KR102217261B1 - Composition for preventing or treating spinal muscular atrophy - Google Patents

Abstract

The present invention relates to a composition for preventing or treating spinal muscular dystrophy, and more particularly, to a use of a compound showing an effect of preventing or treating spinal muscular atrophy by increasing the expression of SMN protein by correcting the splicing defect of SMN2. .
According to the present invention, Rigosertip represented by Chemical Formula 1 exhibits an effect of correcting splicing defects of SMN2 pre-mRNA, thereby increasing the expression of SMN protein, and exhibiting the effect of improving abnormal lesions related to disease It can be very usefully used in the development of therapeutic agents for neuromuscular diseases including muscular dystrophy.

Description

Composition for preventing or treating spinal muscular atrophy}

The present invention relates to a composition for preventing or treating spinal muscular dystrophy, and more particularly, to a use of a compound showing an effect of preventing or treating spinal muscular atrophy by increasing the expression of SMN protein by correcting the splicing defect of SMN2. .

Spinal Muscular Atrophy (SMA) is a typical neuromuscular disease and is classified as a rare disease, but it is a fatal genetic disease that has a high incidence and the number 1 infant mortality rate due to genetic diseases. Among similar neuromuscular diseases, there is Amyotrophic Lateral Sclerosis (ALS), which is more familiar to the general population. Unlike ALS, which mainly begins to show symptoms in adults, spinal muscular dystrophy is considered a more serious disease since it shows symptoms from infants and toddlers. do.

SMA patients gradually lose the motor neuron in the spinal cord, so that normal signals cannot be transmitted to the motor muscles, and eventually the muscles gradually atrophy and degenerate. It is classified into 4 stages (I, II, III, IV) depending on the severity of symptoms, and the most severe SMA type I usually develops within 6 months of birth and dies within 2 years of birth.

In humans, there are two SMN (Survival of Motor Neuron) genes, SMN1 and SMN2. Most SMA patients (96%) lack SMN1, and proteins are expressed only through SMN2. However, most of the mRNAs over 80% transcribed from the SMN2 gene are made in a form deficient in exon 7 due to a defect in splicing, and the protein, which is a product, has a form of SMNΔ 7 whose C-terminus is shortened unlike normal SMN. It has, but is very unstable, is easily degraded in cells. Eventually, in SMA patients, only a small amount of normal full-length SMN (full-length SMN, FL-SMN) protein expressed from the SMN2 gene (20% of normal people) is present.

There is a clear inverse correlation between the amount of SMN protein and the symptoms of SMA disease. In fact, each SMA patient has a different copy number of SMN2, and as the number of copies of SMN2 increases, the amount of SMN expression increases and symptoms are alleviated. Therefore, the most reliable SMA treatment approach is the backup remaining in patient cells. It increases the amount of SMN protein expressed in the gene SMN2.

Therefore, in order to increase the amount of protein expressed from the SMN2 gene of SMA patients, there may be various therapeutic approaches such as increasing the transcriptional activity of the SMN2 gene, correcting the splicing defect (exon7 skipping) of SMN2, and inhibiting proteolysis, but splicing defects. It is a major target in that it is a direct cause of this protein decline.

Many multinational pharmaceutical companies, including Biogen, Norvatis, Roche, and Trophos, have tried various approaches to develop a treatment for spinal muscular dystrophy, and SPINRAZA, an oligonucleotide-based SMN2 splicing improvement drug, recently developed by Biogen at the end of 2016. As the first new drug, it was approved by the US FDA and is currently being administered to patients. However, SPINRAZA has very limited clinical efficacy, and is administered to infants under 6 months of age in a very invasive manner called spinal cord injection, and is administered 6 times a year, and the drug price is close to 150 million won. There is a limitation of being a very unrealistic treatment for patients. Accordingly, there is an urgent need to develop alternative drugs with improved clinical efficacy, non-invasive administration, and more realistic drug prices, and low-molecular drugs are drawing attention as the most appropriate alternative.

