Quantitative colocalization assays for assessing the efficacy and effectiveness of therapies targeting muscle disorders

Abstract

The present invention relates to methods of using quantitative colocalization assays to assess the functionality of cellular molecules in muscle cells, to assess the efficacy of a compound to modulate the functionality of cellular molecules in muscle cells, to predict the ability of a compound to treat a muscle disease, to monitor the response of a patient suffering from a muscle disease to a therapeutic compound, to select a patient suffering from a muscle disease for treatment with a therapeutic compound, or to determine whether a patient suffering from a muscle disease is likely to benefit from treatment with a therapeutic compound.

Description

[Technical Field]

[0001] The present invention relates to methods for assessing the functionality of cellular molecules in muscle cells, assessing the efficacy of a compound to modulate the functionality of cellular molecules in muscle cells, predicting the ability of a compound to treat a muscle disease, monitoring the response of a patient suffering from a muscle disease to a therapeutic compound, selecting a patient suffering from a muscle disease for treatment with a therapeutic compound, or determining whether a patient suffering from a muscle disease is likely to benefit from treatment with a therapeutic compound. [Background technology]

[0002] Over the past few decades, genetic analyses have identified over 500 genes involved in the pathogenesis of neuromuscular disorders. The identification of these genes has greatly advanced our understanding of muscle function and disease and opened up many opportunities for therapeutic intervention. Despite these advances, for many neuromuscular disorders, the disease mechanisms remain unclear and effective therapies have yet to be identified.

[0003] The unique structural and functional properties of muscle pose significant challenges for the functional characterization of muscle disorders and the discovery of therapies. Particularly challenging is striated muscle, which is formed by the fusion of mononuclear precursor cells called myoblasts into multinucleated myotubes. These myotubes then differentiate into muscle fibers containing the contractile apparatus (sarcomeres) that enables them to contract upon stimulation from attached motor neurons. The ability of muscle fibers to differentiate and contract depends on interactions between muscle fibers and the connective tissue (basal lamina) that surrounds them. These interactions are critical for the muscle fiber's ability to escape damage, differentiate, and respond to mechanical forces during contraction. Each muscle fiber is innervated by a single motor neuron through the neuromuscular junction (NMJ). A process called excitation-contraction coupling converts motor neuron-mediated neuronal excitation into Ca ions that induce muscle contraction. 2+Skeletal muscle fibers are terminally differentiated, but specific processes are implemented to repair contraction-induced damage to the plasma membrane or to replace damaged muscle fibers through activation of muscle stem cells.

[0004] Neuromuscular disorders are generally classified as dystrophies, myopathies, and myasthenic syndromes depending on the morphology of the pathological muscle fibers and the functions affected by the disease. The underlying defects in these diseases are diverse and can directly or indirectly affect muscle fiber-matrix interactions, excitation-contraction coupling, the contractile apparatus, muscle metabolic activity, or regenerative capacity (Dowling et al., Nat Rev Mol Cell Biol. 2021 Nov;22(11):713-732). In addition to muscle cells themselves, neuromuscular disorders can also be caused by mutations in motor neurons (e.g., spinal muscular dystrophy, Kennedy's disease) or the basal lamina (e.g., Ullrich's myopathy). Furthermore, muscle structure and function can be metabolically impaired in response to malnutrition, immobility, advanced age, or acute and chronic diseases. In these cases, changes in muscle structure and function are often caused by alterations in protein turnover mediated by the ubiquitin / proteasome system and the autophagic-lysosomal pathway, by inhibition of growth factor pathways and / or activation of inflammatory pathways.

[0005] Among the most well-characterized dystrophies are Duchenne muscular dystrophy and Becker muscular dystrophy (DMD, BMD). In DMD patients, mutations in the DMD gene prevent the expression of functional dystrophin and the assembly of the dystrophin-glycoprotein complex (DGC) at the plasma membrane (Gao & McNally, Compr Physiol. 2015 July 1;5(3):1223-39). This complex is involved in regulating muscle cell signaling and acts as a shock absorber to prevent muscle cell damage during contraction. Boys who do not express dystrophin typically lose the ability to walk by around age 10 and have a shortened lifespan due to pulmonary and cardiac complications. In contrast to DMD, BMD patients express partially functional dystrophin, which is usually associated with less severe symptoms. Another well-studied example of a neuromuscular disease is myotonic dystrophy (DM1). In DM1, disease is caused by an expansion of a CUG repeat in the DMPK pre-mRNA, which forms a stem-loop that is recognized by RNA splicing factors, including MBNL1. The interaction of these splicing factors with the mutant DMPK transcript competes with the accurate splicing of other RNAs, many of which are involved in essential muscle function. It remains to be determined which of the misspliced ​​RNAs is responsible for the DM1-associated phenotype and whether other mechanisms are at play.

[0006] Advances in understanding the cellular processes involved in muscle function have opened the door to therapeutic discovery. A variety of therapies have been developed and are currently in clinical development for a growing number of disorders (Dowling et al., 2021, supra). The most promising are gene and RNA therapies that seek to replace mutated disease-promoting factors or correct disease-associated alterations in the post-transcriptional processing of disease-promoting factors. However, even for well-understood neuromuscular disorders, the discovery and development of these therapies has been challenging.

[0007] In the case of DMD, a major challenge is the size of the DMD gene, the largest gene in the human genome, encoding a 420-kDa protein. Current gene therapy approaches rely on viral vectors with limited payload capacity, so DMD-targeted gene therapies can only deliver severely truncated, functionally impaired dystrophin. Similarly, therapies that alter the splicing of mutant DMD pre-mRNA induce the expression of partially functional dystrophin. Clinical data indicate that restored dystrophin expression alone does not correlate with clinical efficacy of these therapies. Therefore, functional assays are needed to guide the discovery and clinical development of these therapies. The clinical success of these therapies is further hindered by the lack of appropriate animal models that represent the diverse genetic backgrounds found in DMD patients. Similar challenges exist for many neuromuscular disorders.

[0008] Many muscle diseases are rare or ultra-rare. With an increasing number of therapies in development, small patient populations are required to support clinical trials of these therapies and to choose among them. Functional assays performed on patient-derived cells can provide guidance for identifying the most beneficial therapeutic options for each individual.

[0009] Because of the complex processes involved in manufacturing gene and RNA therapies and the high doses required to achieve therapeutic effect, potency assays are required early in clinical development to support batch release. This requirement has led to several moratoriums imposed by the FDA during recent clinical trials targeting neuromuscular disorders.

[0010] Many neuromuscular diseases have myocardial manifestations that often determine a patient's life expectancy. Therefore, evaluation of therapies that target neuromuscular disorders in cardiomyocytes is important. Furthermore, an increased understanding of the molecular mechanisms regulating myocardial function has opened opportunities for targeted therapies for cardiomyopathies. [Prior art documents] [Patent documents]

[0011] [License 1] International Licensed Trademark WO 2015 / 091593 [License 2] WO 2016 / 202850 [License 3] WO 2016 / 139312 [License 4] EP 1 882 736 [Patent Document 5] EP 2 180 042 [License 6] EP 1 664 266 [License 7] US 2008 / 299086 [Non-licensed literature]

[0012] [Non-licensed Document 1] Dowling, Nat Rev Mol Cell Biol. 2021 Nov;22(11):713~732 [Non-licensed Document 2] Gao & McNally, Compr Physiol. July 1, 2015; 5(3): 1223~39 [Non-licensed Document 3] Singanら, 2011(BMC Bioinformatics 12, 407) [Non-licensed Document 4] Ribeiroら、2015(Proc Natl Acad Sci US A.2015;112(41):12705~12710) [Non-licensed Document 5] Funakoshiら、2021(Nat Commun 12、3155(2021)) [Non-licensed Document 6] Leeら、2017(Cell Stem Cell.2017;21(2):179~194.e4) [Non-licensed Document 7] Jerome Chal and Olivier Pourquie, 2017 (Development 2017 June 15;144(12):2104-2122) [Non-patent document 8] Yamamoto et al., 2008, J.Histochem.Cytochem 56, 881–892 [Non-Patent Document 9] Rao et al., Biomaterials.2013;34(10):2399~2411 [Non-Patent Document 10] Junkin et al., 2011, Journal of Cell Science 124, 4213~4220 [Non-Patent Document 11] Bajaj et al., 2011, Integrative Biology 3, 897–909 [Non-Patent Document 12] Young et al., SLAS Discov. 2018 Sep;23(8):790~806 [Non-Patent Document 13] Bray et al. 2008. Cell motility and the cytoskeleton 65(8):641 [Non-Patent Document 14] Ribeiro et al., 2015.Proc Natl Acad Sci US A.2015;112(41):12705~12710 [Non-Patent Document 15] 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, March 17-19, 2017. Neuromuscul Disord. 2018 Aug;28(8):702-710 [Non-Patent Document 16] Lapidos et al., Circulation Research.2004;94:1023~1031 [Non-Patent Document 17] Quan Gao and Elizabeth M. McNally, Compr Physiol.2015;5(3):1223~1239 [Non-Patent Document 18] Rahimov et al., J Cell Biol (2013) 201(4):499~510 [Non-Patent Document 19] Masayuki Nakamori et al., Muscle Nerve. 2007 August;36(2):251-7 [Non-Patent Document 20] Vihola et al., Neuropathol Appl Neurobiol. 2013 June;39(4) [Non-Patent Document 21] Hershberger et al., Genet. Med, 12 (2010), pp. 655-667 [Non-Patent Document 22] Cirino AL, Ho C. Hypertrophic Cardiomyopathy Overview. August 5, 2008 [Updated July 8, 2021]. In: Adam MP, Everman DB, Mirzaa GM, et al. (eds.), GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2023 [Non-Patent Document 23] McNally E, MacLeod H, Dellefave-Castillo L. Arrhythmogenic Right Ventricular Cardiomyopathy. April 18, 2005 [Updated May 25, 2017]. In: Adam MP, Everman DB, Mirzaa GM, et al. (eds.), GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2023. [Non-Patent Document 24] ;Ciarambino et al., Int J Mol Sci. 2021 Jul 19;22(14):7722 [Non-Patent Document 25] Linke et al., Circ Res. 2014 Mar 14;114(6):1052~68 [Non-Patent Document 26] Higashikuse et al., Dis Model Mech. 2019 Nov 15;12(11):dmm041103 [Non-Patent Document 27] Carpenter et al., 2006, Genome Biology, 7(10) [Non-patent document 28] Costes et al., Biophysical Journal, Vol. 86, June 2004, pp. 3993-4003 [Non-Patent Document 29] Zinchuk et al., Acta Histochem. Cytochem.40(4):101–111, 2007 [Non-Patent Document 30] Wu et al., Biophysical Journal, Vol. 98, February 2010, pp. 493-504 [Non-Patent Document 31] Mulders et al., 2009, Cell, 106(33), 13915-13920 [Non-Patent Document 32] Cullen et al., J Histochem Cytochem.1998 Aug;46(8):945~54 [Non-Patent Document 33] Ilsley et al., 2001, Cell Signal. 2001 September;13(9):625-32 [Non-Patent Document 34] Ervasti et al., Biochim Biophys Acta. February 2007;1772(2):108~17 [Non-Patent Document 35] Mankodi et al., 2001;Fardaej et al., 2001;Fardaej et al., 2002 Summary of the Invention [Problem to be solved by the invention]

[0013] Taken together, these examples highlight the critical need in the discovery and development of therapies for muscle disorders for quantitative functional assays that can be performed on 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. [Means for solving the problem]

[0014] In a first aspect, the present invention provides an in vitro method for assessing the functionality of a cellular molecule of interest in muscle cells, comprising: (i) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell has been stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and has been stained with at least one labeling agent that reveals 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; (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis. Including, The degree of co-localization correlates with functionality of the cellular molecule of interest in said at least one muscle cell.

[0015] Preferably, the muscle cells are myotubes or cardiomyocytes.

[0016] In some embodiments, the muscle cells are cardiomyocytes, and said at least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof.

[0017] In some preferred embodiments, the muscle cells are myotubes, and the at least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof.

[0018] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0019] In a second aspect, the present invention provides an in vitro method for assessing the efficacy of a compound to modulate the functionality of a cellular molecule of interest in a muscle cell, comprising: (i) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, The method also relates to a method wherein a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that the compound is capable of modulating the functionality of a first cellular molecule of interest in the at least one muscle cell.

[0020] Preferably, the muscle cells are myotubes or cardiomyocytes.

[0021] In some embodiments, the muscle cells are cardiomyocytes, and said at least one region of interest (ROI) is selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof.

[0022] In some preferred embodiments, the muscle cells are myotubes, and the at least one region of interest (ROI) is selected from the group consisting of individual myotubes, cellular structures of myotubes, and any combination thereof.

[0023] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0024] In another aspect, the present invention provides an in vitro method for predicting the ability of a compound to treat a muscle disease of interest, comprising: (i) providing at least one image comprising at least one in vitro cultured muscle cell, said at least one muscle cell being contacted with a test compound, stained for a first cellular molecule of interest and a second cellular molecule that interacts with said first cellular molecule of interest, and stained with at least one labeling agent that reveals 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, that exhibits characteristics of a muscle disease of interest (a "pathological muscle cell"); and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured diseased muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; Including, The present invention also relates to a method in which a positive correlation between the concentration of the compound and a desired statistically significant variation in the degree of colocalization compared to a reference degree of colocalization indicates that the compound is useful for treating said muscle disease.

[0025] Preferably, the muscle cells are myotubes or cardiomyocytes.

[0026] Preferably, the muscle disease is a neuromuscular disease or a cardiomyopathy.

[0027] In some embodiments, the muscle cells are cardiomyocytes, the at 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 muscle disease is a neuromuscular disease or a cardiomyopathy.

[0028] In some preferred embodiments, the muscle cells are myotubes, the at 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 muscle disease is a neuromuscular disease.

[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 a further aspect, the present invention provides an in vitro method for monitoring the response of a patient suffering from a muscular disorder to a therapeutic compound, comprising: (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained from a sample of a patient suffering from a muscle disease after administration of a therapeutic compound, wherein the at least one muscle cell is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and is stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained from a sample of the patient prior to administration of a therapeutic compound; Including, The method relates to a method in which a desired statistically significant variation in the degree of colocalization compared to a reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0031] Preferably, the muscle cells are myotubes or cardiomyocytes.

[0032] Preferably, the muscle disease is a neuromuscular disease or a cardiomyopathy.

[0033] In some embodiments, the muscle cells are cardiomyocytes, the at 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 muscle disease is a neuromuscular disease or a cardiomyopathy.

[0034] In some preferred embodiments, the muscle cells are myotubes, the at 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 muscle disease is a neuromuscular disease.

[0035] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0036] In another aspect, the present invention provides an in vitro method for selecting a patient suffering from a muscular disorder for treatment with a therapeutic compound, or for determining whether a patient suffering from a muscular disorder will likely benefit from treatment with a therapeutic compound, comprising: (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained from said patient sample, said at least one muscle cell being contacted with a therapeutic compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with said first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound. Including, The present invention also relates to a method in which a positive correlation between the concentration of a therapeutic compound and a statistically significant desired variation in the degree of co-localization indicates that said patient is likely to benefit from treatment with said therapeutic compound.

[0037] Preferably, the muscle cells are myotubes or cardiomyocytes.

[0038] Preferably, the muscle disease is a neuromuscular disease or a cardiomyopathy.

[0039] In some embodiments, the muscle cells are cardiomyocytes, the at 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 muscle disease is a neuromuscular disease or a cardiomyopathy.

[0040] In some preferred embodiments, the muscle cells are myotubes, the at 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 muscle disease is a neuromuscular disease.

[0041] Preferably, the first molecule is a protein or a nucleic acid and the second molecule is a protein or a nucleic acid.

[0042] Preferably, in the method of the present invention, the quantitative colocalization analysis is performed using pixel-based colocalization analysis.

[0043] Preferably, in the methods of the present invention, the muscle cells are derived from primary cells or from immortalized cells.

[0044] Preferably, in the methods of the present invention, the muscle cells are cultured under restrictive conditions that allow for the generation of a homogeneous population of muscle cells.

[0045] The method of the present invention further comprises, before step (i), - culturing muscle cells, in particular myoblasts or cardiomyocytes, under restricted conditions that allow the generation of a homogeneous population of muscle cells, in particular myotubes or cardiomyocytes, optionally in the presence of the test compound; - staining the muscle cells for the first molecule, for the second molecule, and with the at least one labeling agent; and - acquiring at least one image of at least one stained muscle cell; It may further include:

[0046] Preferably, in the method of the present invention, in step (iii), the degree of colocalization is quantitatively determined by calculating one or several colocalization readouts selected from the group consisting of Pearson's Colocalization Coefficient (PCC), Mander's Colocalization Coefficient (MCC), Rank-based Intensity Weighting Coefficient (RWC), and any combination thereof, and optionally applying a mathematical function to said coefficients.

[0047] In some embodiments of the method of the invention, the first molecule is a protein and the second molecule is a protein, and in step (iii) the degree of colocalization is quantitatively determined by calculating a Pearson's colocalization coefficient (PCC), and the method further comprises the step of defining a threshold for a high PCC value.

[0048] In some embodiments of the methods of the present invention, at least one of the first molecules and at least one of the second molecules is a nucleic acid, and in step (iii), the degree of colocalization is quantitatively determined by calculating the Mander's Colocalization Coefficient (MCC).

[0049] Preferably, in the methods of the present invention, the muscle disease of interest is a neuromuscular disease selected from the group consisting of muscular dystrophy, myopathy, congenital myasthenic syndrome, motor neuron disease and metabolic myopathy.

[0050] In some embodiments of the methods of the present invention, the muscle disease is Duchenne muscular dystrophy or myotonic dystrophy type 1 (DM1), and 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, α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin, more preferably selected from the group consisting of dystrophin, α-sarcoglycan, and β-dystroglycan. In particular, the first molecule may be dystrophin and the second molecule may be α-sarcoglycan, or vice versa, or the first molecule may be dystrophin and the second molecule may be β-dystroglycan, or vice versa.

