Selective HDAC6 inhibitors for use in the treatment of myotonic dystrophy type 1

Abstract

Myotonic Dystrophy type 1 (DM1) is an inherited disease characterized by multi-systemic symptoms, particularly in skeletal muscles (progressive weakness and atrophy, myotonia). The inventors screened 7 500 bioactive compounds for their ability to improve the myogenic fusion and reduce the molecular hallmarks of DM1 skeletal muscle cells. The inventors found that five compounds, all inhibiting HDAC6, a cytoplasmic histone deacetylase, were effective at the micromolar range in normalizing the myogenic fusion, reducing the number of nuclear foci of mutant DMPK mRNA, decreasing the expression level of DMPK mRNA, restoring the splicing of several genes, and increasing the acetylation of SMAD3, a transcription factor involved in muscle development. The effects of HDAC6 inhibitors were observed both in immortalized and hiPSC-derived skeletal muscle cells from DM1 patients, and in different stages of differentiation. Thus, the present invention relates to the use of selective HDAC6 inhibitors for the treatment of DM1.

Description

[0001] SELECTIVE HDAC6 INHIBITORS FOR USE IN THE TREATMENT OF MYOTONIC DYSTROPHY TYPE 1

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular genetic diseases.

[0004] BACKGROUND OF THE INVENTION:

[0005] Myotonic dystrophy type 1 (DM1) is the most common form of adult muscular dystrophy of genetic origin, with a prevalence of 1 / 8,000 worldwide, and remains a disease for which there is no treatment. Management primarily includes monitoring for complications and supportive care (assistive devices, hormone therapy, pain medication). Its multisystemic symptoms, which include myotonia, muscle wasting, cardiac conduction defects, insulin resistance, cataracts, and cognitive dysfunction, are linked to disrupted regulation of alternative splicing, mRNA translation and mRNA stability that affect hundreds of genes (Du, Hongqing, et al. "Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy." Nature structural & molecular biology 17.2 (2010): 187-193'). The origin of this dysregulation lies in changes to the bioavailability of RNA-binding proteins, in particular MBNL1, which is sequestered in intranuclear ribonucleoprotein aggregates (called foci) and triggered by a CTG repeat expansion in the 3ZUTR of the DMPK (dystrophic myotonia protein kinase) gene (Kanadia, Rahul N., et al. "A muscleblind knockout model for myotonic dystrophy, "science 302.5652 (2003): 1978-1980). Accordingly, there are major ongoing efforts to identify therapeutic drugs for the treatment of DM1.

[0006] HDAC6 is a unique member of the histone deacetylase (HD AC) family, mainly residing in the cytoplasm and belonging to class lib HDACs. HDAC6 deacetylates several cytoplasmic substrates such as a-tubulin, cortactin and HSP90, whereas other HDACs are typically central regulators of gene expression in the cell nucleus. Considerable efforts to develop selective HDAC6 inhibitors have been made to treat a variety of diseases. In particular, several anticancer treatments targeting HDAC6 have been proposed (He, Xingrui, et al. "Novel selective histone deacetylase 6 (HDAC6) inhibitors: a patent review (2016-2019). " Recent Patents on Anti-Cancer Drug Discovery 15.1 (2020): 32-48). Furthermore, HDAC6 deletion or inhibition has been shown to be beneficial for some neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth disease (Shen, Sida, and Alan P. Kozikowski. "A patent review of histone deacetylase 6 inhibitors in neurodegenerative diseases (2014- 2019). " Expert opinion on therapeutic patents 30.2 (2020): 121-136). More recently, the pharmacological inhibition of HDAC6 was shown to improve muscle phenotypes in dystrophin-deficient mice (Osseni, Alexis, et al. "Pharmacological inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulating TGF-fi via Smad3 acetylation." Nature Communications 13.1 (2022): 7108).