Accordingly, the inventors of the present invention have conducted extensive research to develop a novel substance capable of preventing or treating spinal muscular dystrophy through selective splicing control of SMN2 pre-mRNA. As a result, Rigosertib represented by Formula 1 When selective splicing of SMN2 pre-mRNA, it was confirmed that the expression of the exon 7-containing mRNA and the expression of the SMN protein was significantly increased, finding that the lesion of spinal muscular dystrophy could be alleviated, and the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Another object of the present invention is to provide a food composition for preventing or improving neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In order to achieve the above object of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Spinal muscular dystrophy (SMA) is known as a genetic disease caused by insufficient production of functional SMN proteins due to deletion or mutation of the SMN1 gene. In humans, there are two SMN (Survival of Motor Neuron) genes, SMN1 and SMN2. Most SMA patients (96%) lack SMN1, and proteins are expressed only through SMN2. However, most of the mRNAs over 80% transcribed from the SMN2 gene are made in a form deficient in exon 7 due to a defect in splicing, and the protein, which is a product, has a form of SMNΔ 7 whose C-terminus is shortened unlike normal SMN. It has, but is very unstable, is easily degraded in cells. Eventually, in SMA patients, only a small amount (20% of normal people) normal SMN protein expressed from the SMN2 gene exists.

Accordingly, the present inventors screened dozens of low-molecular compounds in order to search for substances exhibiting the effect of preventing or treating neuromuscular diseases through selective splicing control of SMN2 pre-mRNA. As a result, it was confirmed that rigsertib represented by the structure of Formula 1 increased the expression of the functional SMN protein by increasing the production of exon 7-containing mRNA when selectively splicing SMN2 pre-mRNA.

In particular, the role of the compound of Formula 1 as an SMN2 splicing modulator occurs regardless of the PLK inhibitory efficacy of igosertip, which is well known. In addition, it has been confirmed that the compound of Formula 1 does not act at all on the transcription of SMN2 or the stability of the SMN protein other than SMN2 splicing.

The term "exon 7-containing mRNA" as used herein refers to an mRNA containing exons 1 to 8 formed through splicing from pre-mRNA transcribed from SMN1 or SMN2 gene. The exon 7-containing mRNA encodes a functional SMN protein.

Meanwhile, according to the present invention, the compound of Formula 1 increases the formation of exon 7-containing mRNA when selective splicing of SMN2 pre-mRNA, thereby generating a large amount of functional SMN protein including exon 7 It is possible to prevent and treat spinal muscular dystrophy that occurs due to the large amount of the missing form of non-functional SMN protein being produced.

In the present invention, the neuromuscular disease may be Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS), Duchenne Muscular Dystrophy, or Mytonic Dystrophy, but preferably The lower extremity may be spinal muscular atrophy.

In the present invention, Rigosertip represented by Formula 1 is a low-molecular compound that blocks the activation of Ras effector protein, and is being developed as a cancer treatment, but it has been reported for its use in the treatment of neuromuscular diseases as a splicing modulator of SMN2. none.

In the composition according to the present invention, the compound of Formula 1 may be used by itself or in the form of a pharmaceutically acceptable salt. In the above,'pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not usually cause an allergic reaction or a similar reaction when given to humans, and the salt is a pharmaceutically acceptable glass Acid addition salts formed by acid (free acid) are preferred. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid. In addition, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The pharmaceutical composition according to the present invention may contain the compound of Formula 1 alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration Can be

The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. I can. For example, in oral preparations, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In the case of a formulation for parenteral administration, it can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a formula generally known for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per 1 kg of patient body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient, as well as the formulation method, route of administration and number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

The term'prevention' in the present invention refers to a therapy that protects against the onset of a disease or disorder so that clinical symptoms of the disease do not develop. Thus,'prevention' refers to administering a therapy to a subject (e.g., administering a therapeutic substance) (e.g., administering a therapeutic substance) (e.g., a detectable infectious agent in the subject (e.g., Virus) to a subject in the absence of). A subject may be an individual at risk of developing a disease or disorder, such as an individual having one or more risk factors known to be associated with the development or onset of the disease or disorder.

The term'treatment' in the present invention means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state. Amelioration or temporal alleviation and alleviation of (which may be partial or total), detectable or undetectable. 'Treatment' refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. 'Palliating' a disease means that the extent of the disease state and/or undesirable clinical signs are reduced or the temporal transition of progression is slowed or prolonged, compared to without treatment.

The present invention also provides a food composition for preventing or improving neuromuscular diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be prepared and consumed in the form of tea, juice and drink, or may be ingested by granulating, encapsulating and powdering. In addition, it may be prepared in the form of a composition by mixing with known substances or active ingredients known to have an effect of preventing and improving sepsis or septic shock.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (eg, ham, sausage corn beef, etc.) ), breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, Retort foods, frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) may be prepared by adding the compound of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention.