[0051] In some embodiments of the methods of the present invention, the muscle disease is myotonic dystrophy type 1 (DM1), the first molecule is DMPK RNA, and the second molecule is an RNA-binding protein trapped by CTG repeats in the DMPK gene, preferably MBNL1 protein, or vice versa. [Brief explanation of the drawings]

[0052] [Figure 1A] Figure 1 shows characterization of DMD donor myotubes in MyoScreen micropattern plates. Myotube differentiation and morphology of healthy and DMD donor cells under MyoScreen conditions were assessed using Hoechst as a nuclear dye and myosin heavy chain (MHC) as a differentiation marker to separate myoblasts and myotubes. Morphology of healthy donors (HV #1 and #2) and DMD donors (DMD #1, #4, #5, and #6) used in this study. Scale bar, 100 μm. [Figure 1B]Figure 1 shows characterization of DMD donor myotubes in MyoScreen micropattern plates. Myotube differentiation and morphology were assessed for healthy and DMD donor cells under MyoScreen conditions using Hoechst as a nuclear dye and myosin heavy chain (MHC) as a differentiation marker to separate myoblasts and myotubes. Quantification of nuclei number, fusion index (ratio of the number of nuclei in myotubes to the total number of detected nuclei), and myotube mean area for HV and DMD donors. DMD donors #5 and #6 showed 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. [Figure 2A] Figure 1 shows validation of the dystrophin antibody used in this study. A. Dystrophin expression assessed using an N-terminal domain-targeting antibody in two healthy donors (HV#1 and #2) and four DMD donors (DMD#1, #4, #5, and #6) (left panel). MHC and Hoechst staining demonstrates the presence and morphology of myotubes (right panel). Scale bar, 100 μm. As expected, no dystrophin expression could be detected in the four DMD donors. [Figure 2B] Figure 1 shows validation of the dystrophin antibodies used in this study. Dystrophin expression was assessed using a C-terminal domain-targeting antibody in healthy donors (HV #1 and #2) and DMD donors (DMD #5 and #6) (left panel). MHC and Hoechst staining demonstrates the presence and morphology of myotubes (right panel). Scale bar, 100 μm. Unlike antibodies targeting the N-terminal domain, the C-terminal domain-targeting dystrophin antibody exhibits detectable levels of background staining in DMD donors. [Figure 2C]Figure 1 shows validation of the dystrophin antibodies used in this study. Evaluation of the specificity of dystrophin antibodies was performed using RNAi-mediated knockdown of dystrophin in two healthy donors (HV #1 and #2). Dystrophin staining decreased in a dose-response manner for both the N-terminally targeted antibody (top panel) and the C-terminally targeted antibody (bottom panel). Scale bar, 100 μm. [Figure 2D] Figure 1 shows validation of the dystrophin antibodies used in this study. Quantitative assessment of dystrophin levels using N-terminally or C-terminally targeted antibodies in healthy and DMD donors. In healthy donors treated with high levels of DMD siRNA, both antibodies showed staining similar to that observed in DMD donors, suggesting that the residual staining exhibited by the antibody targeting the dystrophin C-terminus is due to nonspecific activity of this antibody, which is primarily cytosolic. ANOVA with post-hoc multiple comparison test, *, p<0.05; **, p<0.01; ***, p<0.001 compared to mock condition. [Figure 2E] Figure 1 shows validation of the dystrophin antibodies used in this study. Quantitative assessment of dystrophin signal as a function of siRNA dose. Correcting for nonspecific staining of antibodies targeting the C-terminal domain of dystrophin, both dystrophin antibodies monitor changes in dystrophin expression with similar sensitivity. [Figure 3A]Characterization of 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: β-dystroglycan (b-DG), a transmembrane protein that directly interacts with dystrophin; α-sarcoglycan (a-SG), a transmembrane protein that indirectly interacts with dystrophin; and α-dystroglycan (a-DG), an extracellular protein that interacts with the DGC via b-DG. Dysferlin was included as a transmembrane protein not directly related to the DGC. Expression of α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin in myotubes from two healthy donors (HV#1 and #2) and four DMD donors (DMD#1, #4, #5, and #6). Scale bar, 100 μm. [Figure 3B] Characterization of 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: β-dystroglycan (b-DG), a transmembrane protein that directly interacts with dystrophin; α-sarcoglycan (a-SG), a transmembrane protein that indirectly interacts with dystrophin; and α-dystroglycan (a-DG), an extracellular protein that interacts with the DGC via b-DG. Dysferlin was included as a transmembrane protein not directly involved in the DGC. Expression of β-dystroglycan, α-sarcoglycan, α-dystroglycan, and dysferlin in healthy donors (HV#1 and #2) after dystrophin (DMD) siRNA knockdown. Scale bar, 100 μm. [Figure 3C]Characterization of 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: β-dystroglycan (b-DG), a transmembrane protein that directly interacts with dystrophin; α-sarcoglycan (a-SG), a transmembrane protein that indirectly interacts with dystrophin; and α-dystroglycan (a-DG), an extracellular protein that interacts with the DGC via b-DG. Dysferlin was included as a transmembrane protein not directly related to the DGC. Quantitative evaluation of β-dystroglycan, α-sarcoglycan, α-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; #, p<0.05 compared to HV #2. [Figure 4A] General workflow for quantifying colocalization between labeled imaging markers. Cytoplasmic or membrane-localized markers. Top. Myotube segmentation is performed with a myosin heavy chain marker. Rows 2 and 3. Masks of imaging marker 1 (IM1)- and imaging marker 2 (IM2)-positive regions are obtained from their respective images after thresholding the staining of each secondary antibody. [Figure 4B] General workflow for quantifying colocalization between labeled image markers. Mask overlay defines four regions: a region of myotubes, a region of colocalization, a region with IM1 signal above threshold, and a region with IM2 signal above threshold. [Figure 4C] General workflow for quantifying colocalization between labeled imaging markers. Nuclear imaging marker. Top. Segmentation of nuclei in myotubes is performed with a myosin heavy chain marker and a HOECHST 33342 nuclear marker. Rows 2 and 3. Masks of imaging marker 1 (IM1)- and imaging marker 2 (IM2)-positive areas are obtained from their respective images after thresholding the staining of each secondary antibody. [Figure 4D]General workflow for quantifying colocalization between labeled image markers. The overlaid masks define four regions: the region of nuclei in myotubes (dotted line), the region of colocalization (hatched region), the region with IM1 signal above threshold (light gray), and the region with IM2 signal above threshold in nuclei in myotubes (dark gray). [Figure 4E] General workflow for quantifying colocalization between labeled image markers. Three IM1 / IM2 colocalization readout formulas: 1. Pearson's Colocalization Coefficient (PCC), 2. Mander's Colocalization Coefficient (MCC), and 3. Rank-based Intensity Weighting Coefficient (RWC). IIM2 = IM2-related intensity for a given pixel; IIM1 = IM1-related intensity for a given pixel; Mean IIM2 = average IM2 intensity in the region indicated on the right side of the formula; Mean IIM1 = average IM1 intensity in the region indicated on the right side of the formula; W = pixel weight, which increases according to the correlation between the intensity ranks of IM2 and IM1 for a given pixel. For the RWC calculation, an intensity rank is calculated for each pixel, D = absolute difference between the ranks, and R = maximum rank of the pixel (in either the dystrophin channel or the IM2 channel). See Singan et al., 2011 (BMC Bioinformatics 12, 407). 1. PCC is calculated for the region where at least one of the two protein expressions is above a certain threshold, i.e., the junction of the two masks. 2-3. For MCC and RWC, pixel summation is performed in the zone highlighted next to the sum. In the numerator, the region is the colocalized region, and in the denominator, the region is the zone of IM2 expression. [Figure 4F] General workflow for quantifying colocalization between labeled imaging markers. An imaging marker, referred to herein as IMCTL, is used as a control protein that does not colocalize with IM1. 1-3. The readout in Figure 4.D is plotted for IMCTL and IM1 proteins, where ICTL is the IMCTL-associated intensity for a given pixel, and mean ICTL is the average IMCTL intensity in the area indicated on the right side of the equation. [Figure 5A]Figure 1 shows the process for defining the threshold for high PCC values. One of the readouts developed is the percentage of ROIs showing strong colocalization for the two imaging markers defined in the ROI. PCC between dystrophin and four selected colocalization partners: β-dystroglycan, α-sarcoglycan, α-dystroglycan, and dysferlin for all myotubes from DMD donors in untreated and mock conditions. [Figure 5B] Figure 1 shows the process of defining a threshold for high PCC values. One of the readouts developed is the percentage of ROIs showing strong colocalization defined for two imaging markers in the ROI. PCC between dystrophin and MHC for all myotubes from healthy and DMD donors in untreated and mock conditions. In this example, MHC is used as a control protein that does not colocalize with dystrophin. A satisfactory threshold is one in which 99% of PCC values ​​between dystrophin and DGC proteins for DMD donors or MHC for all donor myotubes are below the threshold. In the example shown, 0.6 is determined as the satisfactory threshold for all tested imaging markers and DMD donors, because only outliers located at the 99th percentile exceed this threshold. High PCC% is the percentage of myotubes with PCC values ​​above a threshold of 0.6 among the total number of myotubes:

number

[0053] The discovery and development of therapies targeting muscle disorders has been hampered by the lack of in vitro systems that allow for the culture of differentiated muscle cells, particularly myotubes; the difficulty of generating animal models that exhibit the diverse genetic backgrounds found in patients with neuromuscular disorders; and the lack of quantitative cell-based functional assays to evaluate therapy efficacy and effectiveness. Many neuromuscular disorders require gene or RNA therapy. Because of the complex processes involved in the manufacture of these drug products and the high doses required to achieve therapeutic efficacy, multiple batches of these drug products are required to support even early-stage clinical trials. The release of these drug products requires efficacy assays that measure the activity of disease-promoting factors as a function of the therapeutic compound. However, the lack of suitable assays has led to several moratoriums imposed by the FDA during recent clinical trials targeting neuromuscular disorders. Furthermore, many muscle diseases are rare or ultra-rare. As the number of therapies in development increases, small patient populations are required to support clinical trials of these therapies and select among them. Functional assays performed on patient-derived cells can provide guidance for identifying the most beneficial therapeutic option for each individual patient.

[0054] In this application, we provide quantitative colocalization assays that monitor interactions between cellular components important for muscle function. These assays quantify the spatial overlap between two cellular components labeled in situ with specific labeling agents. Unlike co-immunoprecipitation assays or biochemical isolation of molecular complexes, these assays do not rely on physical interactions but can monitor direct, transient, or indirect interactions between cellular components and correlated changes in the subcellular localization of the labeled entities.

[0055] As proof of concept, we demonstrate herein that our quantitative colocalization assays can be used to quantitatively monitor the colocalization of dystrophin with β-dystroglycan (b-DG), a component of the dystrophin-glycoprotein complex (DGC) that directly interacts with dystrophin, and with α-sarcoglycan (a-SG), a component of the DGC that does not directly interact with dystrophin, in in vitro cultured myotubes and cardiomyocytes. We also show that these assays can be used to quantitatively monitor the colocalization, and therefore the interaction, between proteins and nucleic acids, particularly between the splicing factor MBNL1 and mutated DMPK RNA in DM1 patients. We also demonstrated the ability of dystrophin / b-DG and dystrophin / a-SG colocalization assays to quantitatively monitor the restoration of active dystrophin in myotubes derived from primary and immortalized cells from DMD patients treated with exon-skipping therapy and gene therapy, as well as to distinguish the response of DMD patients to exon-skipping therapy, and to quantitatively monitor the reversal of disease phenotype in myotubes from DM1 patients treated with antisense oligonucleotides (ASOs).

[0056] Taken together, these examples demonstrate that the colocalization assay we have developed fulfills the need for quantitative functional assays that contribute to the mechanistic understanding of muscle disorders, support the clinical development of therapies, particularly gene and RNA therapies, and enable evaluation of the benefit of novel therapies for individual disease genotypes.

[0057] Quantitative colocalization analysis The methods of the present invention are based on quantitative colocalization analysis, i.e., the in situ quantification of the spatial overlap between two cellular molecules labeled with specific labeling agents. These methods use at least one image of at least one in vitro cultured muscle cell to quantify the degree of colocalization between the two molecules by performing quantitative colocalization analysis.

[0058] In particular, the method typically comprises: (a) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell is stained for a first cellular molecule of interest and for a second cellular molecule that interacts / is known to interact with the first cellular molecule of interest; and (b) quantitatively determining the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing a quantitative co-localization analysis. Includes.

[0059] The cell types, culture conditions (e.g., the presence or absence of a particular compound), and molecules considered for quantitative colocalization analysis may vary depending on the method and disease of interest.

[0060] 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 generated by an in vitro culture technique.

[0061] The muscle cells are preferably mammalian muscle cells, particularly human muscle cells. Preferably, the muscle cells are striated muscle cells, i.e., skeletal muscle cells or cardiac muscle cells. More preferably, the muscle cells are myotubes or cardiac muscle cells. In preferred embodiments, the muscle cells are myotubes, particularly human myotubes. In some other embodiments, the muscle cells are cardiac muscle cells, particularly human cardiac muscle cells.

[0062] Depending on the method of the present invention, the muscle cells imaged can be healthy or pathological muscle cells.

[0063] Healthy muscle cells can be obtained by culturing muscle cells from at least one healthy subject, i.e., a subject not suffering from the disease of interest, preferably a subject not suffering from any muscle disease affecting said muscle cells, in particular a subject not suffering from any neuromuscular disease or cardiomyopathies as defined below.

[0064] Pathological muscle cells are muscle cells that exhibit at least one characteristic of a disease of interest.Pathological muscle cells can be obtained by culturing muscle cells derived from at least one patient suffering from the disease of interest, particularly from at least one patient suffering from a neuromuscular disease or cardiomyopathy as defined below, or by culturing muscle cells that have been chemically or genetically modified to not express or to express a mutant form of a molecule known to be a disease-promoting factor.For example, for DMD, pathological muscle cells can be myotubes that have been modified to not express dystrophin or to express a truncated form of dystrophin.Such modifications can be obtained by any method known to those skilled in the art, for example, RNA interference using siRNA that specifically targets the gene of interest, or by genetic alteration of the gene of interest.Preferably, pathological muscle cells are obtained by culturing muscle cells derived from at least one patient suffering from the disease of interest.

[0065] In particular, healthy myotubes can be obtained by culturing myoblasts from at least one healthy subject, preferably a subject not suffering from any muscle disease, in particular a subject not suffering from any neuromuscular disease as defined below. Myoblasts are a mononuclear cell type that mature by fusing with other myoblasts to give rise to myotubes that eventually develop into muscle fibers.

[0066] Pathological myotubes can be obtained by culturing myoblasts derived from at least one patient suffering from the neuromuscular disease of interest, or by culturing myoblasts that have been chemically or genetically modified to not express or to express mutant forms of molecules known to be disease promoters of the neuromuscular disease of interest. Preferably, pathological myotubes are obtained by culturing myoblasts derived from at least one patient suffering from the neuromuscular disease of interest.

[0067] Similarly, healthy cardiomyocytes can be obtained by culturing cardiomyocytes from at least one healthy subject, preferably a subject not suffering from any muscular disease, in particular a subject not suffering from any neuromuscular disease or cardiomyopathies as defined below.

[0068] The diseased cardiomyocytes can be obtained by culturing cardiomyocytes derived from at least one patient suffering from the neuromuscular disease or cardiomyopathy of interest, or by culturing cardiomyocytes that have been chemically or genetically modified so as not to express, or to express mutant forms of, molecules known to be disease promoters of the neuromuscular disease or cardiomyopathy of interest. Preferably, the diseased cardiomyocytes are obtained by culturing cardiomyocytes derived from at least one patient suffering from the neuromuscular disease or cardiomyopathy of interest.

[0069] As used herein, the terms "subject," "individual," and "patient" are interchangeable and refer preferably to animals, more preferably mammals, and even more preferably humans, including adult, pediatric, neonatal, and fetal humans.

[0070] The muscle cells used in the present invention can be obtained by culturing muscle cells derived from primary cells or immortalized cells.

[0071] In some specific embodiments, the muscle cells used in the present invention are obtained by culturing muscle cells derived from primary cells, in particular from primary cells obtained from healthy subjects or from patients suffering from the neuromuscular disease or cardiomyopathy of interest. In particular, the muscle cells can 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 for generating cardiomyocytes or myoblasts from induced pluripotent stem cells are known to those skilled in the art, see, for example, Ribeiro et al., 2015 (Proc Natl Acad Sci US 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 Chal and Olivier Pourquie, 2017 (Development 2017 Jun 15; 144(12): 2104-2122). Cardiomyocytes and myoblasts derived from induced pluripotent stem cells are also commercially available; see, for example, iCell Cardiomyocytes2 (human iPSC-derived cardiomyocytes) and ioSkeletal Myocytes—human iPSC-derived skeletal muscle cells (ab277612, Abcam) from FUJIFILM Cellular Dynamics. In some other specific embodiments, the muscle cells used in the present invention are obtained by culturing immortalized cells, particularly immortalized cells derived from a healthy subject or from a patient suffering from the neuromuscular disease or cardiomyopathies of interest. In embodiments in which two types of cells are used, e.g., healthy and pathological cells, one type may be derived from primary cells and the other type may be derived from immortalized cells. Furthermore, muscle cells of the same type, e.g., healthy or pathological cells, can be obtained from a unique source, i.e., from the same subject or from a unique immortalized cell line, or 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 or diseased cells, are obtained from a unique source, i.e., from the same subject or from a unique immortalized cell line.

[0072] Prior to being imaged, muscle cells are cultured / produced in vitro on a suitable substrate and under suitable conditions known to those skilled in the art.

[0073] Methods for culturing muscle cells, in particular for generating myotubes by culturing myoblasts, or for generating cardiomyocytes, are well known to those skilled in the art and include culturing on patterned or unpatterned substrates, on substrates with or without topological constraints, on soft substrates (e.g., synthetic hydrogel materials, e.g., poly(hydroxyethyl methacrylate), polyacrylamide, polyethylene glycol, polyacrylic acid, poly(vinyl alcohol), polyvinylpyrrolidone, polyimides, and polyurethanes, natural hydrogel materials, e.g., agarose, dextran, gelatin, and matrigel, and silicone materials), or on hard substrates (e.g., glass, silicone, or plastics, e.g., 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 application).