[0007] It has also been shown that vorinostat improves DM1 splicing abnormalities in DM1 muscle cell lines and skeletal muscle from a DM1 mouse model (Neault, Nafisa, et al. "Vorinostat improves myotonic dystrophy type 1 splicing abnormalities in DM1 muscle cell lines and skeletal muscle from a DM1 mouse model. "International Journal of Molecular Sciences 24.4 (2023): 3794).

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention is defined by the claims. In particular, the present invention relates to the use of selective HDAC6 inhibitors for the treatment of myotonic dystrophy type 1.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] Main definitions:

[0012] As used herein, the term “myotonic dystrophy type 1” or “DM1” has its general meaning in the art and refers to a rare genetic multi-system disorder characterized by a wide range of muscle-related manifestations (muscle weakness, myotonia, early onset cataracts (before age 50) and systemic manifestations (cerebral, endocrine, cardiac, gastrointestinal tract, uterus, skin and immunologic involvement) that vary depending on the age of onset. The very wide clinical spectrum ranges from lethal presentations in infancy to mild, late-onset disease. The term is also known as “Steinert myotonic dystrophy”. Diagnosis is suspected on the characteristic clinical manifestations and a consistent family history and confirmed by molecular genetic testing of the causative gene expansion.

[0013] As used herein, the term "patient" refers to a warm-blooded animal, preferably a mammal (including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc...), and more preferably a human. In some embodiments, the patient is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease. In some embodiments, the patient is an adult (for example a subject above the age of 18). In some embodiments, the patient is a child (for example a subject below the age of 18). In some embodiments, the patient is a male. In some embodiments, the patient is a female.

[0014] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0015] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0016] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. As used herein, the term “HD AC” has its genera meaning in the art and refers to histone deacetylases. There are currently 18 known histone deacetylases, which are classified into four groups. Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs, which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HD AC 10, are related to the yeast Hdal gene. Class III HDACs, which are also known as the sirtuins are related to the Sir2 gene and include SIRT1-7. Class IV HDACs, which contains only HDAC11, has features of both Class I and II HDACs.

[0017] As used herein, the term “inhibitor” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule (e.g. HDAC6) compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, aptamers, antisense oligonucleotides, interfering RNA, carbohydrates or small organic molecules.

[0018] As used herein, the term “selective HDAC6 inhibitor” means that the compound inhibits the activity or expression of HDAC6. In particular, a selective HDAC6 inhibitor inhibits the activity or expressions of HDAC6 to a substantially greater extent, such as 5x, 10x, 15x, 20x greater or more, than to any other type of HD AC enzyme, such as HDAC1 or HDAC2. In some embodiments, the selectivity ratio of HDAC6 over HDAC1 is from about 5 to about 30,0000, e.g., about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, about 25,000, or about 30,000, including all values and ranges therebetween. For example, a HDAC6 inhibitor may be at least 100-fold selective against HDAC6 compared to all other isozymes of HD AC. In some embodiments, selectivity may be determined by reference of another HD AC inhibitor, such as a pan-HDAC inhibitor — that is an inhibitor that inhibits HDACs other than HDAC6 in addition to HDAC6. Givinostat is an example of a pan- HD AC inhibitor. In some embodiments, a selective HDAC6 inhibitor inhibits HDACs other than HDAC6 at least 100-fold less effectively than givinostat.

[0019] Methods of the present invention:

[0020] The present invention relates to a method of treating myotonic dystrophy type 1 (DM1) in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a selective HDAC6 inhibitor.

[0021] According to the present invention, the HDAC6 inhibitor of the present invention is a selective HDAC6 inhibitor. Thus, the use of pan-HDAC inhibitors (e.g. vorinostat) is thus not encompassed in the teaching of the present invention.