The preferred content of the compound of Formula 1 or a pharmaceutically acceptable salt thereof in the food composition of the present invention is not limited thereto, but may be, for example, 0.001 to 30% by weight of the finally prepared food. Preferably, it may be 0.01 to 20% by weight of the finally prepared food.

In addition, in order to use the compound of Formula 1 or a salt thereof in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

According to the present invention, Rigosertip represented by Chemical Formula 1 exhibits an effect of correcting splicing defects of SMN2 pre-mRNA, thereby increasing the expression of SMN protein, and exhibiting the effect of improving abnormal lesions related to disease It can be very usefully used in the development of therapeutic agents for neuromuscular diseases including muscular dystrophy.

1, including FIGS. 1A to 1C, is a diagram showing a method and result of searching for an SMN2-specific splicing modulator.
1A shows a schematic diagram of an SMA drug screening platform using a luciferase-based screening system and patient-specific iPSCs.
1B shows a schematic diagram of the SMN2 mini gene reporter and two protein products produced by selective splicing.
Figure 1c shows the results of the first screening of the small molecule compound using the SMN2-Luc stable cell line. The percentage of the relative luciferase activity of the group treated with the small molecule compound was calculated by setting the activity of the DMSO-treated cells to 100%. C33A cells without reporter plasmid were included as negative controls. Mean and standard deviation were obtained from 3 independent experiments.
Figure 2, including Figures 2a to 2d, is a result of confirming the effect of increasing the expression of the SMN protein through the insertion of Rigosertip (rigosertib, No. 65) exon 7.
2A shows the amount of SMN protein in fibroblasts of SMA patients after treatment with candidate compounds (No. 53, No. 65 (rigosertib)). MG132 was used as a positive control, and normal human-derived fibroblasts were included as a cell control. Relative protein content (%) was calculated by setting the amount from DMSO treated cells to 100%.
Figure 2b is a result of confirming the effect of five types of PLK inhibitors on the splicing of SMN2-Luc and SMN1-Luc stable cell lines. Cells were treated with the indicated concentration of the test substance for 24 hours, and then luciferase activity was measured. The percentage of relative luciferase activity was calculated by setting the activity of DMSO-treated cells to 100%.
FIG. 2C is a diagram illustrating an analysis of the effect of rigosertib, SAHA and SB on the activation of SMN2 transcription. 293T cells were transformed with the pGL4.14-SMN2 promoter-Luc-PEST plasmid and treated with the indicated concentrations of Rigoseritip, SAHA and SB for 24 hours, and then luciferase activity was measured.
Figure 2d is a result of analyzing the effect of Rigosertip on the protein stability of SMNΔ7. The FLUC-SMNΔ7 stable cell line was treated with the indicated concentration of Ligosertip for 10 hours in the presence of CHX (0.1 mg/ml), and then luciferase activity was measured. MG132 (10 μM) was included as a positive control. Relative activity was calculated by setting the activity of cells treated simultaneously with DMSO and CHX to 100%.
FIG. 3 including FIGS. 3A to 3D is a diagram for an experiment in which SMA induced pluripotent stem cells (iPSCs) are differentiated into pMN (motor neuron progenitor) and MN (motor neuron) .
3A is a schematic diagram of a differentiation strategy for MN differentiation. pMNs were derived from iPSCs through the NEP step and incubated for 12 days with pMN inducers. pMN was finally differentiated into MN.
3B is a diagram showing the results of immunocytochemistry of lineage-specific markers for identifying each cell type during the differentiation of MN.
3C is a result showing the ratio of OLIG2+ and ChAT+ cells per total DAPI+ cells of differentiated pMN and MN (ns; not significant).
3D is a diagram confirming that pMNs passaged 5 times (p5) were also differentiated into MNs. Nuclei were counterstained with DAPI.
Figure 4, including Figures 4a to 4e, is a result of analyzing the phenotypes of pMN and MN derived from WT ( normal ) and SMA iPSC.
4A shows the results of RT-PCR analysis on the levels of full-length SMN and SMNΔ7 transcripts of pMNs and MNs. The relative expression level of the FL transcript in SMA cells relative to the WT control was quantified and shown as a graph.
4B is a result of analyzing the expression level of SMN protein in pMNs and MNs by Western blotting. The relative expression level of SMN protein in SMA cells relative to the WT control was quantified and shown as a graph.
4C is a result of analyzing the expression levels of SMN proteins in pMN and MN by immunocytochemistry. Gem body (arrow) was hardly found in SMA cells. Scale bar, 20 μm.
Figure 4d is a result of quantitative analysis of the ratio of EthD-1+ apoptotic cells in pMN and MN derived from WT and SMA iPSC. SMA cells showed an increased EthD-1 signal compared to WT cells (n = 3).
Figure 4e is the result of analyzing and quantifying the length of the neurite (n=50)
5, including FIGS. 5A to 5G, is a result of confirming that the restoration of SMN2 exon 7 splicing by treatment with igosertips showed improvement of the SMA-like phenotype in SMA iPSC-pMNs.
5A is a result of confirming the remarkable migration of the SMN2 transcript from SMNΔ7 to SMN-FL by RT-PCR analysis.
5B shows the results of quantitative analysis of SMN-FL expression using qRT-PCR (n = 3). The expression of SMN-FL was increased by igosertip treatment in pMNs induced by SMA iPSC.
5c and 5d show the results of analyzing the expression level of SMN protein by Western blotting (5c) and immunocytochemistry (5d) after treating pMN with 10 nm of Rigosertip for 72 hours.
5E is a result of analyzing the viability of cells through flow cytometry showing live cells stained with Calcein-AM (green) and dead cells stained with EthD-1 (red). The proportion of dead cells decreased in 10 nM igosertip treated SMA iPSC-pMN.
Figure 5f is a result of expressing the superoxide level in the WT and SMA iPSC-derived pMN labeled with MitoTracker Green and MitoSOX as a MitoSOX / MitoTracker ratio (superoxide signal / total mitochondrial signal). Mitochondrial superoxide overproduction (MitoSOX, red) of SMA iPSC-pMNs was reduced by treatment with 10 nM igosertip (n=3).
5G is a result of evaluating the effect of Rigosertip on the neurite length of MNs induced in iPSC.
6, including FIGS. 6A to 6D, is a result of confirming that Rigosertip increases the amount of SMN protein in the spinal cord of SMA mice.
6A shows the spinal cord was isolated from heterozygous, vehicle-treated, and ligosertip (10 mg/kg)-treated SMA mice (each n = 3) and analyzed by Western blotting using an anti-SMN antibody. α-Tubulin was also analyzed as a loading control.
6B is a result of quantification of the protein band by setting the band intensity of the vehicle-treated SMA to 100%.
6C is a result of confirming the expression of SMN protein in the spinal cord section by immunocytochemistry (green background staining indicates capillaries),
6D is a graph showing this by quantifying the relative number of SNM-positive cells for the heterozygous group.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

