ataxia, dystonia, and gaze palsy, childhood-onset (NADGP) and Pompe disease.

15. The method of any of claims 3 to 14, wherein the neuromuscular disease is Duchenne muscular dystrophy or myotonic dystrophy type 1 (DM1), the first protein or nucleic acid is dystrophin and the second protein or nucleic acid 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 |3- dystroglycan, or vice versa.

16. The method of any of claims 3 to 14, wherein the neuromuscular disease is Duchenne muscular dystrophy, the first protein or nucleic acid is dystrophin and the second protein or nucleic acid is selected from the group consisting of a-sarcoglycan and |3- dystroglycan, or vice versa.

17. The method of any of claims 3 to 14, wherein the neuromuscular disease is myotonic dystrophy type 1 (DM1), the first protein or nucleic acid is dystrophin and the second protein or nucleic acid is selected from the group consisting of a-sarcoglycan and |3- dystroglycan, or vice versa.Ill18. The method of any of claims 3 to 14, wherein the neuromuscular disease is myotonic dystrophy type 1 (DM1), the first protein or nucleic acid is DMPK RNA and the second protein or nucleic acid is a RNA binding protein trapped by CTG repeats in the DMPK gene, preferably is MBNL1 protein, or vice-versa.

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