[0022] Various selective HDAC6 are known in the art. In addition, using known methods it is routine to screen compounds to identify further selective HDAC6 inhibitors. In particular, given a known HDAC6 inhibitor, a person of skill in the art can identify which analogs of the compound have selective HDAC6 activity. In some embodiments, illustrative HDAC6 inhibitors are provided in:

[0023] - He, Xingrui, et al. "Novel selective histone deacetylase 6 (HDAC6) inhibitors: a patent review (2016-2019)." Recent Patents on Anti-Cancer Drug Discovery 15.1 (2020): 32- 48)

[0024] Shen, Sida, and Alan P. Kozikowski. "A patent review of histone deacetylase 6 inhibitors in neurodegenerative diseases (2014-2019)." Expert opinion on therapeutic patents 30.2 (2020): 121-136

[0025] - Peng, Jie, et al. "Recent development of selective inhibitors targeting the HDAC6 as anti-cancer drugs: structure, function and design." Bioorganic Chemistry (2023): 106622.

[0026] - Li, Yunheng, et al. "Inhibition of Histone Deacetylase 6 (HDAC6) as a therapeutic strategy for Alzheimer's disease: A review (2010-2020)." European Journal of Medicinal Chemistry 226 (2021): 113874.

[0027] T Tavares, Mauricio, and Sida Shen. "Recent innovative advances in the discovery of selective HDAC6 inhibitors." Future Medicinal Chemistry 13.12 (2021): 1017-1019. Further illustrative HDAC6 inhibitors are provided in U.S. Patent Publications Nos. US8227516B2, US20100292169A1, US20070207950A1, US8222423B2,

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[0041] US20190270744A1, US20200022966A1, and US20210094944A1, which are incorporated herein for purposes of identifying HDAC6 inhibitors that may be used in the methods disclosed herein. In some embodiments, the HDAC6 inhibitor is a fluoroalkyl-oxadiazole derivative. Illustrative fluoroalkyl-oxadiazole derivatives that may be used as HDAC6 inhibitors include those described herein and those described in Int’l Pat. Appl. No. PCT / US2020 / 066439, published as WO2021127643A1 the content of which is incorporated by reference herein in its entirety. PCT / US2020 / 066439, published as WO2021127643A1, also describes methods of synthesis of such compounds, which are specifically incorporated by reference herein.

[0042] In some embodiments, the HDAC6 inhibitor is CAY10603, tubacin, ricolinostat (ACY- 1215), citarinostat (ACY-241), ACY-738, QTX-125, CKD-506, nexturastat A, tubastatin A, or HPOB, or an analog thereof. Typically, the drugs herein disclosed are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0043] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0044] FIGURES:

[0045] Figure 1 : The effect of HDAC6 inhibitors were screened in vitro on the fusion of DM1 and SMA immortalized skeletal muscle cells. A. Scheme of the fusion screening protocol. B. Quantification of Fusion Area index of DM1 immortalized myotubes treated with HDAC6 inhibitors. Data represent the mean ± SD values of the Fusion Area index calculated over three individual technical replicates. Figure 2 : HDAC6 inhibitors normalize the fusion defect of in vitro DM1 hiPSC-derived skeletal muscle cells. A. Scheme of fusion assay protocol. B. Quantification of Fusion Area index of hiPSCs derived myotubes treated with HDAC6 inhibitors. Data represent the mean ± SD of fusion area Index calculated over four individual technical replicates (histogram, left axis). The normalized cell viability is also indicated (curve, right axis). Statistics were calculated using an ordinary two-ways ANOVA tests * : p-values < 0.05.

[0046] Figure 3 : HDAC6 inhibitors reduce the number of nuclear foci in DM1 hiPSC-derived skeletal muscle cells. A. Scheme of foci quantification in differentiating hiPSCs-derived skeletal muscle cells protocol. B. Representative images of the mask used to quantify foci of mutant DMPK mRNA detected by fluorescent in situ hybridization in nuclei of differentiating hiPSCs-derived skeletal muscle cells untreated and treated with HDAC6 inhibitors. Nuclei, detected by Hoescht staining, are represented as their outlines. C. Quantification of the number of foci per nuclei of differentiating hiPSCs-derived skeletal muscle cells treated with HDAC6 inhibitors. Data are represented as the average ± Standard Deviation (SD) values of the number of foci per nuclei, calculated over four individual technical replicates. The normalized cell viability is plotted above (right axis). Statistics were calculated using an ordinary two-ways ANOVA tests, * : p-values < 0.05, only conditions with cell viability > 60% were considered.