Experiment method

1. Cell culture

Primary fibroblasts from SMA type I patients (GM09677, GM00232) were obtained from Coriell Cell Repositories (Camden, NJ, USA). Human WT fibroblasts (CRL-2097 and GM08333) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and Coriell Cell Repositories, respectively. These cells were prepared with minimal essential media (MEM, Gibco, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 1% penicillin-streptomycin (P/S). , USA) as previously described. C33A cells stably expressing SMN1- or SMN2- luciferase splicing reporters (SMN1-Luc and SMN2-Luc stable cell lines) and 293T cells stably expressing the full length (FL)-fusion SMNΔ7 protein (FLuc-SMNΔ7) Stable cell line) was cultured in Dulbecco's modified Eagles medium (DMEM, Gibco) to which 10% FBS, 2 mM L-glutamine, and 1% P/S were added. A mouse myoblast line (myoblast, C2C12) was purchased from ATCC and cultured in a maintenance medium consisting of DMEM high glucose (Gibco) supplemented with 1% P/S and 10% FBS. C2C12 differentiation was induced by switching to a differentiation medium containing DMEM high glucose (Gibco) with 1% P/S and 2% FBS. The medium was changed every 2 days.

2. Establishment and management of human iPSCs (induced pluripotent stem cells)

As previously described, healthy control (CRL2097, CRL2097, Episomal iPSC reprogramming vector (Cat. No. A14703, Invitrogen)) encoding OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA-p53 by electroporation. IMR90) and SMA patients (GM09677, GM00232) fibroblasts were reprogrammed. Five days after electroporation was performed, cells were subjected to 20% knockout serum replacement (Gibco), 1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 10 6-coated with Matrigel (BD Bioscience, San Diego, USA) with iPSC medium containing DMEM/F12 medium (Gibco) supplemented with ng/ml basic fibroblast growth factor (R&D systems, Minneapolis, Minnesota) Re-seeded at 1 X 10 ⁵ /well on a well plate. After 14-20 days, human embryonic stem cell (hESC)-like colonies were isolated and expanded for further experiments. iPSCs were passaged by clump dissociation using 1 mg/ml collagenase IV (Gibco).