[0074] In a preferred embodiment, the in vitro cultured muscle cells are not organized into tissue. In particular, the in vitro cultured muscle cells are not included in organized muscle tissue structures that include connective tissue such as endomysium, perimysium, and epimysium, capillaries, or adipocytes. Thus, the image provided in step a) is not an image of a tissue or biopsy sample.

[0075] Preferably, the muscle cells are cultured under constrained conditions, i.e., on a substrate that allows for the generation of a homogeneous population of muscle cells. Preferably, all muscle cells (e.g., healthy, pathological, or reference myotubes) used in the methods of the invention are cultured under the same constrained conditions, e.g., on a substrate with the same topological constraints or adhesive pattern.

[0076] A homogeneous population of muscle cells is a population of muscle cells that exhibit similar morphological parameters.

[0077] In particular, myotubes of a homogeneous myotube population may exhibit similar fusion index (ratio to the total number of nuclei within a myotube), maturation index (number of nuclei per myotube), and / or myotube area (or width / length ratio, width and / or length). Preferably, myotubes of a homogeneous myotube population exhibit less than 30%, preferably less than 20%, variation in fusion index, less than 30%, preferably less than 20%, variation in maturation index, and / or less than 30%, preferably less than 20%, variation in myotube area (or width / length ratio, width and / or length). More preferably, myotubes of a homogeneous myotube population exhibit less than 30%, preferably less than 20%, variation in fusion index, less than 30%, preferably less than 20%, variation in maturation index, and less than 30%, preferably less than 20%, variation in myotube area (or width / length ratio, width and / or length).

[0078] Cardiomyocytes of a homogeneous cardiomyocyte population may exhibit similar cell area (or width / length ratio, width and / or length). Preferably, cardiomyocytes of a homogeneous cardiomyocyte population exhibit a variation in cell area (or width / length ratio, width and / or length) of less than 30%, preferably less than 20%.

[0079] Homogeneity is assessed independently for each population of muscle cells used in the methods of the invention, i.e., muscle cells derived from a particular source (e.g., muscle cells derived from a healthy subject) and cultured under particular conditions (e.g., cultured in the presence of a test compound).

[0080] Typically, the culture under constrained conditions is culture on a substrate with topological constraints or adhesive patterns. Such substrates that allow the generation of homogeneous populations of muscle cells, and in particular, homogeneous populations of myotubes and cardiomyocytes, are well known to those skilled in the art. Examples of such substrates include, but are not limited to, substrates with linear grooves formed on the surface of the substrate by etching techniques (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, geometric shapes, e.g., circles, squares, and Y-shapes (Junkin et al., 2011, Journal of Cell Science 124, 4213-4220), substrates with adhesive "hybrid" patterns consisting of a combination of linear elements and arcuate elements with a linear element at the center (Bajaj et al., 2011, Integrative Biology 3, 897-909), or substrates with adhesive patterns forming lines, geometric shapes, e.g., circles, squares, and Y-shapes (Junkin et al., 2011, Journal of Cell Science 124, 4213-4220), and substrates with adhesive "hybrid" patterns consisting of a combination of linear elements and arcuate elements with a linear element at the center (Bajaj et al., 2011, Integrative Biology 3, 897-909), or substrates with adhesive patterns forming lines, geometric shapes, e.g., circles, squares, and Y-shapes (Bajaj et al., 2011, Integrative Biology 3, 897-909), as described 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), Bray et al., 2008 (Bray et al., 2008. Cell motility and the cytoskeleton 65(8):641), or Ribeiro et al., 2015 (Ribeiro et al., 2015. Proc Natl Acad Sci US A. 2015;112(41):12705-12710). Preferably, myoblasts are cultured on a substrate comprising an adhesive pattern, preferably an adhesive pattern as disclosed in WO 2015 / 091593, WO 2016 / 202850 and WO 2016 / 139312, in particular as disclosed in FIG. 2A of International Patent Application WO 2015 / 091593 and Young et al., 2018 (Young et al., SLAS Discov. 2018 Sep;23(8):790-806). The adhesive properties of the pattern can be obtained by coating the pattern with one or several extracellular matrix proteins, preferably fibronectin.

[0081] In a particular embodiment, myoblasts are cultured on a substrate as disclosed in WO 2015 / 091593, in particular in FIG. 2A of WO 2015 / 091593 or in FIG. 14 of the present application, i.e., a substrate comprising at least one cell adhesive pattern; - the pattern (1) has an elongated surface comprising a central region (1C) and two side regions (1L) extending in both directions from the central region along the longitudinal axis of the pattern, with a contour discontinuity between the central region (1C) and each side region (1L), the length (L) of the pattern being comprised between 100 and 1000 μm, and the maximum width (W) of the pattern being C ) is between 50 and 500 μm, - Maximum width of the central area (1C) (W C ) and the maximum width (W L ) is greater than or equal to 2, - The length (L) and maximum width (W) of the pattern (1) C ) is less than or equal to 4, - the pattern consists of three overlapping elliptical surfaces: a first elliptical surface defining a central region of the pattern, and second and third elliptical surfaces having major axes coinciding with the major axis of the first ellipse defining side regions of the pattern; The second and third elliptical surfaces intersect the first elliptical surface along their transverse axes.

[0082] Preferably, the pattern is symmetrical along its longitudinal axis (X) and a horizontal axis perpendicular to the longitudinal axis.

[0083] Preferably, the length (L) and maximum width (W) of the pattern C ) is 2.5, and / or the area of ​​the pattern is 5,000 to 500,000 μm 2 is included between.

[0084] Preferably, the method for culturing muscle cells, in particular myoblasts / myotubes or cardiomyocytes, is adaptable to high-throughput platforms and to the performance of high-throughput assays.

[0085] Prior to being imaged, the muscle cells are stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest, i.e., the muscle cells are stained with a first labeling agent that reveals the first molecule of interest and a second labeling agent that reveals the second molecule.

[0086] In a preferred embodiment, the muscle cells are also stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual muscle cells, cellular structures of muscle cells, including, but not limited to, nuclei, vacuoles, mitochondria, lysosomes, cell membranes, and cytoskeleton.

[0087] In particular, myotubes can be stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes, myotube cellular structures, and any combination thereof. Cardiomyocytes can be stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes, cardiomyocyte cellular structures, and any combination thereof. In a preferred embodiment, myocytes are stained with at least one labeling agent that reveals individual cells, in particular individual myotubes or cardiomyocytes. Optionally, myocytes, in particular myotubes or cardiomyocytes, can be further stained with at least one labeling agent that reveals nuclei.

[0088] Examples of labeling agents that can be used to reveal individual muscle cells include, but are not limited to, antibodies against troponin-T or myosin heavy chain (MHC). Examples of labeling agents that can be used to reveal nuclei include, but are not limited to, Hoechst and DAPI dyes. Examples of labeling agents that can be used to reveal mitochondria include, but are not limited to, Mitotracker™ dyes.

[0089] The selection of ROI depends on the disease of interest and can be easily selected by those skilled in the art based on general knowledge. In the case of a disease caused by a mutation in a gene associated with the function of an organelle or inducing a change in the structure of said organelle, those skilled in the art can select said organelle as the ROI.

[0090] DM1 is known to induce RNA foci, therefore, in a specific embodiment, the disease of interest is DM1 and myotubes are stained for two ROIs, i.e., individual myotubes and nuclei, to perform quantitative colocalization analysis in myotube nuclei.

[0091] In another specific embodiment, the disease of interest is DMD and the myotubes are stained for one ROI, i.e., individual myotubes. Optionally, the myotubes can be stained for another ROI, i.e., nuclei, to perform colocalization analysis in the nuclei of the myotubes.

[0092] Staining can be performed during or after culturing the muscle cells. Methods for staining / labeling cellular targets are well known to those skilled in the art.

[0093] As used herein, the term "labeling agent" refers to any agent used to specifically detect and label a first or second molecule or ROI. The agent emits a signal that is visible in an acquired image of the stained muscle cell. The labeling agent can specifically recognize a target (i.e., a first or second molecule or ROI) and emit a detectable signal, such as a fluorescent, luminescent, chemiluminescent, or radioactive signal, preferably a fluorescent signal.

[0094] A labeling agent can include a moiety capable of specifically recognizing a target, i.e., an antibody or nucleic acid moiety, and a moiety that emits a detectable signal, i.e., a fluorescent dye. In some embodiments, these moieties are covalently bound, for example, an antibody or nucleic acid that specifically recognizes the target and has a fluorescent dye. In some other preferred embodiments, these moieties are provided by two or more separate molecules, for example, a primary antibody or nucleic acid that specifically recognizes the target and a secondary antibody or nucleic acid that specifically recognizes the primary antibody or nucleic acid and emits a detectable signal, for example, has a fluorescent dye. Thus, the term "labeling agent" encompasses one or several molecules depending on the embodiment.

[0095] Each labeling agent emits a detectable and unique signal, i.e., a signal that can be distinguished from the signals of other labeling agents used to stain cells. Preferably, each labeling agent comprises a different fluorescent label with a different emission and / or excitation wavelength. For example, such a labeling agent may be or include an antibody or nucleic acid probe conjugated to a fluorescent dye (i.e., immunostaining). Alternatively, the labeling agent may be a molecule that naturally binds to a target and emits a detectable signal, such as a fluorescent dye, e.g., DAPI, which naturally binds to DNA.

[0096] Accurate colocalization determination in fluorescence microscopy can be achieved when the emission spectra of fluorescent dyes are sufficiently separated.To achieve this, fluorescent labels can be selected so that the emission wavelengths are sufficiently separated and can be resolved by the imaging device used.Depending on the spectral resolution of the detection device used, those skilled in the art will be able to select appropriate labels that allow accurate colocalization determination.Conversely, when a specific set of labels is to be used, those skilled in the art will be able to select appropriate imaging devices so that the labels can be separated and determined.

[0097] The labeling agent used to stain muscle cells can be easily selected by those skilled in the art depending on the nature of the molecule to be labeled. Preferably, when the molecule to be labeled is a protein, the labeling agent may be an antibody against the wild-type or mutant form of the protein, the antibody comprising a moiety that emits a detectable signal, preferably a fluorescent signal, or a primary antibody against the wild-type or mutant form of the protein and a secondary antibody that recognizes the primary antibody and emits a detectable signal, preferably a fluorescent signal. Preferably, when the molecule to be labeled is a nucleic acid, the labeling agent may be a nucleic acid probe that specifically hybridizes with the wild-type or mutant form of the nucleic acid and comprises a moiety that emits a detectable signal, preferably a fluorescent signal, or a first nucleic acid probe that specifically hybridizes with the wild-type or mutant form of the nucleic acid and a second nucleic acid probe that specifically hybridizes with the first nucleic acid probe and emits a detectable signal, preferably a fluorescent signal.

[0098] The first molecule and the second molecule are cellular molecules, i.e., molecules naturally present in healthy or pathological 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 a preferred embodiment, 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 molecule and the second molecule are different, for example, two different proteins, one protein and one nucleic acid, or two different nucleic acids.

[0099] The molecules considered for colocalization analysis depend on the disease of interest. Significant advances in our molecular understanding of muscle function have led to the identification of protein complexes and protein-protein or protein-nucleic acid interactions that are important for muscle function and that are involved in the pathogenesis of muscle diseases, particularly neuromuscular diseases or cardiomyopathies.

[0100] The first and second molecules are preferably selected from a group of molecules associated with a cellular molecular complex or cellular function affected by the disease of interest. Preferably, the first or second molecule is a disease promoter of the disease of interest, i.e., a protein or nucleic acid identified as a known genetic or physiological cause of the disease, or a protein or nucleic acid whose expression and / or activity is known to be specifically altered in the disease of interest.

[0101] The second molecule is a molecule known to interact with the first molecule in healthy or pathological muscle cells, i.e., directly or indirectly. Preferably, the first and second molecules are known to interact in healthy muscle cells and to have no, little, or impaired interaction in pathological muscle cells, or vice versa. A direct interaction involves physical contact between two molecules. For example, dystrophin directly interacts with β-dystroglycan (b-DG). An indirect interaction involves an intermediate molecule between the first and second molecules. In particular, the first and second molecules may belong to the same protein complex but are not in direct physical contact. For example, dystrophin and α-sarcoglycan (a-SG) interact indirectly; they are components of the dystrophin-glycoprotein complex (DGC) but do not directly interact together.

[0102] Examples of first and second molecules that can be selected depending on the disease of interest include, but are not limited to, the molecules provided in Tables 1 and 2. For each disease in the columns "Neuromuscular Disease" or "Cardiomyopathy," the first molecule can be selected from the molecules in the column "Examples of Molecules Useful as First Molecules" and the second molecule can be selected from the molecules in the column "Examples of Molecules Useful as Second Molecules," or the first molecule can be selected from the molecules in the column "Examples of Molecules Useful as Second Molecules," and the second molecule can be selected from the molecules in the column "Examples of Molecules Useful as First Molecules." In a preferred embodiment, the first molecule is selected from the molecules in the column "Examples of Molecules Useful as First Molecules," and the second molecule is selected from the molecules in the column "Examples of Molecules Useful as Second Molecules." These tables also include examples of appropriate ROIs depending on the disease and the molecules used in the colocalization analysis. Preferably, in embodiments in which muscle cells are stained with at least one labeling agent that defines at least one region of interest (ROI), said at least one ROI includes an ROI from Table 1 or 2 corresponding to the first molecule and the second molecule and the disease. By way of example, in embodiments in which 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 a myotube.

[0103] [Table 1A]

[0104] [Table 1B]

[0105]

Table 1C

[0106]

Table 1D

[0107] Table 1E

[0108] Table 1F

[0109]

Table 1G

[0110] Table 1H

[0111] Table 2A

[0112] Table 2B

[0113] Table 2C

[0114]

Table 2D

[0115] [Table 2E]

[0116] [Table 2F]

[0117] [Table 2G]

[0118] Dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and arrhythmogenic cardiomyopathy (ARCV) are a large group of cardiac pathologies that can be caused by various genetic mutations. Each cardiomyopathic cardiomyopathy in Table 2 can be further characterized by a mutation in a gene encoding a molecule shown in the column "Examples of molecules useful as first molecules." By way of example, the cardiomyopathy can be dilated cardiomyopathy (DCM) caused by a mutation in the gene encoding ACTC1 (actin alpha cardiac muscle). In this case, the first molecule can be ACTC1, and the second molecule can 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.

[0119] In some embodiments, the muscle 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 molecule and the second molecule are independently selected from the group consisting of DMD, SNTA, SNTB, ANK1, ANK2, DAG1, SSPN, SGCA, SGCB, SGCD, SGCG, DTNA, and FLNC proteins.

[0120] In some embodiments, the muscle disease is selected from the group consisting of LGMD D5, LGMD R22, and Ullrich myopathy (UM), and the first molecule and the second molecule are independently selected from the group consisting of COL6A1, COL6A2, COL6A3, DAG1, LAMA2, ITGA7, DES, FKTN, FKPR, POMT1 / 2, POMGNT1, and LARGE1 proteins.

[0121] In some embodiments, the muscle 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 molecule and the second molecule are independently selected from the group consisting of DAG1, POMT1 / 2, POMGNT1, LARGE1, FKTN, FKRP, LAMA2, ITGA7, and DES proteins.

[0122] In some embodiments, the muscle disease is selected from the group consisting of centronuclear myopathy (CNM), distal myopathy (DM), inclusion body myopathy (IBM), LGMD R10, myofibrillar myopathy (MFMP), nemaline myopathy (NEM2, NEM3), protein aggregation myopathy (PAM), rimmed vacuolar myopathy (RVM), and tibial muscular dystrophy (TMD), and the first molecule and the second molecule are independently selected from the group consisting of TTN, MYH2, DES, ACTA1, NEB, MYH7, MYOT, TNNT3, and MURF1 proteins.

[0123] In some embodiments, the muscle disease is selected from the group consisting of congenital muscular dystrophy (CMD) and Emery-Dreifuss muscular dystrophy (EDMD), and the first molecule and the second molecule are independently selected from the group consisting of LMNA, EMD, SYNE1 / 2, TNPO3, and BAF1 proteins.

[0124] In some embodiments, the muscle disease is selected from the group consisting of centronuclear myopathy (CNM), congenital myopathy (CM), LGMD R1, malignant hyperthermia syndrome (MHS), tubular aggregate myopathy (TAM), X-linked myotubular myopathy (XLMTM), Becker muscular dystrophy (BMD), facioscapulohumeral muscular dystrophy (FSHD), and myotonic dystrophy type 1 (DM1), and the first molecule and the second molecule are independently selected from the group consisting of BIN1, DNM2, MTM1, SPEG, RYR1, STAC3, CACNA1S, CACNB1, CAPN3, CASQ1, STIM1, ORAI1, TRDN, SERCA1, and CLCN1 proteins.

[0125] In some embodiments, the muscle disease is selected from the group consisting of LGMD R2, LGMD R12, centronuclear myopathy (CNM), and Miyoshi myopathy (MM), and the first molecule and the second molecule are independently selected from the group consisting of DYSF, ANO5, BIN1, TRIM72, EHD1, EHD2, ANXA1, ANXA2, and ANXA5 proteins.

[0126] In some embodiments, the muscle disease is selected from the group consisting of LGMD D1, Marinesco-Sjögren's syndrome (MSS), and myofibrillar myopathy (MFMP), and the first molecule and the second molecule are independently selected from the group consisting of DNAJB6, SIL1, BAG3, and HSPB5 proteins.

[0127] In some embodiments, the muscle disease is selected from the group consisting of LGMD R8, nemaline myopathy 8 (NEM8), Danon disease (DD), distal myopathy with rimmed vacuoles (DMRV), neurodegeneration with ataxia, dystonia and gaze palsy, childhood onset (NADGP), Pompe disease, protein aggregation myopathy (PAM), Vici syndrome (VS), and X-linked myopathy with excessive autophagy (xMEA), and the first molecule and the second molecule are independently selected from the group consisting of TRIM32, KLH40, KLH41, LAMP1, LAMP2, SQSTM1, GAA, MURF1, EPG5, and VMA21 proteins.