[0047] Figure 4 : HDAC6 inhibitors reduce the expression of DMPK mRNA and normalize alternative splicing defect in DM1 hiPSC-derived skeletal muscle cells. A. Scheme of foci quantification in differentiating hiPSCs-derived skeletal muscle cells protocol. B. Quantification by RTqPCR of DMPK mRNA expression in differentiating hiPSCs derived hiPSC-derived skeletal muscle cells treated with HDAC6 inhibitors. C. Quantification exon 78 in DMD mRNA in differentiating hiPSC-derived skeletal muscle cells treated with HDAC6 inhibitors. D. Quantification exon 11 in ISR1 mRNA in differentiating hiPSC-derived skeletal muscle cells treated with HDAC6 inhibitors. E. Quantification of MBNL1 and MBNL2 mRNA expression in differentiating hiPSC-derived skeletal muscle cells treated with HDAC6 inhibitors. Data represent and the mean±SD calculated over three individual technical replicates. Statistics were calculated using an ordinary two-ways ANOVA tests, Ns : Not significant, * : p-values < 0.05, ** : p-values < 0.01, *** : p-values < 0.001, **** : p-values < 0.0001. Figure 5 : Inhibition of HDAC6 induce an increase of SMAD3 acetylation in differentiating hiPSC-derived skeletal muscle cells. A. Scheme of SMAD3 quantification in differentiating hiPSCs-derived skeletal muscle cells protocol. B. Quantification of SMAD3 acetylated and phosphorylated in nuclei of differentiating hiPSC-derived skeletal muscle cells. Data represent the average of fluorescence intensity of phospho-SMAD3 (curve labelled 2, right axis) and acetyl-SMAD3 (curve labelled 1, left axis) immunostaning in nuclei of hiPSCs- derived skeletal muscle cells± SD for four technical replicates.

[0048] Figure 6 : Inhibition of HDAC6 normalize the fusion defect and reduce the number of nuclear foci of in vitro DM1 hiPSC-derived skeletal myotubes. A. Scheme of fusion and foci quantification in advanced differentiating hiPSCs-derived skeletal muscle cells protocol. B. Quantification of Fusion Area index of hiPSCs derived myotubes treated with HDAC6 inhibitors. Data represent the mean ±SD of fusion area index calculated over four individual technical replicates. The normalized cell viability, quantified as the total nuclei area , is also indicated (curve, right axis). Statistics were calculated using an ordinary two-ways ANOVA tests * : p-values < 0.05, only conditions with cell viability > 60% were considered.

[0049] Figure 7 : Inhibition of HDAC6 reduce the number of nuclear foci and the expression of DMPK mRNA and normalize alternative splicing defect in in vitro DM1 hiPSC-derived skeletal myotubes. A. Scheme of fusion and foci quantification in advanced differentiating hiPSCs-derived skeletal muscle cells protocol. B. Quantification of the number of foci per nuclei in hiPSCs derived myotubes treated with HDAC6 inhibitors. Data represent the mean ±SD of the number of foci per nuclei, calculated over four individual technical replicates. The normalized cell viability is plotted as a curve (right axis). C. Quantification by RTqPCR of DMPK mRNA expression in hiPSCs derived myotubes treated with HDAC6 inhibitors. D. Quantification exon 11 in BINI mRNA in hiPSCs derived myotubes treated with HDAC6 inhibitors. Statistics were calculated using an ordinary two-ways ANOVA tests * : p-values < 0.05, ** : p-values < 0.01, *** : p-values < 0.001, **** : p-values < 0.0001. Only conditions with cell viability > 60% were considered.