3. Compound

Rigosertip (rigosertib, Selleckchem, Houston, TX, USA), suberoylanilide hydroxamic acid (SAHA) (Selleckchem), cycloheximide (CHX, Sigma-Aldrich), MG132 (Calbiochem, San Diego, CA, USA) sodium butyrate (SB, Sigma-Aldrich), BI2536 (Sigma-Aldrich), GSK461364 (Selleckchem), HMN214 (Selleckchem), and BI6727 (Selleckchem) were used.

4. Analysis of alternative splicing of SMN pre-mRNA

SMN1-Luc and SMN2-Luc cells were generated by introducing SMN1 and SMN2 splicing mini gene reporter plasmids into C33A cells, respectively. The SMN splicing mini gene reporter was designed by the following procedure. Briefly, in order to inactivate a definite translation stop codon at the 3'end of exon 7, a single nucleotide'C' was inserted just before the stop codon (CUAA). The FL reporter gene was modified by fusing 21 nucleotides downstream from the 5'end of exon 8 and removing the'A' at the 5'end initiation codon of the reporter gene to reduce undesirable internal translation initiation and background expression. SMN2-Luc cells were dispensed in a 96-well plate at a density of 1×10 ⁵ cells/well to screen for small molecule compounds that regulate the splicing of SMN2 pre-mRNA. Luciferase analysis was performed 24 hours after treatment with a low molecular weight compound using the One-Glo Luciferase Assay System (Promega, Madison, WI, USA).

5. Cell-based SMN immunoassay

Fibroblasts (8×10 ³ cells/well) of SMA patients were dispensed into a black-well transparent bottom 96-well plate (Greiner, Wemmel, Belgium) and treated with a small molecule compound (10 μM) for 24 hours. MG132, a proteasome inhibitor previously characterized with an SMN-elevating compound, was also included as a positive control. Cells were washed and fixed with 4% paraformaldehyde (PFA). Cells were infiltrated with 0.5% Triton X-100 and 1% bovine serum albumin (BSA) in PBS, and 2 with a mouse monoclonal antibody against SMN (Cat.No.610647, BD Transduction Laboratories, Franklin Lakes, NJ, USA). Reacted for hours. Then, the cells were cultured for 1 hour with Alexa Fluor 488-conjugated goat secondary antibody against mouse IgG (Invitrogen) and stained with Hoechst 33342 dye (Invitrogen). Cells were analyzed with a Cellomics ArrayScan device (Cellomics, Pittsburgh, PA, USA). For each sample, more than 300 cells were analyzed to obtain the mean and standard deviation.

6. Analysis of SMN2 promoter activity

To prepare the SMN2 promoter-induced firefly luciferase (FLuc) reporter plasmid, the promoter and the 5'-UTR end portion of the SMN2 gene (~3.4kb) were cloned into the pGL4.14 vector (Promega). To enhance the reporter's responsiveness to changes in transcription, a PEST sequence was added to the C-terminus of the FL protein. 293T cells were transformed with 2 μg of pGL4.14-SMN2 promoter-Luc-PEST reporter plasmid using X-tremeGENE siRNA Transfection Reagent (Roche, Indianapolis, IN, USA), and the density of 1 x 10 ⁴ cells/well Was dispensed into 96-well plates. After 12 hours, cells were treated with Ligosertip, SAHA and SB for 24 hours and luciferase activity was measured using the One-Glo Luciferase Assay System (Promega). Cell viability was measured under the same conditions by CellTiter-Glo Luminescent Cell Viability Analysis (Promega).

7. Stability analysis of SMNΔ7 protein

To construct the FLuc-SMNΔ7 protein expression plasmid, the FL gene was cloned into the pcDNA3.1 vector, and SMNΔ7 was inserted after the Fluc of the pcDNA3.1-Fluc vector. 293T cells were transformed with 2 μg plasmid and treated with 300 μg/ml hygromycin B for 2 weeks for stable selection. The stable cell line was dispensed at a density of 1 × 10 ⁴ cells/well in a 96-well plate, and after 12 hours, the cells were treated with various concentrations of Ligosertip in the presence of CHX (0.1 mg/mL). Proteasomal inhibitor MG132 (10 μM) was also included as a positive control. Luciferase assay was performed 10 hours after treatment using the One-Glo Luciferase Assay System. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability analysis under the same conditions.