[0128] In some preferred embodiments, particularly when the disease of interest is Duchenne muscular dystrophy or myotonic dystrophy type 1, the first molecule and the second molecule 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, α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin, more preferably selected from the group consisting of dystrophin, α-sarcoglycan, and β-dystroglycan. In particular, the first molecule may be dystrophin, and the second molecule can be selected from the group consisting of proteins belonging to the dystrophin glycoprotein complex (DGC) and dysferlin, preferably selected from the group consisting of α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin, more preferably selected from the group consisting of α-sarcoglycan and β-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 α-sarcoglycan, β-dystroglycan, α-dystroglycan and dysferlin, more preferably α-sarcoglycan and β-dystroglycan.

[0129] In some other preferred embodiments, particularly when the disease of interest is myotonic dystrophy type 1, the first molecule is DMPK RNA and the second molecule is an RNA-binding protein, preferably MBNL1 protein, that is trapped by CTG repeats in the DMPK gene. Alternatively, the second molecule may be DMPK RNA and the first molecule may be an RNA-binding protein, preferably MBNL1 protein, that is trapped by CTG repeats in the DMPK gene.

[0130] Those skilled in the art can readily select combinations of first and second molecules that can be used in the methods of the present invention. (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 (a "pathological muscle cell"), preferably at least one in vitro cultured pathological myotube or cardiomyocyte, wherein the muscle cell has been stained for a first cellular molecule and a second cellular molecule, preferably selected as described above; and (b) quantitatively determining the degree of co-localization of the first cellular molecule and the second cellular molecule in the at least one healthy muscle cell and the at least one diseased muscle cell by performing a quantitative co-localization analysis. Including, If the degree of co-localization in the at least one healthy muscle cell is significantly different from the degree of co-localization in the at least one diseased muscle cell, the combination of first and second molecules can be used in the methods of the invention, particularly in methods related to a disease of interest. The method can be used.

[0131] Healthy and diseased cells are cultured under the same conditions to provide comparable degrees of co-localization. Preferably, the numbers of healthy and diseased cells are selected to provide statistically significant results.

[0132] As mentioned previously, the possibility of using a high-throughput platform to culture myocytes, stain myocytes, and acquire images of myocytes makes this method easily repeatable without undue burden to those skilled in the art.

[0133] After staining the myocytes, images of these stained myocytes are acquired.

[0134] This step can be performed using any device suitable for acquiring microscopic images. The microscopic images can be taken, for example, 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 selected to provide an image with a resolution in the range of 1 μm to 10 nm, preferably in the range of 800 nm to 100 nm, more preferably in the range of 600 nm to 200 nm.

[0135] The choice of imaging technique also depends on the nature of the signal emitted by the labeling agent. Preferably, the labeling agent emits a fluorescent signal, and the microscopic image is taken by fluorescent imaging and acquiring each channel corresponding to the first molecule, the second molecule, and optionally the labeling agent used to reveal at least one ROI.

[0136] Optionally, an illumination function can be applied to the acquired image to correct for uneven illumination.

[0137] As used herein, the measurement of each different label may also be referred to as a detection channel. For example, in fluorescence detection, each channel corresponds to one label with a specific emission or excitation wavelength. In some embodiments, different channels are acquired as different images. The colocalization of two different labels / signals may also be referred to as the colocalization of two channels.

[0138] In preferred embodiments, the imaged myocytes are stained with at least one labeling agent for a first molecule of interest, a second molecule that interacts with the first molecule of interest, and at least one region of interest (ROI) selected from the group consisting of individual cells, cellular structures, and any combination thereof. In these embodiments, prior to quantitative colocalization analysis, image segmentation is performed using an algorithm on the appropriate staining channel to identify the ROI. Each image segmented in this manner can contain one or more ROIs. The appropriate staining channel corresponds to the labeling agent used to define the ROI. For example, segmentation of individual myotubes and nuclei can be performed using the channel for the labeling agent that defines troponin T or myosin heavy chain and the channel for Hoechst dye, respectively. Preferably, the segmentation threshold is set to avoid detecting background noise and to exclude abnormal small cellular structures, particularly abnormal small myotubes or cardiomyocyte structures. Segmentation of the ROI can be performed by any method known to those skilled in the art, for example using the open source software Cell Profiler (Carpenter et al., 2006, Genome Biology, 7(10)) or suitable software applications such as Matlab or Fiji, or programming languages ​​such as Python, Java, C++.

[0139] An image of stained in vitro cultured muscle cells is provided, and optionally image segmentation is performed to identify an ROI, followed by quantitative colocalization analysis to determine the degree of colocalization of a first molecule of interest with a second molecule in the muscle cells or the ROI.

[0140] To determine the degree of colocalization, each myocyte or each ROI is analyzed individually. To provide statistically significant results, one skilled in the art can easily adjust the number of myocytes and ROIs considered in determining the degree of colocalization. In particular, the degree of colocalization is preferably determined in at least 30 myocytes, more preferably at least 60 myocytes. In a preferred embodiment, the degree of colocalization is determined in at least 30 ROIs (preferably at least one ROI per myocyte), and preferably at least 60 ROIs (preferably at least one or two ROIs per myocyte).

[0141] Quantitative colocalization analysis can be performed by any method known to those skilled in the art, such as quantitative pixel-based colocalization analysis or machine learning-based analysis. Preferably, the method of the present invention does not include any step of using machine learning-based analysis.

[0142] Preferably, quantitative colocalization analysis is performed using quantitative pixel-based colocalization analysis, where colocalization refers to the presence of a signal from a label of a first molecule and a signal from a label of a second molecule at the same pixel location.

[0143] Methods for quantitatively determining the degree of colocalization between two molecules, particularly by performing quantitative colocalization analysis using pixel intensity-based colocalization analysis, are well known to those skilled in the art (see, for example, Costes et al., Biophysical Journal, Vol. 86, June 2004, 3993-4003; Zinchuk et al., Acta Histochem. Cytochem. 40(4):101-111, 2007; Wu et al., Biophysical Journal, Vol. 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 an image or a selected region of interest (ROI).

[0144] When two molecules are spatially distributed across the region of the cell being analyzed, there will be some degree of random overlap, but if their spatial distributions are independent of each other, there will be no specific colocalization between the two molecules. Conversely, if two molecules have some kind of specific colocalization, then superposition of the two spatial distributions will show a level of correlation that exceeds random overlap.

[0145] Therefore, proper assessment of colocalization typically requires background correction by thresholding. Quantitative colocalization analysis using thresholding quantifies the colocalized portion of each molecular species but also requires a threshold for each signal / channel, which is then used as a cutoff between specific and nonspecific staining. The threshold is the intensity threshold at which a labeled molecule is considered to be present at an image location (pixel). The colocalization region can therefore be defined by the region where the signal from the first molecule exceeds threshold T1 and the signal from the second molecule exceeds threshold T2. Typically, the determination of the threshold is assisted / performed by computer software. Typically, the threshold is determined with respect to a control sample in which the signal of interest is absent; for example, the threshold for the signal of the first molecule can be determined with respect to an image of muscle cells that are not stained for that molecule. In some preferred embodiments, where the labeling agents for the first and second molecules comprise the primary and secondary antibodies disclosed above, the threshold can be determined with respect to an image of muscle cells that have been contacted with the secondary antibody but not the primary antibody. The threshold may also be determined by quantile statistics or by any other known method.

[0146] Optionally, one or more filtering steps can be applied to the images before calculating the colocalization readout. In particular, a respective threshold value for each labeled molecule can be subtracted from its corresponding image. Pixel values ​​below zero can be clipped to zero.

[0147] The degree of colocalization between a first molecule and a second molecule can be quantitatively determined by calculating a colocalization metric between two detection channels, i.e., a channel corresponding to the signal of the first molecule and a channel corresponding to the signal of the second molecule. This can be done by various approaches well known to those skilled in the art. In particular, the degree of colocalization can be quantitatively determined by calculating a value representing the overlap coefficient of the two detection channels.

[0148] Preferably, the degree of colocalization is quantitatively determined by calculating an overlap coefficient for each cell or ROI and, optionally, applying a mathematical function to the coefficient. In particular, the overlap coefficient can be selected from the group consisting of Pearson's colocalization coefficient (PCC), Mander's colocalization coefficient (MCC), rank-based intensity weighting coefficient (RWC), Mander's overlap coefficient (MOC), and any combination thereof. Preferably, the overlap coefficient is selected from the group consisting of Pearson's colocalization coefficient (PCC), Mander's colocalization coefficient (MCC), rank-based intensity weighting coefficient (RWC), and any combination thereof. Tools for quantifying PCC, MCC, and RWC are provided in almost all image analysis software packages.

[0149] In some embodiments, determining the degree of colocalization comprises calculating the Pearson's colocalization coefficient (PCC) for each myocyte or ROI, preferably for each myocyte.

[0150] PCC measures the pixel-by-pixel covariance of signal levels in two images / channels. Typically, PCC values ​​range from 1 for two images / channels whose signal intensities are perfectly linearly correlated to -1 for two images / channels whose signal intensities are perfectly but inversely related to each other. Values ​​near zero reflect uncorrelated signal distribution. The formula for calculating PCC, exemplified in the case of colocalization of a first molecule, IM1 (imaging marker 1), and a second molecule, IM2 (imaging marker 2), is illustrated in Figure 4.E.1. PCC can be calculated for all cells, all ROIs, or within the threshold range above. This latter option is more rigorous because regions without any molecule positively influence the PCC. If arbitrary image culling is selected, certain ROIs may not have pixel intensities above zero for one molecule-related channel. In this case, the denominator value is equal to zero, resulting in an undefined PCC. Since the correlation between the signal associated with the other molecule and this invariant signal is null, the PCC can be set to 0.

[0151] In some embodiments, determining the degree of colocalization includes calculating the Mander's Colocalization Coefficient (MCC) for each myocyte or ROI, preferably for each myocyte. The MCC is independent of pixel intensity correlation. In effect, the MCC is a measure of co-occurrence. It measures the proportion of signals associated with one molecule that overlap with other molecular signals. The formula for calculating the MCC, exemplified in the case of co-localization of a first molecule IM1 (imaging marker 1) and a second molecule IM2 (imaging marker 2), is illustrated in Figure 4.E.2.

[0152] In some embodiments, determining the degree of colocalization involves calculating a rank-based intensity weighting coefficient (RWC) for each myocyte or ROI, preferably for each myocyte. RWC attempts to add the concept of correlation to MCC. In effect, the algorithm uses a nonparametric ranking of pixel intensities in each channel and weights the pixel intensities of a molecule using the difference in rank of colocalized pixel locations in the two channels (see Figure 4.E.3). The closer the pixel ranks of the two intensities, the higher the intensity weight at this pixel. This weighting is applied to co-occurring pixels, thereby combining both co-occurrence and correlation. The formula for calculating RWC, exemplified in the case of co-localization of a first molecule IM1 (imaging marker 1) and a second molecule IM2 (imaging marker 2), is illustrated in Figure 4.E.3.

[0153] In some embodiments, the degree of colocalization is quantitatively determined by calculating, for each myocyte or ROI, preferably for each myocyte, an overlap coefficient as defined above and applying a mathematical function to said coefficient. In particular, the degree of colocalization can be quantitatively determined by calculating, for each myocyte or ROI, preferably for each myocyte, an overlap coefficient selected from the group consisting of Pearson's Colocalization Coefficient (PCC), Mander's Colocalization Coefficient (MCC), Rank-Based Intensity Weighting Coefficient (RWC), and any combination thereof, and applying a mathematical function to said coefficient.

[0154] The mathematical function can be selected to calculate any meaningful value derived from the overlap coefficients obtained from individual cells or ROIs, such as a mean value (e.g., the mean overlap coefficients of myocytes or ROIs, e.g., mean PCC, mean MCC, mean RWC), a quantile value, e.g., a median, or a ratio, e.g., the ratio between the colocalization value of a first ROI, e.g., myotubes, and a second ROI, e.g., nuclei, a mean / max coefficient value, e.g., mean / max PCC. The mathematical function is selected to reflect the degree of colocalization and to preserve the ability to compare different assays. One skilled in the art can easily select such a function.

[0155] Optionally, in embodiments where determining the degree of colocalization includes calculating the Pearson's Colocalization Coefficient (PCC), the quantitative colocalization analysis can further include defining a threshold for high PCC values, i.e., the threshold at which the PCC is considered to reflect a strong correlation between two signals in the region under consideration. To define this threshold, the PCC values ​​of each myocyte and the negative control PCC values ​​can be plotted. A satisfactory threshold, for example, is for 99% of the PCC values. In this case, only outliers located at the 99th percentile exceed this threshold. High PCC% is the percentage of myocytes with PCC values ​​above this threshold among the total number of myocytes:

[0156]

number

[0157] Optionally, to enhance the reliability of the colocalization readout, the method can further include repeating steps (a) and (b) while i) replacing the first molecule of interest with a negative control molecule known not to colocalize with the second molecule, and / or ii) replacing the second molecule with a negative control molecule known not to colocalize with the first molecule. In this case, the colocalization readout considers only regions where the signal of the first molecule or the second molecule exceeds the signal obtained with the negative control. In particular, the negative control molecule can be a molecule that is uniformly present throughout the muscle cell. For example, in embodiments where the muscle cell is a myotube, the negative control molecule can be myosin heavy chain (MHC).

[0158] The method for determining the degree of colocalization can be easily selected depending on the pair of first / second molecules and / or muscle cells and / or the disease of interest. In some embodiments, the first molecule and the second molecule are proteins, and determining the degree of colocalization comprises calculating a Pearson's colocalization coefficient (PCC), and optionally determining a threshold for a high PCC value. In some other embodiments, at least one of the first molecule and the second molecule is a nucleic acid, and determining the degree of colocalization comprises calculating a Mander's colocalization coefficient (MCC).

[0159] Methods of the Invention The inventors herein demonstrate that the quantitative colocalization assay of the present invention can be used to quantitatively monitor the colocalization of two cellular molecules that are known to interact directly or indirectly in healthy or diseased muscle cells, thereby quantitatively monitoring the functionality of these molecules.

[0160] In a first aspect, the present invention therefore relates to an in vitro method for assessing the functionality of a cellular molecule of interest in muscle cells.

[0161] The method comprises: (a) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing a quantitative co-localization analysis. Including, The degree of co-localization correlates with the functionality of the cellular molecule of interest in said at least one muscle cell.

[0162] In a preferred embodiment, the at least one muscle cell is also stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0163] Thus, in a preferred embodiment, the method comprises: (i) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell has been stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest, and has been stained with at least one labeling agent that reveals 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 image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis. Including, The degree of co-localization correlates with the functionality of the cellular molecule of interest in said at least one muscle cell.

[0164] Steps (a), (b), (i), (ii) and (iii) are described in detail above in the section "Quantitative Colocalization Analysis." All of the above embodiments of these steps are also encompassed by this aspect.

[0165] The muscle cells used in this method can be healthy or diseased.

[0166] In one embodiment, the method comprises: (a) providing at least one image of at least one in vitro cultured myotube, wherein the at least one myotube is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing a quantitative co-localization analysis. Including, The degree of co-localization correlates with the functionality of the cellular molecule of interest.

[0167] In particular, the method (i) providing at least one image of at least one in vitro cultured myotube, wherein the at least one myotube has been stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest, and has been stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes and myotube cellular structures, and any combination thereof; and (ii) performing image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis. and The degree of co-localization correlates with the functionality of the cellular molecule of interest in said at least one myotube.

[0168] Preferably, said at least one in vitro cultured myotube is at least one human healthy or pathological myotube, in particular at least one myotube exhibiting features of a neuromuscular disease as defined below.

[0169] In another embodiment, the method comprises: (a) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein the at least one cardiomyocyte is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing a quantitative co-localization analysis. Including, The degree of co-localization correlates with the functionality of the cellular molecule of interest.

[0170] In particular, the method (i) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein the at least one cardiomyocyte is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and is stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second molecule in at least one ROI by performing a quantitative colocalization analysis. and The degree of co-localization correlates with the functionality of the cellular molecule of interest in said at least one cardiomyocyte.

[0171] Preferably, said at least one in vitro cultured cardiomyocyte is at least one human healthy or pathological cardiomyocyte, in particular at least one cardiomyocyte exhibiting features of a neuromuscular disease or cardiomyopathies as defined below.

[0172] In this method, the degree of co-localization correlates with the functionality of the first cellular molecule of interest, preferably a protein or nucleic acid, in the muscle cell of interest.

[0173] Depending on the first molecule and the second molecule, the degree of colocalization can be positively or negatively correlated with the functionality of the molecule of interest. A positive correlation is the relationship between two variables that move in tandem, i.e., in the same direction. A positive correlation exists when one variable decreases, the other variable also decreases, or when one variable increases, and the other also increases. Conversely, a negative correlation is the relationship between two variables when one variable increases, the other decreases (or vice versa).

[0174] In some embodiments, the degree of colocalization positively correlates with the functionality of the first cellular molecule of interest in the muscle cell of interest, where the higher the degree, the more functional the molecule. In some other embodiments, the degree of colocalization negatively correlates with the functionality of the first cellular molecule of interest in the muscle cell of interest, where the lower the degree, the more functional the molecule.

[0175] Those skilled in the art will know whether the functionality of the molecule of interest is positively or negatively correlated with the degree of colocalization depending on the first molecule and the second molecule.Indeed, when two molecules are known to interact in healthy cells, the functionality of the molecule of interest is positively correlated with the degree of colocalization.For example, in an embodiment where the first molecule is dystrophin and the second molecule is a protein of the dystrophin-associated protein complex (DGC), the functionality of dystrophin is positively correlated with the degree of colocalization, because these proteins interact in healthy cells.In contrast, when two molecules are known to have no or little interaction in healthy cells, but are known to interact in pathological muscle cells, the functionality of the molecule of interest is negatively correlated with the degree of colocalization. For example, in embodiments where the first molecule is DMPK RNA and the second molecule is an RNA-binding protein, e.g., MBNL1 protein, that is trapped by CTG repeats in the DMPK gene, or vice versa, the functionality of the molecule of interest, i.e., DMPK RNA or MBNL1 protein, will negatively correlate with the degree of co-localization because these molecules have no or little interaction in healthy cells compared to diseased cells.

[0176] By performing the method on different muscle cells or series of muscle cells, for example on healthy and diseased cells, it is therefore possible to compare the functionality of the molecules in said cells or series of cells. In particular, if the degree of colocalization determined in the muscle cells of interest is significantly different from the degree of colocalization determined under 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 cells of interest.