[0050] Material & Methods Obtention of human induced pluripotent stem cells (hiPCS)

[0051] Human iPSC were generated by reprogramming skin fibroblasts derived from non-affected and DM1 affected patients as previously described1. The repeats expansion length was estimated to be approximately -2500 CTG repeats1. Informed consents were obtained from all the patients included in this study, complying with the ethical guidelines of the institutions and with the legislation requirements. All the different hiPSC lines were grown on culture dishes coated with vitronectine (Gibco) and maintained in iPS-Brew XF medium (Miltenyi Biotec). Cell passaging was performed using accutase (Life Technologic) every 5 days and culture medium was changed every 2 days as previously described1.

[0052] Generation of myoblasts and myotubes from hiPSCs

[0053] Myoblasts were derived from hiPSCs using the STEMdiff™ Myogenic Progenitor Supplement Kit (STEMCELL technologies). Briefly, hiPSCs were seeded at different densities on Matrigel (Corning), in iPS-Brew XF medium (Miltenyi Biotec) supplemented with lOpM Y-27632 (BioTechne) and grown for 24h at 37 °C, 5% CO2. The densities resulting in the recommended confluency of 30% with colonies of 10 to 30 cells were selected to pursue differentiation. The cells were kept in culture for 30 days, in DMEM / F12 without HEPES medium (ThermoFisher Scientific) complemented with the different supplements of the kit. Medium was changed every day. Myoblasts weree expanded and frozen for banking in expansion medium [DMEM with lOOOmg / L D-Glucose (STEMCELL technologies) supplemented with MyoCult™-SF Expansion Supplement Kit (STEMCELL technologies)]. Their myogenic identity was assessed by myogenic markers immunostaining (Desmin, PAX7, MF20, MyoD, MyoG, Table 1).

[0054] To evaluate the fusion capacity of hiPSC s-derived myoblasts, cells were seeded between 15 000 and 25 000 cells per cm2on Matrigel (Coming) and let grown for 72h, in expansion medium at 37 °C, 5% CO2. When the cells reached a confluency of 80 to 90%, medium was switched into the MyoCult™ Differentiation Kit (Human) (STEMCELL technologies) and the cells were grown for 7 more days minimum, with medium refreshment every 3 to 4 days. The fusion of myotubes was assessed by Desmin and MF20 immunostaining.

[0055] Differentiation of immortalized myoblasts

[0056] Two lines of immortalized myoblasts (kindly gifted of Denis Furling, Institute of Myology, Paris) were used: the AB 1167 control line and the DMl lcl5 DM1 line2. Immortalized myoblasts were expanded on gelatin coating (Sigma), in immortalized expansion medium DMEM+GlutaMAX medium (Life Technologic) supplemented with 16% of Medium 199 + GlutaMAX (Life Technologic), 20% FBS (Life Technologic), 25pg / mL of fetuin (ThermoFisher Scientific), 5pg / mL of insulin solution human (Sigma), 0.2pg / mL of dexamethasone (Sigma), 5 ng / mL of EGF human recombinant protein (Life Technologic), 0.5 ng / mL of bFGF human recombinant protein (Life Technologic), and 1 pM of penicillin / streptomycin (Life Technologic).

[0057] For myogenic differentiation, myoblasts were seeded at 100 000 cells per cm2on collagen I coating (Gibco), in immortalized expansion medium and let grown for 24h. To induce myogenic fusion, cells were shifted in DMEM+GlutaMAX medium supplemented with 10 pg / mL of insulin solution human and 1 pM of penicillin / streptomycin, for 7 days minimum. The fusion of myotubes was assessed by Desmin and MF20 immunostaining.

[0058] Primary screening

[0059] Immortalized myoblasts were seeded as previously described for myogenic differentiation, in immortalized myoblasts expansion medium at 37 °C, 5% CO2. Cells were switched in differentiation medium after 24h. Chemical compounds were added at one day later at 8 different concentrations ranging from 0.01 pM to 10 pM. Each chemical compound was dissolved in pure DMSO for which the final concentration was calculated to not exceed 0.1%.