8. Total-RNA preparation and PCR analysis

Total RNA isolation was performed with the RNeasy Mini Kit (Cat. No. 74104, Qiagen, Hilden, Germany) according to the manufacturer's instructions and Superscript IV First-Strand Synthesis System Kit (Cat. No. 18091200, Thermo Fisher Scientific, Waltham, MA). , USA) was used for cDNA synthesis. Semi-quantitative RT-PCR was performed under the following conditions: 94° C. for 3 minutes; 32-35 cycles extending for 30 seconds at 94°C, 40 seconds at 55°C, 40 seconds at 72°C, 5 minutes at 72°C. Each PCR product was loaded onto a 2% agarose gel containing SYBR Safe DNA gel stain (Cat No. S33102, Invitrogen) and Gel-Doc (Biorad, Hercules, California, USA) and used for imaging. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green PCR Master mix (Applied Biosystems, Foster City, CA, USA), and the reaction was detected in a 7500 Fast Real-time PCR system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard and the CT value of the target gene was calculated using the provided software.

9. Alkaline phosphatase (AP) and immunocytochemistry

AP staining was performed using a Naphthol/Fast Red Violet solution (Sigma-Aldrich) as previously described. For immunocytochemical staining, cells were immobilized in 4% PFA and infiltrated with 0.1% Triton X-100. After blocking, the cells were reacted with each of the primary antibodies overnight at 4°C, and then incubated for 2 hours in a fluorescently-conjugated secondary antibody. The slides were observed under an Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) or a confocal microscope (Cat. No. FV1000 Live, OLYMPUS, Tokyo, Japan).

10. Karyotype identification and STR (short tandem repeat) analysis

Chromosome G-band karyotyping was performed by GenDix Inc. (Seoul, Korea). The preparation of gDNA of fibroblasts and iPSCs for STR analysis was performed using DNeasy Blood & Tissue Kits (Cat. No. 69504, Qiagen) according to the manufacturer's instructions. STR genotyping is owned by HumanPass Inc. (Seoul, Korea).

11. EB (embryoid body) and teratoma formation assay

For in vitro differentiation capacity analysis, iPSCs were isolated using 1 mg/ml collagenase type IV (Gibco) and non-in EB medium supplemented with 10% knockout serum replacement (Gibco) based on DMEM/F12 (gibco). Re-plated on coated dishes. After 5 days, EBs were attached to Matrigel coated LabTek chamber slides (Nunc, Rochester NY, USA) and incubated for 10 days. 6 weeks old BALB/c-nude mice (Orient Bio Inc, Seoul, Korea) were injected with iPSCs (1 × 10 ⁶ cells) subcutaneously. The resulting teratomas were fixed with 10% formaldehyde, paraffin-embedded, serially sectioned, and stained using hematoxylin and eosin staining (H & E) solution (Sigma-Aldrich). Teratoma assay was performed with the approval of KRIBB's Institutional Animal Care and Use Committee (approval number: KRIBB-AEC-17014).

12. Differentiation into motor neurons (MN)

MN differentiated as previously described. Nervous system induction medium is 1% P/S, 1% GlutaMAX (Gibco), 100nM L-ascorbic acid (Santacruz Biotechnology, Delaware, CA, USA), 0.5 X N-2 supplement (Gibco) and 0.5x B-27 supplement A 1:1 mixed medium of DMEM/F12 medium and neurobasal medium supplemented with (Gibco) was used. iPSCs were isolated using 1 mg/ml collagenase type IV and 1 mg/ml dispase (Invitrogen). The isolated iPSCs were cultured in Petri dishes not coated with NEP medium containing 3 μM CHIR99021 (Torcris, Ballwin, MO, USA), 2 μM DMH-1 (Stemgent, Texas, USA) and 2 μM SB431542 (Stemgent). . The culture medium was changed every other day. After 6 days, the NEPs were separated with 1 mg/ml dispase, supplemented with 1 μM CHIR99021, 2 μM DMH-1, 2 μM SB431542, 0.1 μM retinoleic acid (RA, Stemgent) and 0.5 μM purmorphamine (Pur, Stemgent). It was differentiated into pMN (motor neuron progenitors) using a nerve induction medium. The pMN induction medium was changed every other day. pMN was subcultured once a week using a McIlwainTM tissue cutter (Mickle Engineering, Gomshall, UK), and cultured in pMN induction medium supplemented with 0.5 mM VPA (Stemgent). The medium was changed every other day. pMN was frozen with Recovery™ cell culture freezing medium (Gibco) and stored in liquid nitrogen. For further differentiation into MN, HB9 ⁺ MN was generated by culturing dissociated pMN in MN induction medium supplemented with 0.5 μM RA and 0.1 μM Pur, and the medium was replaced every other day. After 6 days, the induction of ChAT ⁺ and SMI32 ⁺ MNs was dissociated into single cells using Accumax (eBioscience, San Diego, CA, USA), dispensed into matrigel coated-plates, and 0.1 mM compound E (Calbiochem, USA). Darmstadt, Germany) in MN medium supplemented for 10 days. All differentiation experiments were performed independently at least three times.