[0177] As used herein, the term "functionality" refers to the activity of a molecule in healthy muscle cells. In particular, the functionality of a nucleic acid can be its ability to provide a sufficient amount of the encoded functional protein. For example, the functionality of DMPK RNA can be its ability to provide a sufficient amount of the encoded functional DMPK protein. In fact, mutant mRNA transcripts containing CUG expansions are retained in the nucleus and aggregate as nuclear foci, negatively affecting the ability of DMPK RNA to produce sufficient amounts of DMPK protein. These expansions form stem loops that are recognized by RNA splicing factors, including MBNL1, which can be used as a second molecule. The functionality of a protein can be its activity or one of its activities in muscle cells, particularly in relation to its interaction with a second molecule. For example, the functionality of dystrophin can be its activity as an essential component of the dystrophin-associated protein complex (DGC).

[0178] The inventors have also demonstrated that the quantitative co-localization assay of the present invention can be used to quantitatively monitor the effect of a compound on the functionality of a molecule of interest.

[0179] Thus, in another aspect, the present invention relates to an in vitro method for assessing the efficacy of a compound to modulate the functionality of a molecule of interest in muscle cells.

[0180] The method comprises: (a) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing a quantitative co-localization analysis; (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that the compound is capable of modulating the functionality of the first cellular molecule of interest in the at least one muscle cell.

[0181] In a preferred embodiment, the at least one muscle cell is also stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0182] Thus, in a preferred embodiment, the method comprises: (i) providing at least one image of at least one in vitro cultured muscle cell, wherein the at least one muscle cell has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest, and stained with at least one labeling agent that reveals 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 image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that the compound is capable of modulating the functionality of the first cellular molecule of interest in the at least one muscle cell.

[0183] Steps (a), (b), (i), (ii) and (iii) are described in detail above in the section "Quantitative Colocalization Analysis." All of the above embodiments of these steps are also encompassed by this aspect.

[0184] The muscle cells used in this method can be healthy or diseased.

[0185] In embodiments where the method is used to test the toxicity of a compound to muscle cells, the muscle cells used in the assay and used as reference muscle cells are preferably healthy muscle cells. In embodiments where the method is used to test the ability of a compound to treat a muscle disease or ameliorate a feature of a muscle disease, the muscle cells used in the assay and used as reference muscle cells are preferably diseased muscle cells, particularly muscle cells that exhibit features of the neuromuscular disease or cardiomyopathy of interest.

[0186] In one embodiment, the method comprises: (a) providing at least one image of at least one in vitro cultured myotube, wherein the at least one myotube has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing a quantitative co-localization analysis; (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference myotube, wherein the at least one reference myotube is the at least one in vitro cultured myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that the compound is capable of modulating the functionality of the first cellular molecule of interest in the at least one myotube.

[0187] In particular, the method (i) providing at least one image of at least one in vitro cultured myotube, wherein the at least one myotube has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes and myotube cellular structures, and any combination thereof; and (ii) performing image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; and A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that said compound is capable of modulating the functionality of a molecule of interest in said at least one myotube.

[0188] The myotubes used in this method can be healthy or pathological myotubes, particularly myotubes that exhibit characteristics of a neuromuscular disorder of interest.

[0189] In another embodiment, the method comprises: (a) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein the at least one cardiomyocyte has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing a quantitative co-localization analysis; (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured cardiomyocyte that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that the compound is capable of modulating the functionality of the first cellular molecule of interest in the at least one cardiomyocyte.

[0190] In particular, the method (i) providing at least one image of at least one in vitro cultured cardiomyocyte, wherein the at least one cardiomyocyte has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm on the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured cardiomyocyte that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. and A statistically significant difference between the degree of co-localization and the reference degree of co-localization indicates that the compound is capable of modulating the functionality of the first cellular molecule of interest in the at least one cardiomyocyte.

[0191] The cardiomyocytes used in this method can be healthy cardiomyocytes or diseased cardiomyocytes, particularly cardiomyocytes that exhibit characteristics of the cardiomyopathy of interest.

[0192] The test compound can be of any nature, for example, 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, the compound can be a drug approved by a regulatory agency, such as the FDA or EMA.

[0193] The muscle cells may be contacted with the test compound before, during or after culturing the cells, preferably during culturing. Preferably, the muscle cells are contacted with the test compound before being stained with a labeling agent (for the first molecule and the second molecule, and optionally for the ROI).

[0194] 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 cell is cultured under the same conditions (same culture medium / substrate, same incubation parameters, etc.) as the muscle cells contacted with the test compound, i.e., the conditions differ only in the concentration or presence / absence of the test compound.

[0195] The reference muscle cells can be healthy or pathological muscle cells. The muscle cells used in the assay and used as the reference muscle cells are of the same type, in particular myotubes or cardiomyocytes, preferably of the same state, i.e., healthy or pathological cells, and more preferably obtained from the same source, e.g., from the same donor. The reference muscle cells can be easily selected by those skilled in the art based on the properties of the muscle cells, in particular myotubes or cardiomyocytes, and the test compound.

[0196] The method can further comprise determining a reference degree of co-localization. - 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 the at least one reference muscle cell is not contacted with a test compound or is contacted with a determined concentration of the compound and is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest; and - quantifying a reference degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one reference muscle cell by performing a quantitative co-localization analysis. It may further include:

[0197] In embodiments in which cells are stained for a ROI, the method comprises: - 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 the at least one reference muscle cell is not contacted with a test compound or is contacted with a determined concentration of the compound, is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first molecule of interest, and is stained with at least one labeling agent that reveals 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 - performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and - quantitatively determining a reference degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis. It may further include:

[0198] Preferably, the ROIs used to determine the reference degree of colocalization are the same as the ROIs used in the assay, eg, individual myocytes and nuclei.

[0199] To be comparable, the degree of colocalization and the reference degree of colocalization are determined by performing the same calculation, preferably by calculating the same overlap coefficient, e.g., PCC, for each cell or ROI and, optionally, applying the same mathematical function to said coefficients.

[0200] A statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that the compound can modulate the functionality of the first cellular molecule of interest in the muscle cells used in the method. No difference or no statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that the compound cannot 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.

[0201] The statistical significance of difference can be evaluated by any method known to those skilled in the art.In particular, the statistical significance of difference can be evaluated by carrying out statistical testing to determine the P value between colocalization degree and reference colocalization degree.Usually, the P value of less than 0.05 indicates that the difference is significant.

[0202] Optionally, the methods of the present invention for assessing compound efficacy can be performed several times using different concentrations of the test compound, particularly to assess dose-response effects of the test compound.

[0203] The inventors have also demonstrated that the quantitative colocalization assay of the present invention can be used to quantitatively monitor the restoration of cellular function, in particular the restoration of active dystrophin, in pathological muscle cells, in particular in myotubes from DMD patients treated with exon skipping therapy and from DM1 patients treated with antisense oligonucleotides (ASOs).

[0204] Thus, in another aspect, the present invention also relates to an in vitro method for predicting the ability of a compound to treat a muscle disorder of interest.

[0205] The method comprises: (a) providing at least one image of at least one in vitro cultured diseased muscle cell, i.e., a muscle cell exhibiting characteristics of a muscle disease of interest, wherein the at least one muscle cell has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased muscle cell by performing a quantitative co-localization analysis; (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured diseased muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; Including, A positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said muscle disease.

[0206] In a preferred embodiment, the at least one pathological muscle cell is stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0207] Thus, in a preferred embodiment, the method comprises: (i) providing at least one image comprising at least one in vitro cultured pathological muscle cell, wherein the at least one muscle cell has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes, myotube structures, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured diseased muscle cell that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disorder.

[0208] Steps (a), (b), (i), (ii), and (iii) are described in detail above in the section "Quantitative Colocalization Analysis." All embodiments of these steps described above are also encompassed by this aspect. All embodiments described above relating to methods for assessing the functionality of a cellular molecule of interest or methods for assessing the efficacy of a compound to modulate the functionality of a cellular molecule of interest are also encompassed by this aspect.

[0209] The muscle cells used in this method are pathological muscle cells, ie, muscle cells that exhibit the characteristics of a muscle disease.

[0210] As used herein, the term "muscle disease" refers to a neuromuscular disease or a cardiomyopathic disease.

[0211] As used herein, the terms "neuromuscular disorder" and "neuromuscular disease" are used interchangeably and cover disorders that impair muscle function either directly (pathology of voluntary muscles) or indirectly (pathology of nerves, neuromuscular junctions, or extracellular matrix). This term includes, but is not limited to, muscular dystrophies, such as 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 dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophies. myopathy, such as those selected from the group consisting of merosin-deficient congenital muscular dystrophy, scapuloperoneal muscular dystrophy, tibial muscular dystrophy, and autosomal recessive muscular dystrophy; myopathy, such as Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, distal myopathy with rimmed vacuoles (DMRV), centronuclear myopathy (CNM), X-linked myotubular myopathy (XLM), MTM), tubular aggregate myopathy, malignant hyperthermia syndrome, inclusion body myopathy, protein aggregation myopathy, nemaline myopathy, congenital myopathy (CM), vacuolar aggregate myopathy, Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, dystonia and gaze palsy, childhood onset (NADGP), Pompe disease and primary myopathy It encompasses a wide range of disorders, including chondriamyopathies; congenital myasthenic syndromes, e.g., myasthenia gravis and other myasthenic syndromes caused by mutations in the CHAT, COLQ, RAPSN, CHRNE, DOK7 and / or GFPT1 genes; or motor neuron diseases, e.g., spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Kennedy's disease, cachexia, sarcopenia and muscle atrophy.

[0212] Preferably, the neuromuscular disease is 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 dystrophy (LGMD) (preferably LGMD). R1, R2, R3, R4, R5, R6, R8, R10, R12, R16, R22, R23, D1 and D5), Walker-Warburg syndrome, muscle-eye-brain disease, congenital muscular dystrophy, tibial muscular dystrophy, Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, distal myopathy with rimmed vacuoles (DMRV), centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), tubular aggregate myopathy, The myopathy is selected from the group consisting of: chronic hyperthermia syndrome, inclusion body myopathy, protein aggregation myopathy, nemaline myopathy (preferably nemaline myopathy 2 or 8), congenital myopathy (CM), vacuolar aggregate myopathy, Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, Friedreich's ataxia, dystonia and gaze palsy, childhood onset (NADGP), and Pompe disease.In particular, the neuromuscular disease may be Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), myotonic dystrophy 1 (DM1), myotonic dystrophy 2 (DM2), facioscapulohumeral muscular dystrophy (FSHD), Emery-Dreifuss muscular dystrophy, or limb-girdle muscular dystrophy (LGMD) (preferably LGMD). R1, R2, R3, R4, R5, R6, R8, R10, R12, R16, R22, R23, D1 and D5), Walker-Warburg syndrome, muscle-eye-brain disease, congenital muscular dystrophy, tibial muscular dystrophy, Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, distal myopathy with rimmed vacuoles (DMRV), centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), tubular aggregate myopathy The myopathy is selected from the group consisting of malignant hyperthermia syndrome, inclusion body myopathy, protein aggregation myopathy, nemaline myopathy (preferably nemaline myopathy 2 or 8), congenital myopathy (CM), vacuolar aggregate myopathy, Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, dystonia and gaze palsy, childhood onset (NADGP), and Pompe disease.

[0213] In one embodiment, the neuromuscular disease of interest is selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes, motor neuron diseases, and metabolic myopathies. Preferably, the neuromuscular disease of interest is selected from the group consisting of muscular dystrophies, myopathies, congenital myasthenic syndromes, and motor neuron diseases.

[0214] In a preferred embodiment, 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, limb-girdle muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, congenital muscular dystrophy, scapuloperoneal muscular dystrophy, tibial muscular dystrophy and autosomal recessive muscular dystrophy, preferably Duchenne muscular dystrophy. The muscular dystrophy 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 dystrophy, 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).

[0215] In another specific embodiment, the neuromuscular disease of interest is Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), tubular aggregate myopathy, malignant hyperthermia syndrome, inclusion body myopathy, myofibrillar myopathy, protein aggregation myopathy, nemaline myopathy, congenital myopathy (CM), Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, dystonia and gaze palsy, childhood onset (NADGP), Pompe disease, and primary mitochondrial myopathy. and preferably the myopathy is selected from the group consisting of Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, centronuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), tubular aggregate myopathy, malignant hyperthermia syndrome, inclusion body myopathy, myofibrillar myopathy, protein aggregation myopathy, nemaline myopathy, congenital myopathy (CM), Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, dystonia and gaze palsy, childhood-onset (NADGP) and Pompe disease.

[0216] In another specific embodiment, the neuromuscular disease of interest is a congenital myasthenic syndrome selected from the group consisting of myasthenia gravis and other myasthenic syndromes caused by mutations in the CHAT, COLQ, RAPSN, CHRNE, DOK7 and / or GFPT1 genes.

[0217] 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.

[0218] In a further embodiment, the neuromuscular disease of interest is a metabolic myopathic disorder selected from the group consisting of cachexia, sarcopenia, and muscle atrophy.

[0219] In a preferred embodiment, the neuromuscular disease is selected from the group consisting of Duchenne muscular dystrophy (DMD) and myotonic dystrophy 1 (DM1).

[0220] As used herein, the term "cardiomyopathies" refers to diseases of the heart muscle in which the walls of the heart chambers become stretched, thickened, or stiffened. This affects the heart's ability to pump blood throughout the body and can lead to heart failure. Cardiomyopathy can 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 the cardiomyopathy is caused by a mutation in a gene listed in the "Examples of molecules useful as the first molecule" column of Table 2. By way of example, the cardiomyopathy can be dilated cardiomyopathy (DCM) caused by a mutation in the gene encoding ACTC1 (actin alpha cardiac muscle).

[0221] In embodiments in which the disease is a neuromuscular disease, the muscle cells are preferably myotubes or cardiomyocytes, more preferably myotubes. In embodiments in which the disease is a cardiomyopathy, the muscle cells are preferably cardiomyocytes.

[0222] In one embodiment, the muscle disease is a neuromuscular disease and the method comprises: (a) providing at least one image of at least one in vitro cultured diseased myotube, i.e., a myotube exhibiting characteristics of a neuromuscular disease of interest, wherein the at least one myotube has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased myotube by performing a quantitative co-localization analysis; (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a) and (b) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured diseased myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; Including, A positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disease.

[0223] In a preferred embodiment, the at least one pathological myotube is also stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes and myotube cellular structures and any combination thereof, and image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0224] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease and the method comprises: (i) providing at least one image comprising at least one in vitro cultured pathological myotube, wherein the at least one myotube has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes, myotube structures, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured diseased myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, A positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disease.

[0225] 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.

[0226] In another embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (a) providing at least one image of at least one in vitro cultured diseased cardiomyocyte, i.e., a cardiomyocyte exhibiting characteristics of a neuromuscular disease or cardiomyopathy of interest, wherein the at least one cardiomyocyte has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one diseased cardiomyocyte by performing a quantitative co-localization analysis; (c) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured diseased cardiomyocyte that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; Including, A positive correlation between the concentration of the compound and the desired statistically significant variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disease or cardiomyopathy.

[0227] In a preferred embodiment, the at least one diseased cardiomyocyte is stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI before step (b), and in step (b), the degree of colocalization is quantitatively determined in at least one ROI.

[0228] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (i) providing at least one image comprising at least one in vitro cultured diseased cardiomyocyte, wherein the at least one cardiomyocyte has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes, cardiomyocyte structures, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured diseased cardiomyocyte that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound; Including, A positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disease or cardiomyopathy.

[0229] 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.

[0230] The test compound may be as defined above.

[0231] The muscle cells may be contacted with the test compound before, during or after culturing the cells, preferably during culturing. Preferably, the muscle cells are contacted with the test compound before being stained with a labeling agent (for the first molecule and the second molecule, and optionally for the ROI).

[0232] The reference degree of colocalization is obtained by performing steps (a) and (b) or (i)-(iii) on at least one reference muscle cell. Preferably, the reference muscle cells are cultured under the same conditions (same culture medium / substrate, same incubation parameters, etc.) as the muscle cells used in the assay, i.e., the conditions differ only in the concentration or absence of the test compound.

[0233] Preferably, the reference muscle cells are diseased muscle cells. The muscle cells used in the assay and as the reference muscle cells are of the same type, in particular myotubes or cardiomyocytes, and are preferably 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 selected by one skilled in the art based on the disease of interest and the test compound.

[0234] The method may further comprise determining a reference degree of co-localization as described above.

[0235] A positive correlation between the concentration of the compound and the statistically significant desired change in the degree of colocalization compared with the reference degree of colocalization indicates that the compound is useful for treating the muscle disease.As explained above, a positive correlation exists when one variable decreases, the other variable also decreases, or when one variable increases and the other variable also increases.In this embodiment, the first variable is the concentration of the compound, and the second variable is the desired change, whether positive or negative, i.e., the absolute value of the desired change.Therefore, a positive correlation 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 change in the degree of colocalization increases (i.e., the absolute value of the desired change increases) compared with the reference degree of colocalization, 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 change in the degree of colocalization decreases (i.e., the absolute value of the desired change decreases) compared with the reference degree of colocalization.

[0236] The statistical significance of the variation of colocalization degree compared with the reference degree of colocalization can be evaluated by any method known to those skilled in the art, particularly by carrying out a statistical test to determine the P value between the colocalization degree and the reference degree of colocalization.Typically, a P value of less than 0.05 indicates that the variation is significant.

[0237] The desired variation is a variation toward the normal state. This direction of variation can be easily determined by those skilled in the art depending on the concentration of the compound used to determine the reference degree of colocalization, i.e., higher or lower than that used in the assay, and the selected combination of the first molecule and the second molecule.