[0060] HDAC6 inhibitors treatment assays

[0061] Immortalized myoblasts were seeded as previously described for myogenic differentiation, in immortalized myoblasts expansion medium at 37 °C, 5% CO2. Cells were switched in differentiation medium after 24h. The compounds, dissolved in pure DMSO (MilliporeSigma), were added to the immortalized myotubes differentiation medium after 24h. The final concentration of DMSO did not exceed 0.1% and a solution of 0.1% DMSO was used as a negative control. Differentiation medium was refreshed after 3 days of treatment. After 7 days of treatment, the fusion was assessed by immunostaining of MF20. hiPCS-derived myoblasts were seeded as previously described for myogenic differentiation, and let grown for 72h, in expansion medium at 37 °C, 5% CO2. When the cells reached a confluency of 80 to 90%, medium was switched into the MyoCult™ Differentiation Kit (Human) (STEMCELL technologies). After 24h, the compounds, dissolved in DMSO, were added in the medium. A solution of 0.1% DMSO (MilliporeSigma) was used as a negative control. Differentiation medium was refreshed after 3 days of treatment. After 7 days of maturation, the fusion was assessed by immunostaining of MF20. For the test on more mature myotubes, cells were differentiated for 5 days before adding the treatment for 48h. Immunostaining

[0062] Cells were fixed with 4% paraformaldehyde (PF A) (Electron Microscopy Sciences) for 10 min at room temperature (RT) and washed with PBS. Cells were then incubated overnight at 4 °C with primary antibodies (listed in Table 1). The cells were washed 3 times in PBS buffer and incubated for 2 h at RT with appropriate fluorescent-labeled secondary antibodies and Hoechst (5 pg / mL).

[0063] Table 1: List of primary and secondary antibodies

[0064] FastLane quantification of mRNA expression and splicing defects The quantification of mRNA expression from differentiating hiPSC-derived myoblasts treated in 384-well plates was performed using FastLane Cell probe Kit (Qiagen) according to the manufacturer's protocol. Briefly, samples were collected after 48h of treatment. Briefly, cells were washed and lysed with Fastlane’s reagents. Cell lysates were then used directly as templates in real-time one-step reverse transcription PCR using the QuantiTect Probe from FastLane kit combined with specific primers. For splicing defect quantification, two sets of primers were designed, one flanking the splicing defect location, and another including the exon involved in the defect. The defects (exon inclusion or exclusion) were quantified using the flanking set as reference. For DMPK, MBNL1 and MBNL2 expression, the 18S housekeeping gene was used as reference gene. Fold change variations induced by compound treatments were calculated using the 2-AACt method with DMSO treated control cells as reference.

[0065] Fluorescent in situ hybridization (FISH)

[0066] Toxic mRNA foci were quantified with FISH in differentiating hiPSC-derived myoblasts treated in 384-well plates. FISH experiments were performed as previously described3. Briefly, cells were fixed after 48h of treatment with 4% PF A (Electron Microscopy Sciences) for 10 min at RT, and washed with PBS. Cells were then incubated overnight at 4 °C with 70% Ethanol. Cells were re-hydrated for lOmin at RT, with PBS supplemented with 5mM of MgC12. Cells were then sequentially put in pre-hybridization buffer (50mM phosphate buffer, 40% formamide, 2X SSC) for lOmin at RT, and hybridization buffer (50mM phosphate buffer, 40% formamide, 2X SSC, 0.2% BSA and Img / mL of salmon sperm DNA) containing 300ng / ml of the (CAG)10-Cy5 probe (Operon) overnight at 37 °C. Cells were washed twice for 30min at 37 °C with washing buffer (pre-hybridization buffer + 0.2% BSA). The first wash was supplemented with Hoechst (5 pg / mL). Cells were finally washed twice with PBS at RT. The images acquisition was performed on the automated imaging Cell Insight CX7 HCS Platform (Cellomics Inc), using the 20X objective. The nuclear foci detection was automatically performed on the same platform, using the colocalization pipeline.