[0238] Indeed, as explained above, the degree of colocalization can be positively or negatively correlated with the health of muscle cells, depending on the first and second molecules. When two molecules are known to interact in healthy cells, the degree of colocalization is positively correlated with the health of muscle cells. In this case, the higher the degree, the healthier the cell. For example, this is the case when the first molecule is dystrophin and the second molecule is a protein of the dystrophin-associated protein complex (DGC). When two molecules are known to have no or little interaction in healthy cells but are known to interact in pathological muscle cells, the degree of colocalization is negatively correlated with the health of muscle cells. In this case, the higher the degree, the less healthy the cell. For example, this is the case when the first molecule is DMPK RNA and the second molecule is an RNA-binding protein, such as MBNL1 protein, trapped by CTG repeats in the DMPK gene, or vice versa.

[0239] If the concentration of 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 healthy state of the muscle cells, the desired variation is a decrease in the degree of colocalization compared to the reference degree (the difference between the degree of colocalization and the reference degree is negative).

[0240] If the concentration of compound used to determine the reference degree of colocalization is lower than the concentration used in the assay (especially if the reference muscle cells have not been contacted with the compound), and the degree of colocalization positively correlates with the healthy state of the muscle cells, the desired variation is an increase in the degree of colocalization compared to the reference degree (the difference between the degree of colocalization and the reference degree is positive).

[0241] If the concentration of compound used to determine the reference degree of colocalization is higher than the concentration used in the assay, and the degree of colocalization is negatively correlated with the healthy state of the muscle cells, the desired variation is an increase in the degree of colocalization compared to the reference degree (the difference between the degree of colocalization and the reference degree is positive).

[0242] If the concentration of compound used to determine the reference degree of colocalization is lower than the concentration used in the assay, and the degree of colocalization is negatively correlated with the healthy state of the muscle cells, the desired variation is a decrease in the degree of colocalization (the difference between the degree of colocalization and the reference degree is negative).

[0243] Thus, a positive correlation between the concentration of the compound and a statistically significant desired change in the degree of colocalization compared to the reference degree of colocalization indicates that the compound is useful for treating the disease of interest. If the change is in the opposite direction to the desired change, the assay does not indicate that the compound is useful for treating the disease of interest.

[0244] In a preferred embodiment, the reference degree of co-localization is obtained using at least one reference muscle cell, which is at least one in vitro cultured diseased muscle cell that has not been contacted with the test compound, and a desired statistically significant variation in the degree of co-localization compared to the reference degree of co-localization indicates that the compound is useful for treating said neuromuscular disease.

[0245] Preferably, the method of the present invention for predicting the ability of a compound to treat a muscle disease is performed several times using different concentrations of the test compound, particularly to evaluate the dose-response effect of the test compound. In particular, a positive correlation between the concentration of the compound and a desired statistically significant change in the degree of colocalization compared to the reference degree of colocalization can be obtained at a specific range of compound concentrations, indicating that the compound is useful for treating a muscle disease.

[0246] The inventors have also demonstrated herein that the quantitative co-localization assay of the present invention can be used to discern the response of patients, eg, DMD patients, to therapy, eg, exon skipping therapy.

[0247] Thus, in another aspect, the present invention also relates to an in vitro method for monitoring the response of a patient suffering from a muscle disease to a therapeutic compound.

[0248] The method comprises: (a) providing at least one image of at least one in vitro cultured muscle cell obtained / derived from said patient sample after administration of a therapeutic compound, wherein said at least one muscle cell has been contacted with a test compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with said first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing a quantitative co-localization analysis; (c) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (a) and (b) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained / derived from a sample of the patient prior to administration of a therapeutic compound; Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0249] In a preferred embodiment, the at least one muscle cell is also stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0250] Thus, in a preferred embodiment, the method comprises: (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained / derived from said patient sample after administration of a therapeutic compound, said at least one muscle cell stained for a first cellular molecule of interest and for a second cellular molecule that interacts with said first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained / derived from a sample of the patient prior to administration of a therapeutic compound. Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0251] Steps (a), (b), (i), (ii), and (iii) are described in detail above in the section "Quantitative Colocalization Analysis." All of the above-described embodiments of these steps are also encompassed by this aspect. All of the above-described embodiments of the method for assessing the functionality of a cellular molecule of interest, the method for assessing the efficacy of a compound to modulate the functionality of a cellular molecule of interest, and the method for predicting the ability of a compound to treat a muscle disease are also encompassed by this aspect.

[0252] In one embodiment, the muscle disease is a neuromuscular disease and the method comprises: (a) providing at least one image of at least one in vitro cultured myotube obtained / derived from a sample of a patient suffering from a neuromuscular disease after administration of a therapeutic compound, wherein the at least one myotube has been contacted with a test compound and is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one myotube by performing a quantitative co-localization analysis; (c) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (a) and (b) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained / derived from a sample of the patient prior to administration of a therapeutic compound. Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0253] In a preferred embodiment, the at least one myotube is also stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes and myotube cellular structures and any combination thereof, and image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0254] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease and the method comprises: (i) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from said patient sample after administration of a therapeutic compound, said at least one myotube stained for a first cellular molecule of interest and for a second cellular molecule that interacts with said first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes, myotube cellular structures, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained / derived from a sample of the patient prior to administration of a therapeutic compound; Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0255] 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.

[0256] In another embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (a) providing at least one image of at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient suffering from a neuromuscular disease or cardiomyopathy after administration of a therapeutic compound, wherein the at least one cardiomyocyte has been contacted with a test compound and is stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing a quantitative co-localization analysis; (c) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (a) and (b) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured cardiomyocyte obtained / derived from a sample of the patient prior to administration of a therapeutic compound. Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0257] In a preferred embodiment, the at least one cardiomyocyte is stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), and in step (b), the degree of colocalization is quantitatively determined in at least one ROI.

[0258] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (i) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from said patient sample after administration of a therapeutic compound, said at least one cardiomyocyte stained for a first cellular molecule of interest and for a second cellular molecule that interacts with said first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured cardiomyocyte obtained / derived from a sample of the patient prior to administration of a therapeutic compound; Including, A statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the subject will be responsive to the treatment.

[0259] 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.

[0260] As used herein, the term "sample" refers to any sample containing muscle cells derived from a subject, preferably a sample containing myoblasts or cardiomyocytes. Examples of such samples include biopsy samples, tissue samples, or cell samples. The sample can be processed prior to its use, particularly to obtain isolated muscle cells for cell culture, preferably isolated myoblasts or cardiomyocytes. Muscle cells, particularly myotubes or cardiomyocytes, can be obtained from a sample by isolating one or several myoblasts or cardiomyocytes and culturing them in vitro as described above to obtain in vitro cultured myotubes and cardiomyocytes. They can also be obtained from a sample by isolating cells such as fibroblasts from the sample, generating induced pluripotent stem cells from these cells, and differentiating the iPSCs into muscle cells. All of these methods are well known to those skilled in the art.

[0261] In another aspect, the present invention also relates to an in vitro method for selecting a patient suffering from a muscle disease for treatment with a therapeutic compound or for determining whether a patient suffering from a muscle disease is likely to benefit from treatment with a therapeutic compound.

[0262] The method comprises: (a) providing at least one image comprising at least one in vitro cultured muscle cell obtained / derived from a sample from a patient suffering from a muscle disease, wherein the at least one muscle cell has been contacted with a therapeutic compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one muscle cell by performing a quantitative co-localization analysis; and (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a)-(b) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound. Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0263] In a preferred embodiment, the at least one muscle cell is also stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0264] Thus, in a preferred embodiment, the method comprises: (i) providing at least one image comprising at least one in vitro cultured muscle cell obtained / derived from a sample of a patient suffering from a muscle disease, wherein the at least one muscle cell has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference muscle cell, wherein the at least one reference muscle cell is at least one in vitro cultured muscle cell obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound. Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0265] Steps (a), (b), (i), (ii), and (iii) are described in detail above in the section "Quantitative Colocalization Analysis." All embodiments of these steps described above are also encompassed by this aspect. All embodiments described above relating to methods for assessing the functionality of a cellular molecule of interest, for assessing the efficacy of a compound to modulate the functionality of a cellular molecule of interest, for predicting the ability of a compound to treat a muscular disorder, or for monitoring the response of a patient suffering from a muscular disorder to a therapeutic compound are also encompassed by this aspect.

[0266] In one embodiment, the muscle disease is a neuromuscular disease and the method comprises: (a) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from a sample of a patient suffering from a neuromuscular disease, wherein the at least one myotube has been contacted with a therapeutic compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and a second cellular molecule in said at least one myotube by performing a quantitative co-localization analysis; and (c) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (a)-(b) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound; Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0267] In a preferred embodiment, the at least one myotube is also stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual myotubes and myotube cellular structures and any combination thereof, and image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), in which the degree of colocalization is quantitatively determined in the at least one ROI.

[0268] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease and the method comprises: (i) providing at least one image comprising at least one in vitro cultured myotube obtained / derived from a sample of a patient suffering from a neuromuscular disease, wherein the at least one myotube has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound; Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0269] 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.

[0270] In another embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (a) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient suffering from a neuromuscular disease or cardiomyopathy, wherein the at least one cardiomyocyte has been contacted with a therapeutic compound and stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest; and (b) quantifying the degree of co-localization of the first cellular molecule of interest and the second cellular molecule in said at least one cardiomyocyte by performing a quantitative co-localization analysis; and (c) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (a)-(b) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured myotube obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound. Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0271] In a preferred embodiment, the at least one cardiomyocyte is stained with at least one labeling agent that reveals 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 image segmentation is performed using an algorithm for the appropriate staining channel to identify the ROI prior to step (b), and in step (b), the degree of colocalization is quantitatively determined in at least one ROI.

[0272] Thus, in a preferred embodiment, the muscle disease is a neuromuscular disease or a cardiomyopathy and the method comprises: (i) providing at least one image comprising at least one in vitro cultured cardiomyocyte obtained / derived from a sample of a patient suffering from a neuromuscular disease or cardiomyopathy, wherein the at least one cardiomyocyte has been contacted with a test compound, stained for a first cellular molecule of interest and for a second cellular molecule that interacts with the first cellular molecule of interest, and stained with at least one labeling agent that reveals at least one region of interest (ROI) selected from the group consisting of individual cardiomyocytes, cellular structures of cardiomyocytes, and any combination thereof; and (ii) performing image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of colocalization of the first cellular molecule and the second cellular molecule in at least one ROI by performing a quantitative colocalization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference cardiomyocyte, wherein the at least one reference cardiomyocyte is at least one in vitro cultured cardiomyocyte obtained / derived from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound; Including, A positive correlation between the concentration of the therapeutic compound and a statistically significant desired variation in the degree of colocalization compared to the reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

[0273] 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.

[0274] The method can further comprise administering a therapeutic compound to the patient when the patient is susceptible to benefit from treatment with said therapeutic compound.

[0275] The muscle cells may be contacted with the therapeutic compound to be tested before, during or after culturing the cells, preferably before or during culturing. Preferably, the muscle cells are contacted with the therapeutic compound to be tested before staining with a labeling agent (for the first molecule and the second molecule, and optionally for the ROI).

[0276] The test compound may be as defined above.

[0277] The reference degree of co-localization can be obtained as described above. Preferably, the reference degree of co-localization is determined using muscle cells obtained / derived from the same sample as the muscle cells in the assay.

[0278] The method may further comprise determining a reference degree of co-localization as described above.

[0279] The definition of the desired variation is also as described above and depends on the concentration of compound used to determine the reference degree of colocalization, i.e., higher or lower than that used in the assay, and the selected combination of first and second molecules.

[0280] If the variation is in the opposite direction to the desired variation, the assay does not indicate that the patient is likely to benefit from treatment with the therapeutic compound.

[0281] Preferably, the method is performed several times using different concentrations of the therapeutic compound being tested, in particular to evaluate the dose-response effect of this therapeutic compound. In particular, a positive correlation between the concentration of the therapeutic compound and a desired statistically significant change in the degree of colocalization compared to a reference degree of colocalization can be obtained at a specific range of compound concentrations, indicating that the patient is likely to benefit from treatment with said therapeutic compound.

[0282] Optionally, the method of the present invention further comprises, before step (a) or (i), - culturing muscle cells, in particular myoblasts or cardiomyocytes, preferably under restricted conditions that allow the generation of a homogeneous population of muscle cells, in particular myotubes or cardiomyocytes; - staining these muscle cells, in particular myotubes or cardiomyocytes, with the first molecule, with the second molecule, and optionally with the at least one labeling agent; and - acquiring at least one image of at least one stained muscle cell; It may further include:

[0283] In some preferred embodiments of the methods of the present invention, the muscle disease is Duchenne muscular dystrophy or myotonic dystrophy type 1 (DM1), and the first molecule or the second molecule is dystrophin, and the first molecule or the 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 α-sarcoglycan, β-dystroglycan, α-dystroglycan and dysferlin, more preferably selected from the group consisting of α-sarcoglycan and β-dystroglycan.

[0284] In a particularly preferred embodiment of the method of the present invention, the muscle disease is Duchenne muscular dystrophy, the first molecule is dystrophin, and the second molecule is α-sarcoglycan or β-dystroglycan.

[0285] In some other preferred embodiments of the methods of the present invention, the muscle disease is myotonic dystrophy type 1 (DM1), the first molecule is DMPK RNA, and the second molecule is an RNA-binding protein trapped by CUG repeats in the DMPK gene, preferably MBNL1 protein.

[0286] In the present application, the proteins belonging to the dystrophin glycoprotein complex (DGC) are preferably selected from the group consisting of DMD, SNTA, SNTB, ANK1, ANK2, DAG1, SSPN, SGCA, SGCB, SGCD, SGCG, DTNA and FLNC.

[0287] In a preferred embodiment, the method of the present invention is a computer-implemented method. As used herein, the term "computer-implemented method" refers to a method comprising a programmable device, particularly a computer, a computer network, or a computer-readable medium carrying a computer program, wherein at least one step of the method is performed by using at least one computer program. The computer-implemented method may further comprise at least one step that is not performed by using a computer program, such as a cell culture step. In a preferred embodiment, the method of the present invention does not involve the use of a trained classifier.

[0288] In another aspect, the present invention relates to the use of a first molecule as an imaging marker to assess the functionality of a second molecule in a muscle cell using a quantitative colocalization assay, preferably using the methods of the present invention described above, to evaluate the efficacy of a compound to modulate the functionality of a second molecule in a muscle cell, to predict the ability of a compound to treat a muscle disease, to monitor the response of a patient suffering from a muscle disease to a therapeutic compound, to select a patient suffering from a muscle disease for treatment with a therapeutic compound, or to determine whether a patient suffering from a muscle disease is likely to benefit from treatment with a therapeutic compound.

[0289] All embodiments disclosed above for the method of the present invention are also encompassed in this aspect.

[0290] In one 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 α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin, preferably selected from the group consisting of dystrophin, α-sarcoglycan, and β-dystroglycan, or vice versa. Preferably, the muscle cells are myotubes or cardiomyocytes, and the muscle disease is a neuromuscular disease or a cardiomyopathy. Preferably, the muscle cells are myotubes or cardiomyocytes, and the muscle disease is a neuromuscular disease, more preferably a muscular dystrophy, even more preferably Duchenne muscular dystrophy (DMD) or myotonic dystrophy type 1 (DM1). More preferably, the muscle cells are myotubes and the muscle disease is a neuromuscular disease, more preferably a muscular dystrophy, even more preferably Duchenne muscular dystrophy (DMD) or myotonic dystrophy type 1 (DM1).

[0291] In another embodiment, the first molecule is DMPK RNA and the second molecule is an RNA-binding protein trapped by CTG repeats in the DMPK gene, preferably MBNL1 protein, or vice versa. Preferably, the muscle cells are myotubes and the muscle disease is a neuromuscular disease, more preferably a muscular dystrophy, even more preferably myotonic dystrophy type 1 (DM1).

[0292] All references cited in this description are incorporated herein by reference. Other features and advantages of the present invention will become apparent from the following examples, which are given by way of illustration and not by way of limitation. [Example]

[0293] Materials and Methods Cell Source and Maintenance Primary human skeletal myoblasts from healthy and DMD donors were sourced from various donors (Table 3). Myocytes were expanded to generate master and working cell banks according to the supplier's recommendations.

[0294] [Table 3]

[0295] Cells expanded after patient biopsy collection were subsequently enriched for myoblasts using CD56+ cell sorting. Primary vials were obtained, thawed, and the percentage of desmin+ cells determined. Cells were expanded and cryopreserved to form a master bank (MB), at which point they were characterized using immunostaining (desmin+ cells) and the Myoscreen platform (CYTOO, France, fusion index). Finally, the master bank vial was thawed, expanded, and finally cryopreserved to form a working cell bank (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 of doubling time, percentage of desmin+ cells, and fusion index.

[0296] High-throughput myotube formation All steps were performed automatically using a Freedom EVO150 workstation (Tecan). The growth medium for myoblasts of healthy and DMD cells 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, 5 μg / ml bovine insulin (Sigma), 0.4 μg / ml dexamethasone (Sigma), and 10 ng / ml FGF2 (Miltenyi Biotec).

[0297] For all experiments, on day 0, MyoScreen™ plates (CYTOO, France; International Patent Application WO 2015 / 091593, Young et al., SLAS Discov. 2018 Sep;23(8):790-806) containing microplates coated with 10 μg / ml fibronectin (Invitrogen) were pre-filled with 100 μl / well of growth medium and stored in a 37°C incubator. Human primary myoblasts were detached from flasks, counted, and plated at 15,000 cells per well in 100 μl of growth medium. On day 1, the growth medium was changed to differentiation medium (DMEM / F12 (Invitrogen), 2% horse serum (GE Healthcare), 0.5% penicillin-streptomycin (Invitrogen)), in which the myoblasts initiated differentiation and myotube formation. The myotube formation process was then continued for 8 or 9 days in differentiation medium without changing the medium.

[0298] The MyoScreen platform enables the generation of myotubes from primary and immortalized cells under controlled conditions. These myotubes differentiate, become linear, and exhibit the morphological characteristics required for neuromuscular junction formation. The standardized size and controlled culture conditions facilitate quantitative image-based analysis and are critical for the robustness of colocalization assays.

[0299] High-throughput cardiomyocyte culture iCell Cardiomyocytes2 (human iPSC-derived cardiomyocytes) were purchased from FUJIFILM Cellular Dynamics. On day 0, cells were thawed according to the manufacturer's instructions and seeded into micropattern plates at 30,000 cells per well in 100 μL of plating medium (Fujifilm) containing 1% penicillin-streptomycin (Invitrogen). The micropattern design consisted of 132.3 μm x 18.9 μm rectangles as described (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 4.5 hours, the plating medium was replaced with 100 μL of maintenance medium (Fujifilm) containing 1% penicillin-streptomycin (Invitrogen). On days 2 and 6, the maintenance medium was refreshed.

[0300] siRNA treatment of myotubes and hIPSC-CMs After 4 days of culture, differentiated healthy and DMD myotubes were transfected with DMD siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. A dose response of DMD siRNA was obtained via serial dilutions to final concentrations of 0.0016, 0.008, 0.04, 0.2, 1, and 5 nM.

[0301] [Table 4]

[0302] siRNA treatment of hIPSC-CMs After 2 days of culture in maintenance medium, the medium was refreshed and the cells were transfected with DMD#2 siRNA (Table 4) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. A dose response of DMD siRNA was obtained by serial dilution to final concentrations of 0.0032, 0.016, 0.08, 0.4, 2, and 10 nM.

[0303] VivoPMO, PMO and ASO processing Morpholino oligonucleomers ("PMOs") were used to perform exon skipping of exons 44 or 45 of the DMD transcript. PMOs targeting exon 44 are listed from 1 to 4 according to their distance to the splice 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 octaguanidine dendrimers (vivo groups).

[0304] After 4 days of culture, differentiated healthy and DMD myotubes were transfected according to the manufacturer's instructions. Exon 44 skipping vivo PMO was used at final concentrations of 0.075, 0.15, 0.3, and 0.6 μM. Exon 45 skipping vivo PMO was used at final concentrations of 0.25, 0.5, 1, and 2 μM.

[0305] [Table 5]

[0306] After 4 days of culture, differentiated healthy and DM1 myotubes were transfected with (CAG)7 ASO (antisense oligonucleotide) (Mulders et al., 2009, Cell, 106(33), 13915-13920) at final concentrations ranging from 2.7 to 10 nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0307] Immunofluorescence staining On day 9, myotubes were fixed in 3.3% formalin (Sigma-Aldrich) for 30 minutes, and hIPSC-CMs were fixed in 3.3% formalin for 20 minutes. Subsequent immunofluorescence staining was performed according to Young et al., 2018 (Advancing Life Sciences R&D, 23(8), 790–806). Myotubes and hIPSC-CMs were washed three times in Dulbecco's phosphate-buffered saline (DPBS, Invitrogen) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich). After blocking for 20 minutes in 1% bovine serum albumin (BSA, Sigma-Aldrich), the cells were incubated with the primary antibodies listed in Table 6 in 1% BSA at room temperature for 2 hours (or overnight for NCL-Dys2 antibody) and then washed three times with DPBS. Secondary antibodies (Thermo Fisher Scientific and Jackson ImmunoResearch) were added with Hoechst 33342 (Invitrogen) for 2 hours at room temperature. Cells were washed three times in DPBS before imaging.

[0308] [Table 6]

[0309] Fluorescence in situ hybridization (FISH) On day 9, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes, washed three times in Dulbecco's phosphate-buffered saline (DPBS, Invitrogen), and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich). After washing three times in DPBS, cells were incubated with prehybridization buffer (0.5M phosphate buffer + 40% formamide) for 20 minutes at room temperature. The RNA probe (CAG)5-Cy3 was added to the hybridization buffer (7% dextran, 0.2% BSA in prehybridization buffer) and incubated overnight at 37°C. The next day, cells were washed with wash buffer (0.2% BSA in prehybridization buffer) prewarmed to 37°C, followed by three washes in DPBS. After the FISH procedure, immunofluorescence staining of MBNL1 was performed in the same wells.

[0310] High-content analysis of myotube morphology and DMD-related biomarkers After fixation and immunostaining, quantitative microscopy was performed using an Operetta HCS imaging system (PerkinElmer) with a 10x / 0.3 NA objective. Images were analyzed using scripts developed in Acapella software (PerkinElmer). 11 fields of view were acquired per well. First, myotube and nuclei were segmented using troponin T or myosin heavy chain staining and Hoechst staining, respectively. One to two myotubes were typically identified per micropattern. The segmentation threshold was set to avoid detecting background noise and to exclude aberrant small myotube structures. At the end of this first step, specific morphological readouts, such as nuclei number and fusion index (percentage of nuclei contained in troponin T or MHC staining) and average myotube area, were calculated across the entire well to control myotube viability and differentiation quality. Typically, approximately 50–60 myotubes were detected per well in the control condition. Next, an image cleanup step was performed on the myotube images to remove myotubes bordering the image border. The resulting myotubes were used to extract the expression of specific markers (eg, dystrophin, α-sarcoglycan staining intensity, etc.).

[0311] High-content analysis of dystrophin in cardiomyocytes After fixation and immunostaining, quantitative microscopy was performed using an Operetta-CLS HCS imaging system with a 40x / 1.1 NA confocal water-immersion objective (PerkinElmer). Images were analyzed using scripts developed in Acapella software (PerkinElmer). 64 fields per well were acquired. Myosin heavy chain staining was used to segment myocytes spanning the pattern. An average of four cardiomyocytes per micropattern was typically identified. The segmentation threshold was set to avoid detecting background noise and to eliminate aberrant small structures. Approximately 100–150 micropatterns per well were typically detected and analyzed under control conditions. Myotube images were then subjected to an image cleanup step to remove patterns bordering the image border. The resulting patterns were used to extract the expression of specific markers in cardiomyocytes (e.g., dystrophin staining intensity).

[0312] Colocalization analysis 1. Image Acquisition Cells were grown on MyoScreen plates. Four stainings were performed: HOECHST 33342 for nuclei, DRAQ5 for myosin heavy chain (MHC) or troponin T staining, and Cy3 and Alexa488 channels associated with identified disease biomarkers. For DMD examples, plates were imaged using an Operetta HCS imaging system in confocal mode with a 20x NA objective. For DM1 and cardiomyocyte examples, cells were imaged using an Operetta HCS platform (Perkin Elmer) using a 40x objective. For each condition, image processing and analysis were performed using proprietary algorithms developed by the inventors in Acapella High Content Imaging software (Perkin Elmer), with the goal of determining the level of colocalization between the two proteins of interest.

[0313] 2. Region of Interest (ROI) Segmentation Regions of interest (ROIs) were identified via a segmentation algorithm for the appropriate staining channel. The channel is disease-dependent. In the case of DMD, myotubes are segmented using the troponin T or MHC channel (see, for example, Figure 4A, line 1). Figure 4C shows another example of a region of interest. In this example, nuclei within myotubes were segmented using the troponin T or MHC channel for myotubes and the HOECHST channel for nuclei.

[0314] 3. Protein presence threshold: 3.1.Calculation method To study the colocalization of two proteins, first find the intensity threshold at which the fluorescently labeled protein is considered to be present at an image location (pixel). The threshold is determined in wells where either the protein is not expressed or no primary antibody is added. The threshold is determined by quantile statistics. At the ROI level, the high quantile of the pixel intensity distribution is taken. The ROI values ​​are aggregated at the well level and then at the plate level. For this plate, the final value determines the protein-specific threshold.

[0315] 3.2.Mask The second row on the right of Figure 4A shows the mask obtained by thresholding the imaging marker 1 channel in the myotube region. The quantile was set to 0.99.

[0316] The third row on the right of Figure 4A shows the mask obtained by thresholding the imaging marker 2 channel for the myotube region. The quantile was set to 0.99.

[0317] The intersection of these two masks defines the colocalization zone, the region where both proteins are detected. The junction of the two masks is the threshold range above, where either one protein or the other, or both proteins, are detected (Figure 4B).

[0318] The second row of Figure 4C represents the mask obtained by thresholding the imaging marker 1 channel of nuclei in the myotube region. The third row of Figure 4C represents the mask obtained by thresholding the imaging marker 2 channel of nuclei in the myotube region.

[0319] 3.3. Optional Image Selection Prior to calculation of the colocalization readout, one filtering step can be applied to the proteins in the image of interest: Subtract the threshold of each labeled protein from their corresponding image; Clip pixel values ​​below zero to zero.

[0320] 4. Calculation of Colocalization Readout 4.1. Pearson Correlation Coefficient (PCC) The Pearson's colocalization coefficient (PCC) is one statistical calculation that can be used to quantify colocalization. The formula, exemplified for the case of colocalization of imaging marker 1 (IM1) and imaging marker 2 (IM2), is shown in Figure 4E.1. PCC measures the pixel-by-pixel covariance of the signal levels of two images. When the pixel intensity of one protein image is plotted against the pixel intensity of the other protein, the more linear the relationship, the higher the PCC.

[0321] The PCC can be calculated for all ROIs or within the threshold range above, the latter being more stringent since regions where no protein is present will positively affect the PCC.

[0322] If arbitrary image culling is selected, some ROIs may not have pixel intensities above zero for one protein-related channel. In this case, the denominator value is equal to zero, resulting in an undefined PCC. Since the correlation between other protein-related signals and this invariant signal is null, the PCC is set to 0.

[0323] 4.2. Mander's Colocalization Coefficient (MCC) Mander's measure of colocalization is independent of pixel intensity correlation. In fact, MCC is a measure of co-occurrence. It measures the proportion of one protein-associated signal that overlaps with other protein signals. As illustrated in Figure 4E.2, the MCC for IM1 represents the proportion of IM1-associated intensity that colocalizes with IM2. The numerator takes the sum of IM1 intensity for pixels in the colocalization zone, while the denominator takes the sum of the intensities of all pixels where IM1 is detected.

[0324] 4.3. Rank-based Strength Weighting Factor (RWC) RWC attempts to add the concept of correlation to MCC. In fact, the algorithm uses a non-parametric ranking of pixel intensities in each channel and uses the difference in rank of co-localized pixel locations in the two channels to weight the pixel intensities of a molecule (see Figure 4E.3). The closer the pixel ranks of the two intensities, the higher the intensity weight at this pixel. This weighting is applied to co-occurring pixels, thereby combining both co-occurrence and correlation.

[0325] 4.4. Negative Control Protein To strengthen the reliability of the readout, colocalization between one of the two proteins and a protein known not to colocalize with it is also assessed. In the example case, IM2 is IM CTL The readout calculation is performed by calculating the intensity associated with IM1 as IM CTL In Figure 5B, MHC is replaced by the intensity associated with IM. CTL Since MHC is uniformly present throughout the myotube, the colocalization zone corresponds to the zone where IM1 is above threshold. Such a readout is considered a negative control for the colocalization readout.

[0326] 4.5. Defining the threshold for high PCC values Another readout developed is the percentage of ROIs showing strong colocalization. To define what constitutes strong colocalization, a threshold must be established at which the PCC is considered to reflect a strong correlation between two markers in the ROI. In the case of DGC and dystrophin (Figure 5), the PCC values ​​for each myotube and the negative control PCC values ​​are plotted. Plots are performed for four pairs of dystrophin and α-dystroglycan, β-dystroglycan, α-sarcoglycan, and dysferlin, for untreated and mock DMD donors, and for untreated and mock healthy donors. A satisfactory threshold is one in which 99% of the PCC values ​​between dystrophin and DGC proteins for DMD donors or MHC for all donor myotubes are below the threshold. In the example shown, 0.6 is determined as a satisfactory threshold for all tested imaging markers and DMD donors, because only outliers located at the 99th percentile exceed this threshold. High PCC% is the percentage of myotubes with PCC values ​​above the threshold of 0.6 among the total number of myotubes:

[0327]

number

[0328] 4.6. Significance-corrected readout information The readout can be corrected by subtracting the readout obtained with random colocalization. What we call the significance-corrected readout can be calculated as follows: For each of the two images, the pixels of the zone under consideration are shuffled multiple times. For each shuffle, a readout is calculated and then these values ​​are averaged. For MCC, this average value is expected to correlate with the number of molecules present in the zone. This measurement calculated for the shuffled image is used by subtracting it from the measurement made for the original image.

[0329] result Selection of cellular components to distinguish healthy and diseased cells using immunofluorescence (IF)-based colocalization The following examples of colocalization assays monitored the assembly of the dystrophin-glycoprotein complex (DGC) and the interaction of RNA splicing factors with nuclear RNA foci. These assays were then used to evaluate the efficacy and effectiveness of therapies targeting DMD and DM1.

[0330] The DGC connects the intracellular actin cytoskeleton and sarcomeres to the extracellular matrix. Dystrophin is an essential component of this complex and physically interacts with several proteins, such as β-dystroglycan (b-DG). The interaction between dystrophin and β-dystroglycan is crucial for DGC formation and has been demonstrated using various biochemical and imaging-based assays (Cullen et al., J Histochem Cytochem. 1998 Aug;46(8):945-54; Ilsley 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 for monitoring the restoration of functional dystrophin in DMD patients through gene therapy or exon-skipping RNA therapy. This study monitored the interactions of dystrophin with β-dystroglycan (b-DG), α-dystroglycan (a-DG), and α-sarcoglycan (a-SG). α-DG and α-sarcoglycan (a-SG) are also components of the DGC, but they do not directly interact with dystrophin. These interactions are difficult to monitor using physical isolation methods, but were monitored in situ using a nonquantitative colocalization assay.

[0331] Like the dystrophin / b-DG interaction, the interaction between the splicing factor MBNL1 and mutated DMPK RNA in DM1 patients has been well studied and documented ( Mankodi et al., 2001 ; Fardaej et al., 2001 ; Fardaej et al., 2002 ). There are no published reports characterizing the interaction of components of the DGC with misspliced ​​dystrophin as seen in DM1 patients.

[0332] Characterization of DMD donors and imaging targets To characterize the proliferation and differentiation behavior of healthy and DMD donors under MyoScreen conditions, we evaluated myotube morphology in untreated conditions. Two healthy donors and four DMD donors were labeled using Hoechst as a nuclear dye and myosin heavy chain (MHC) as a differentiation marker to separate myoblasts and myotubes. Figure 1A shows that all donors formed myotubes exhibiting the standard MyoScreen morphology. Healthy donors had a larger average myotube area than DMD donors, but similar numbers of nuclei, resulting in a higher fusion index (Figure 1A, Figure 1B). This result indicates that healthy donors were more differentiated than DMD donors, which is the expected DMD phenotype.

[0333] Functional assays for assessing myocyte structural integrity should demonstrate a good correlation between the level of rescue induced by a therapeutic agent and the assay readout. In the context of DMD pathology, this would translate to a correlation between dystrophin expression and the colocalization of dystrophin with other components of the DGC. Figure 2 shows baseline dystrophin expression in healthy and DMD donors using antibodies targeting either the N-terminal domain (Figure 2A) or the C-terminal domain (Figure 2B). Dystrophin detection was reduced to background noise in DMD donors using an N-terminally targeted dystrophin antibody and to approximately 40% of healthy donor levels using a C-terminally targeted dystrophin antibody (Figure 2C, Figure 2D). To assess the correlation between dystrophin expression levels and the colocalization assay readout, RNAi was used to modulate dystrophin levels in healthy donors (Figure 2D and Figure 2E). As expected, dystrophin signals decreased with increasing siRNA doses. At the highest siRNA concentration, the dystrophin signal observed in healthy donors mimicked that observed in DMD donors. Thus, the 40% signal obtained in siRNA-treated healthy and DMD donors when monitoring dystrophin using a C-terminally targeted antibody suggests the low specificity of this antibody. Despite the high background with the C-terminally targeted dystrophin antibody, both dystrophin antibodies have similar sensitivity for detecting changes in dystrophin expression (Figure 2E).

[0334] The membrane-bound formation of DGC is a biomarker of dystrophin activity restoration in DMD patients treated with therapies that restore dystrophin expression. In the following examples, we assessed the colocalization of dystrophin with a-DG, b-DG, and a-SG to monitor dystrophin restoration upon treatment of myotubes derived from DMD patients with either gene therapy or exon skipping therapy. Dysferlin was included in these studies as a transmembrane protein not directly related to the DGC. Figures 3A and 3B show the expression of a-DG, b-DG, a-SG, and dysferlin in myotubes from healthy and DMD donors. Except for variability in the expression levels of a-DG, b-DG, and a-SG among healthy donors, there were no significant differences in the expression levels of these proteins between healthy and diseased donors. Treatment of healthy donors with DMD siRNA downregulated dystrophin levels by less than 20%. The decrease in dystrophin levels was associated with a decrease in the levels of a-DG, b-DG, and a-SG, but had no significant effect on the expression levels of dysferlin (Fig. 3C).

[0335] Methods for quantifying the colocalization of dystrophin with α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin Four quantification methods were used to quantify the colocalization of dystrophin with α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin in myotubes. We compared the dynamic range, discrimination, and selectivity of the MCC, RCW, PCC, and high PCC% readouts in healthy donors upon modulation of dystrophin levels by RNAi (Figure 6). Dystrophin expression in two healthy donors was modulated by RNAi using DMD-specific siRNA at concentrations ranging from 0.001 to 1 nM. Colocalization was analyzed using the calculated mean PCC, MCC, and RWC, as well as the high PCC% readouts, between α-sarcoglycan, β-dystroglycan, α-dystroglycan, dysferlin, and dystrophin (N-terminal antibody). For HV#1 and HV#2, the colocalization readouts were then plotted against the dose of dystrophin siRNA. For dystrophin and β-dystroglycan colocalization, all four statistical methods yielded similar results. Although all four statistical methods yielded satisfactory results for colocalization between dystrophin and α-sarcoglycan or dysferlin, the high PCC% readouts exhibit a larger dynamic range than PCC, MCC, or RWC.

[0336] Sensitivity of colocalization between dystrophin and β-dystroglycan, α-sarcoglycan, α-dystroglycan, or dysferlin to changes in dystrophin levels Dystrophin expression was modulated in two healthy donors by RNAi using DMD siRNA at concentrations ranging from 0.001 to 1 nM. The resulting dystrophin levels were determined by high-content analysis. Colocalization between dystrophin and α-sarcoglycan, β-dystroglycan, α-dystroglycan, and dysferlin was analyzed using a high PCC% readout and plotted as a function of the percentage of dystrophin in untreated healthy donors (Figure 7). Changes in colocalization between dystrophin and β-dystroglycan or α-sarcoglycan could be detected when dystrophin levels were reduced by more than 40%. The sensitivity of these assays is consistent with the level of dystrophin restoration achieved by therapies targeting DMD. A high PCC% does not indicate significant colocalization between dystrophin and α-dystroglycan. Dysferlin showed significant colocalization with dystrophin, but this colocalization was highly variable between donors and highly sensitive to changes in dystrophin concentration. These results revealed that among the four colocalization pairs tested, colocalization between dystrophin and β-dystroglycan or α-sarcoglycan, analyzed by high PCC%, provided optimal conditions for monitoring dystrophin-dependent assembly of the DGC in the tested donors.