[0067] Results:

[0068] Myotonic Dystrophy type 1 (DM1) is an inherited disease characterized by multi-systemic symptoms, particularly in skeletal muscles (progressive weakness and atrophy, myotonia). Different studies have previously demonstrated that primary and immortalized skeletal muscle cells isolated from DM1 patients display a myogenic fusion defect2’4’5. To identify potential therapeutic candidates capable to normalize this defective myogenic fusion, we developed a high-content drug screening on immortalized differentiated DM1 skeletal muscle cells. For this purpose, DM1 immortalized myoblasts were differentiated for eight days with the addition of each compound at the start of the differentiation process. We based our approach on the use of a chemical library of 7 500 bioactive compounds (Figure 1A). Cultures were characterized at the end of those eight days. ISOX, previously described to be a MBNL1 modulator in DM1 cells6, was shown to partially normalize the in vitro DM1 myogenic fusion defect and thus was used as positive control for the screening. Over the 7 100 tested compounds, 34 induced an improvement of the myogenic fusion of DM1 immortalized myoblasts. Among these compounds, two others HD AC inhibitors, namely Citarinostat and Ricolinostat, were found to be efficient at the micromolar range (Figure IB). Interestingly, these two compounds have been shown to have a high specificity for HDAC6 inhibition7.

[0069] To confirm the effect of HDAC6 inhibition in DM1 myogenic fusion, we performed similar experiments using skeletal muscles cells differentiated from hiPCS derived from DM1 and control (CTL) patients treated with ISOX, Ricolinostat, Cirtarinostat as well as two other specific HDAC6i, namely Nexturastat A and Tubastatin A. As for the immortalized cells, the myotubes generated from DM1 hiPSC-derived skeletal muscle cells display a myogenic fusion defect that is normalized by treatment with the different HDAC6i at the same micromolar range (Figure 2A-B).

[0070] We next sought to evaluate the potential of these HDAC6i on key molecular hallmarks of DM1. The most noticeable DM1 related molecular hallmark is the presence of nuclear aggregates made of toxic DMPK mRNAs called foci8, 9. To explore if HDAC6i were affecting those aggregates, hiPCS-derived skeletal muscle cells were treated with the five selected HDAC6i and observed after 48h of treatment (Figure 3A). Only conditions maintaining at least 60% of cell viability were considered. For each compound, a significate reduction of nuclear foci was observed after treatment with HDAC6i (Figure 3B-C), up to more than 50%. Interestingly, the compounds improved the myogenic fusion and reduced the number of nuclear foci at the same micromolar range concentrations.

[0071] To determine whether the effect of these compounds on the number of foci could be correlated to the level expression of DMPK gene, we measured, by RTqPCR, the expression level of DMPK mRNA in hiPCS-derived skeletal muscle cells treated with three HDAC6i (ISOX, Tubastatin A, Citarinostat, Figure 4A). DM1 cells treated with these compounds displayed a significant reduction of DMPK mRNA when compared to mock-treated cultures (Figure 4B), correlating with the reduced number of nuclear foci. The pathogenesis of DM1 involves an RNA gain-of-function mechanism caused by the expression of mutant mRNAs containing abnormal expansion of hundreds to thousands of CUG repeats. These mutant mRNAs sequester the splicing regulator Muscleblind-like RNA binding proteins (MBNL), resulting in specific misregulation of the alternative splicing of other pre- mRNAs10, n. Therefore, we sought to evaluate the potential of these HDAC6i on the defective inclusion of exon 78 in the dystrophin (DMD) gene and of exon 11 of the insulin receptor 1 (ISR1) gene that have been previously described in DM112. Our results confirmed that HDAC6i treatment normalized the DM1 -altered ratio of DMD transcripts at concentrations similar to the ones active on myogenic fusion (Figure 4C). The normalization of ISR1 transcript was also observed with Citarinostat and Ricolinostat treatment, at similar doses (Figure 4D). We excluded the hypothesis that these normalizations could be related to an effect of HDAC6i on the expression level of MBNL at the mRNA level (Figure 4E).