[0337] These experiments demonstrate that quantitative colocalization assays monitoring dystrophin and b-DG or dystrophin and a-SG, preferably using a high PCC% readout, are both equally suitable as functional assays for assessing the efficacy and effectiveness of DMD therapies that restore dystrophin expression.

[0338] Assessing the efficacy and effectiveness of DMD therapies by monitoring the colocalization of dystrophin with β-dystroglycan or α-sarcoglycan The examples shown in Figures 8 and 9 demonstrate that the colocalization assays of the present invention can be used to quantitatively monitor dystrophin-dependent assembly of the DGC. The following examples provide evidence that these assays can be used as cell-based functional assays to monitor dystrophin activity restored in patient-derived primary and immortalized DMD myotubes by gene or RNA therapy, to predict the ability of therapeutic compounds to treat DMD, and to assess the sensitivity of various DMD patient genotypes to particular therapies.

[0339] Exon skipping therapy in primary myotubes derived from DMD patients Most DMD patients have deletions of various lengths in the DMD gene, which shift the reading frame and prevent the expression of functional dystrophin. In more than 50% of these patients, skipping exons at the boundaries of these deletions can restore the reading frame, allowing the expression of partially functional, but truncated, dystrophin. Skipping specific exons can be achieved with the help of specific oligonucleotides that prevent splicing of specific exons by masking sequences required for spliceosome assembly at specific intron / exon junctions.

[0340] To skip exon 44 in DMD patients suitable for exon 44 skipping, we used a PMO targeting exon 44 fused to an octaguanidine dendrimer (vivo group) for better cellular delivery. As shown in Figure 8B, treatment of myotubes from two DMD donors with this in vivo PMO had no effect on the number of nuclei, fusion index, or average myotube area. The level of dystrophin restoration was assessed using a dystrophin antibody targeting the N-terminal domain of dystrophin and reached 39% (DMD #5 donor) and 22% (DMD #6 donor) of the average dystrophin signal from healthy donors treated with control PMO (Figure 8C). The two DMD donors have the same genotype (exon 45 deletion). Therefore, the observed difference in the level of dystrophin restoration may reflect differences in the uptake of the in vivo PMO by these donors. The expression levels of b-DG and a-SG were not significantly altered by in vivo PMO treatment (Figures 8A and 8C). The high PCC% readout was used to monitor the colocalization of dystrophin with b-DG or a-SG. Both assays yielded comparable results (Figure 8D). Consistent with the low level of dystrophin restoration, DMD donor #6 exhibited lower levels of colocalization between dystrophin and b-DG or a-SG than DMD donor #5. However, when their responses were normalized, both donors exhibited the same relative response to in vivo PMO treatment and the resulting dystrophin restoration (Figure 8D). These results confirm the usefulness of the dystrophin / b-DG and dystrophin / a-SG colocalization assays for monitoring dystrophin restoration and function in patient-derived myotubes during exon-skipping therapy.

[0341] To skip exon 45 in DMD patients suitable for exon 45 skipping, we used an in vivo PMO targeting one of the exon 45 splice junctions. As shown in Figures 9A-9B, treating myotubes from two DMD donors with this in vivo PMO had no effect on the number of nuclei, fusion index, or average myotube area. Both donors responded to exon 45 in vivo PMO treatment and restored dystrophin to similar levels (Figure 9C). However, the restored dystrophin activity in these donors differed when monitoring the colocalization of dystrophin and β-dystroglycan versus α-sarcoglycan (Figure 9D). Although dystrophin restoration was similar between both donors, the high PCC% in donor DMD#1 only reached 30%, while that in DMD#4 reached 70%, indicating that the restored dystrophin from donor#1 was less able to interact with β-dystroglycan than that from donor#4 (Figure 9D). In contrast, the restored dystrophin from both donors interacted with α-sarcoglycan to a similar extent (Figure 9D). Because donors#1 and#4 have different deletions within the DMD gene (see Table 3), the restored dystrophin from these patients likely has different structural properties. This example demonstrates the ability of the developed colocalization assay to detect differences in the functional properties of restored dystrophin.

[0342] Exon skipping therapy in immortalized DMD myotubes Potency assays require a steady supply of key reagents, are limited in supply, and cannot be easily performed in primary cells, which are at risk of outgrowth and loss of their characteristics over time. Figure 10 illustrates a colocalization assay between restored dystrophin and β-dystroglycan in myotubes derived from a DMD-immortalized cell line amenable to exon 44 skipping, demonstrating the ability of this method to detect differences in products with known potency. Using Hoechst as a nuclear dye and myosin heavy chain (MHC) as a differentiation marker to separate myoblasts and myotubes, myotube differentiation and morphology of healthy and DMD-immortalized cell lines under MyoScreen conditions were assessed. The morphology of healthy (HVimm) and DMD (DMDimm) cell lines did not show any significant differences (Figures 10A and 10B). Healthy and DMD myotubes were treated with control PMO (5 μM), and DMD cells were treated with increasing doses of four exon 44-skipping PMOs (0.3–10 μM), listed in order of predicted efficacy (Figure 10C, Figure 10D). Treatment of DMD cell lines with exon 44-skipping PMOs partially restores dystrophin expression in a distance-dependent manner. PMOs targeting proximal splice junctions (PMO1, 2, and 3) exhibit greater dystrophin rescue than PMOs targeting distally from the acceptor site (PMO4). β-dystroglycan expression levels were unchanged by exon 44 PMO treatment (Figure 10D). Colocalization of dystrophin and β-dystroglycan was monitored using the high PCC% readout as a function of exon 44-skipping PMO dose or % dystrophin levels (Figure 10E). PMO4 showed the lowest activity, followed by PMO3. PMO1 and 2 show comparable colocalization levels. This result indicates that the colocalization assay can distinguish between products of different potency.

[0343] Establishment of a quantitative colocalization assay for DM1 Characterization of DM1 donors and assessment of quantitative co-localization of nuclear DMPK RNA foci and splicing factor MBNL1 To characterize the proliferation and differentiation behavior of healthy and DM1 donors under MyoScreen conditions, we evaluated myotube morphology under untreated conditions. Two healthy donors and five DM1 donors were labeled using Hoechst as a nuclear dye and myosin heavy chain (MHC) as a differentiation marker to separate myoblasts and myotubes. Figure 11A shows that all donors formed myotubes exhibiting standard MyoScreen morphology. DM1 #2 donor had a lower fusion index than the other HV and DM1 donors, which may indicate altered differentiation compared to the other donors (Figures 11A and 11B). We assessed the presence of DMPK mRNA foci using a (CAG)5-Cy3 FISH probe, and immunofluorescent staining of MBNL1 was used to determine the presence of MBNL1 protein trapped in DMPK mRNA foci. DMPK ASO was used to remove abnormal DMPK mRNA in DM1 patients. All DM1 donors showed the presence of co-labeled DMPK mRNA and MBNL1 protein, representative of the DM1 phenotype. For the congenital donors (DM1 #4 and #5), the results are also shown on a different scale to make the dose-response effect more visible (Figure 11C). With increasing DMPK ASO dose, the number of MBNL1 / DMPK foci co-labeled spots decreased, indicating the disappearance of RNA foci and the release of MBNL1 protein. The same effect was observed in all donors, but only reached statistical significance for DM1 #3 and #5 due to considerable variability among the other donors (Figure 11C). Colocalization was analyzed using the calculated mean PCC, MCC, and RWC between DMPK foci and MBNL1 as a function of the ASO dose response in DM1 donors, as well as the high PCC % readout (Figure 11D). MCC yielded the highest range among all compared methods and for all DM1 donors, making it the most suitable method for estimating colocalization between DMPK foci and MBNL1 in DM1. Using this selected colocalization method, we further compared the responses of five DM1 donors to treatment with six concentrations of ASO. Colocalization in healthy donors is close to 0 because MBNL1 protein is not captured by nuclear RNA foci. In contrast, colocalization is highest in DM1 donors.ASO treatment releases MBLN1 from nuclear RNA foci and reduces colocalization ( Figure 11E ).

[0344] Monitoring the colocalization of misspliced ​​dystrophin with β-dystroglycan or α-sarcoglycan In DM1 patients, loss of MBNL1 function eliminates exons 71 and 78 from the DMD mRNA, resulting in a different C-terminus of the dystrophin protein. Treatment of DM1 donors with DMPK ASO partially restored the dystrophin C-terminal domain. Antibodies targeting the dystrophin C-terminal domain did not show any specific sarcolemmal staining in DM1 donors. Increasing the dose of DMPK ASO restored the dystrophin C-terminal signal in DM1 donors (the white arrow in Figure 12A indicates the sarcolemmal region positive for dystrophin signal). The response was donor-dependent, with lower recovery in non-congenital donors and higher recovery in congenital donors (Figure 12A). Treatment of DM1 donors with DMPK ASO led to partial rescue of the expression level of the dystrophin C-terminal domain, whereas the expression levels of α-sarcoglycan and β-dystroglycan remained unchanged by DMPK ASO treatment (Figures 12B and 12C). Colocalization was analyzed using the calculated mean PCC, MCC, and RWC between α-sarcoglycan or β-dystroglycan and dystrophin (C-terminal antibody) as a function of ASO dose response in DM1 donors, as well as the high PCC% readout (Figure 12D). High PCC% yielded the highest range among all compared methods and for all DM1 donors, and is therefore the most suitable method for estimating colocalization between α-sarcoglycan, β-dystroglycan, and dystrophin (C-terminal antibody) in DM1 donors. The response of five DM1 donors to treatment with six concentrations of ASO was further compared. The high PCC% readout was used to monitor colocalization of C-terminal dystrophin with β-dystroglycan or α-sarcoglycan. Both assays yielded comparable results (Figures 12D and 12E). Colocalization in healthy donors was close to 100%. In contrast, colocalization was lowest in DM1 donors. Congenital donors show colocalization levels between β-dystroglycan or α-sarcoglycan and dystrophin lying between 30% and 80%, with dystrophin expression levels lying between 20% and 60%.Non-congenital donors show lower colocalization levels: with dystrophin levels of 20%-50%, they show colocalization percentages of 0-70%.

[0345] Colocalization of dystrophin and β-dystroglycan in cardiomyocytes To assess the feasibility of applying the colocalization assay to cardiomyocytes, we measured the expression of b-DG and dystrophin in human cardiomyocytes derived from DMD siRNA-knockdown induced pluripotent stem cells (hIPSC-CMs). Figure 13A shows the expression of b-DG and dystrophin in control cardiomyocytes and cardiomyocytes treated with siRNA. Treatment of cells with DMD siRNA downregulated dystrophin levels by less than 50%. The decrease in dystrophin levels was associated with a decrease in b-DG levels (Figure 13B), as previously observed in myotubes.

[0346] To determine the optimal method for quantifying dystrophin and β-dystroglycan colocalization in hIPSC-CMs, 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 siRNA at concentrations ranging from 0.0032 to 10 nM. Colocalization was analyzed using the calculated mean PCC, MCC, and RWC between β-dystroglycan and dystrophin (N-terminal antibody), as well as the high PCC% readout. For hIPSC-CMs, the colocalization readouts were then plotted against the dose of dystrophin siRNA (Figures 13C and 13D). For hIPSC-CMs, the colocalization between dystrophin and β-dystroglycan, the high PCC% readout, exhibits a greater dynamic range than the PCC, MCC, or RWC, as also measured in myotubes. [Explanation of symbols]

[0347] 1 Pattern(1) 1L Side area W L Maximum width of the side area (1L) W C Maximum width of the central region (1C) L length 1C central area X vertical axis

Claims

1. 1. An in vitro computer-implemented method for assessing the functionality of a cellular protein or nucleic acid of interest in myotubes, comprising: (i) providing at least one image of at least one in vitro cultured myotube, wherein the 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 that interacts with the first cellular protein or nucleic acid of interest, and has been stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing a quantitative co-localization analysis. Including, An in vitro computer-implemented method, wherein the degree of co-localization correlates with the functionality of the cellular protein or nucleic acid of interest in said at least one myotube.

2. 1. An in vitro computer-implemented method for assessing the efficacy of a compound to modulate the functionality of a cellular protein or nucleic acid of interest in myotubes, comprising: (i) providing at least one image of at least one in vitro cultured myotube, wherein the at least one myotube has been contacted with a test compound, stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid that interacts with the first cellular protein or nucleic acid of interest, and stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, An in vitro computer-implemented method, wherein a statistically significant difference between the degree of colocalization and the reference degree of colocalization indicates that the compound is capable of modulating the functionality of a first cellular protein or nucleic acid of interest in the at least one myotube.

3. 1. An in vitro computer-implemented method for predicting the ability of a compound to treat a neuromuscular disorder of interest, comprising: (i) providing at least one image comprising at least one in vitro cultured myotube exhibiting features of a neuromuscular disease of interest ("pathological myotube"), wherein the at least one myotube has been contacted with a test compound, stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid that interacts with the first cellular protein or nucleic acid of interest, and stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured diseased myotube that has not been contacted with the compound or that has been contacted with a higher or lower concentration of the compound. Including, An in vitro computer-implemented method in which a positive correlation between the concentration of the compound and a statistically significant desired variation in the degree of colocalization compared to a reference degree of colocalization indicates that the compound is useful for treating said neuromuscular disease.

4. 1. An in vitro computer-implemented method for monitoring the response of a patient suffering from a neuromuscular disease to a therapeutic compound, comprising: (i) providing at least one image comprising at least one in vitro cultured myotube obtained from a sample of a patient suffering from a neuromuscular disease after administration of a therapeutic compound, wherein the 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 that interacts with the first cellular protein or nucleic acid of interest, and has been stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of co-localization with a reference degree of co-localization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained from a sample of the patient prior to administration of a therapeutic compound; Including, An in vitro computer-implemented method in which a desired statistically significant variation in the degree of colocalization compared to a reference degree of colocalization indicates that the subject is responsive to the treatment.

5. 1. An in vitro computer-implemented method for selecting a patient suffering from a neuromuscular disease for treatment with a therapeutic compound, or for determining whether a patient suffering from a neuromuscular disease is likely to benefit from treatment with a therapeutic compound, comprising: (i) providing at least one image comprising at least one in vitro cultured myotube obtained from said patient sample, said at least one myotube being contacted with a therapeutic compound, stained for a first cellular protein or nucleic acid of interest and for a second cellular protein or nucleic acid that interacts with said first cellular protein or nucleic acid of interest, and stained with at least one labeling agent that reveals 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 image segmentation using an algorithm for the appropriate staining channel to identify the ROI; and (iii) quantitatively determining the degree of co-localization of the first cellular protein or nucleic acid and the second cellular protein or nucleic acid in at least one ROI by performing a quantitative co-localization analysis; and (iv) comparing the degree of colocalization with a reference degree of colocalization obtained by performing steps (i) to (iii) on at least one reference myotube, wherein the at least one reference myotube is at least one in vitro cultured myotube obtained from a sample of the patient that has not been contacted with the therapeutic compound or that has been contacted with a higher or lower concentration of the therapeutic compound. Including, An in vitro computer-implemented method in which a positive correlation between the concentration of a therapeutic compound and a statistically significant desired change in the degree of colocalization compared to a reference degree of colocalization indicates that the patient is likely to benefit from treatment with the therapeutic compound.

6. 6. The method of claim 1, wherein the quantitative colocalization analysis is performed using quantitative pixel-based colocalization analysis.

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

8. 8. The method of any one of claims 1 to 7, wherein the myotubes are cultured under restrictive conditions that allow for the generation of a homogeneous myotube population.

9. Before step (i), - culturing the myoblasts under restrictive conditions, preferably allowing the generation of a homogenous myotube population, optionally in the presence of the test compound; - staining the myotubes for the first protein or nucleic acid, for the second protein or nucleic acid, and with the at least one labeling agent; and - acquiring at least one image of at least one stained myotube.

8. The method of claim 1, further comprising:

10. 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 Pearson's Colocalization Coefficient (PCC), Mander's Colocalization Coefficient (MCC), Rank-based Intensity Weighting Coefficient (RWC), and any combination thereof, and optionally applying a mathematical function to said coefficients.

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

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

13. 13. The method of any one 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 myopathies.

14. The neuromuscular diseases of interest are 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 dystrophy (LGMD), Walker-Warburg syndrome, muscle-eye-brain disease, congenital muscular dystrophy, tibial muscular dystrophy, Ullrich myopathy, myofibrillar myopathy, distal myopathy, rimmed vacuolar myopathy, distal myopathy with rimmed vacuoles (DMRV), and central myopathy.

13. The method of any one of claims 3 to 12, wherein the myopathy is selected from the group consisting of nuclear myopathy (CNM), X-linked myotubular myopathy (XLMTM), tubular aggregate myopathy, malignant hyperthermia syndrome, inclusion body myopathy, protein aggregation myopathy, nemaline myopathy, congenital myopathy (CM), vacuolar aggregate myopathy, Miyoshi myopathy, Vici syndrome, X-linked myopathy with excessive autophagy, Danon disease, Marinesco-Sjogren syndrome, neurodegeneration with ataxia, Friedreich's ataxia, dystonia and gaze palsy, childhood-onset (NADGP), and Pompe disease.

15. 15. The method of any one 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 α-sarcoglycan, β-dystroglycan, α-dystroglycan and dysferlin, more preferably selected from the group consisting of α-sarcoglycan and β-dystroglycan, or vice versa.

16. 15. The method of any one 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 α-sarcoglycan and β-dystroglycan, or vice versa.

17. 15. The method of any one 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 α-sarcoglycan and β-dystroglycan, or vice versa.

18. 15. A method according to any one 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 an RNA-binding protein trapped by CTG repeats in the DMPK gene, preferably MBNL1 protein, or vice versa.

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