[0072] HDAC6 is a unique cytoplasmic member of the histone deacetylase family. It has been recently demonstrated that SMAD3 is a new target of HDAC6 and that inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by SMAD3 acetylation13. We therefor sought to evaluate whether a similar mechanism could be involved in DM1 skeletal muscle cells. The proportions of acetylated and phosphorylated SMAD3 were quantified in nuclei of DM1 hiPCS-derived skeletal muscle cells treated or not with the five HDAC6i (Figure 5). Our results confirmed that the treatment with these five HDAC6i led to an increased proportion of acetylated-SMAD3 that is negatively correlated with a decrease of phophorylated-SMAD3.

[0073] Since the effects of HDAC6i were observed on immature cells that had just begun to differentiate, we sought to determine whether this effect could also be observed on more mature cultures, differentiated for longer time. For this purpose, hiPCS-derived skeletal muscle cells were differentiated for eight days, and the treatment was added for the last 48h of the differentiation process (Figure 6A). This new protocol also led to an improved myogenic fusion of the advanced differentiated hiPCS-derived skeletal muscle cells (Figure 6B). We also looked at the molecular DM1 hallmarks in hiPSC-derived myotubes treated with HDAC6i (Figure 7 A) and observed a reduction of the number of foci (Figure 7B), as well as a significant reduction of DMPK expression (Figure 70). Finally, anormalization of DM1 -mediated defective inclusion of exon 11 in BINI transcript was also observed with Citarinostat and Ricolinostat treatment (Figure 7D). REFERENCES:

[0074] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0075] 1. Merien, A. et al. CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis. Hum Mol Genet 31, 41-56 (2021).

[0076] 2. Arandel, L. et al. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds. Disease models & mechanisms 10, 487-497 (2017).

[0077] 3. Maury, Y. et al. Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1. iScience 11, 258-271 (2019).

[0078] 4. Mondragon-Gonzalez, R. & Perlingeiro, R.C.R. Recapitulating muscle disease phenotypes with myotonic dystrophy 1 induced pluripotent stem cells: a tool for disease modeling and drug discovery. Disease models & mechanisms 11 (2018).

[0079] 5. Kawada, R. et al. Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs. Scientific reports 13, 94 (2023).

[0080] 6. Zhang, F. et al. A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I. Hum Mol Genet 26, 3056-3068 (2017).

[0081] 7. Zhang, Q.Q., Zhang, W.J. & Chang, S. HDAC6 inhibition: a significant potential regulator and therapeutic option to translate into clinical practice in renal transplantation. Frontiers in immunology 14, 1168848 (2023).

[0082] 8. Davis, B.M., McCurrach, M.E., Taneja, K.L., Singer, R.H. & Housman, D.E. Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci U S A 94, 7388-7393 (1997).

[0083] 9. Taneja, K.L. Localization of trinucleotide repeat sequences in myotonic dystrophy cells using a single fluorochrome-labeled PNA probe. BioTechniques 24, 472-476 (1998).

[0084] 10. Fardaei, M. et al. Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11, 805- 814 (2002).

[0085] 11. Miller, J.W. et al. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. Embo J 19, 4439-4448 (2000). 12. Goers, E.S., Purcell, J., Voelker, R.B., Gates, D.P. & Berglund, J.A. MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing. Nucleic Acids Res 38, 2467- 2484 (2010).

[0086] 13. Osseni, A. et al. Pharmacological inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulating TGF-beta via Smad3 acetylation. Nat Commun

[0087] 13, 7108 (2022).

Claims

CLAIMS:

1. A method of treating myotonic dystrophy type 1 (DM1) in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a selective HDAC6 inhibitor.

2. The method of claim 1 wherein the HDAC6 inhibitor is selected from the group consisting of Ricolinostat, Citarinostat, Tubastatin A, Nexturastat A, and ISOX.

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