13. Western Blotting

PMN and MN were harvested at the indicated times. Cells were lysed with RIPA buffer containing 1 mM protease inhibitor and 1xPMSF. 20 μg of cell lysate was analyzed by precast gel (4-15% gradient, Bio-Rad Laboratories, Hercules, CA, USA). After delivery, the membrane was reacted with the appropriate primary antibody, washed, treated with the secondary antibody and left to stand. Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

14. Cell viability and death assay

Wash cells to detect LIVE/DEAD cells using the LIVE/DEAD survival/cytotoxicity kit (Cat.No.L3224, Invitrogen) and live/contain 2 μM Calcein-AM and 4 μM EthD-1 at room temperature. Treated with DEAD assay reagent. After 40 minutes, the cells were washed and visualized under a microscope (IX51, Olympus, Japan). Fluorescence was measured with Calcein-AM and EthD-1 at 494/517 nm and 528/617 nm, respectively. In order to quantify LIVE/DEAD cells, cells were trypsinized and 1-2x10 ⁶ cells were stained with LIVE/DEAD assay reagent mixed with 50 μM Calcein-AM and 4 μM EthD-1. Cells were incubated for 20 minutes at room temperature protected from light and evaluated by flow cytometry (BD Accuri C6; Becton-Dickinson, Mansfield, MA, USA).

15. Mitochondrial detection and mitochondrial superoxide levels

To monitor mitochondrial content and mitochondrial superoxide production, cells were stained with MitoTracker (Cat No. M7514, Invitrogen) and MitoSOX (Cat No. M36008, Invitrogen), respectively. Cells were incubated at 37° C. for 15 minutes using 250nM MitoTracker and 5 μM MitoSOX staining solution protected from light. After staining, cells were captured at 490/516 nm and 510/580 nm, respectively.

16. Analysis of neurite overgrowth

The MN culture solution of the Matrigel coated-plate was fixed with 4% PFA. Neurites were stained with TUJ1 according to immunocytochemistry. Images were taken with a fluorescence microscope (IX51, Olympus, Japan). Neurites were analyzed by NeuronJ (ImageJ add-on software). Even if the curve was made using NeuronJ, each neurite could be observed, and 45 neurites per condition were measured and the length was quantified.

17. Animal testing

All animal experiments were conducted according to the guidelines of the Ministry of Food and Drug Safety. All protocols were reviewed and approved by KRIBB's Institutional Animal Care and Use Committee (Institutional Permit No.: KRIBB-AEC-18132). Heterozygous SMA mice (FVB.Cg-Grm7 ^{Tg (SMN2) 89Ahmb} Smn1 ^tm1Msd Tg (SMN2 * delta 7) 4299Ahmb/J, stock number 005025) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and mated to homozygous SMA. Mice (SMNΔ7 transgenic mice) were obtained. Rigosertip (10 mg/kg) was administered intraperitoneally to SMNΔ7 transgenic mice once a day from 3 to 10 days of age (n = 3 for each group). On the last day of administration, 5 hours after injection, the spinal cord was isolated from each mouse and sonicated in RIPA buffer (LPS solution, Daejeon, Korea) containing protease inhibitor cocktail set III (Calbiochem) to prepare a lysate. Quantitative western blotting analysis was performed as previously described. Quantification of the SMN expressing cell population in spinal cord tissue was performed as previously described. The mouse spinal cord was collected for histological analysis, fixed with 4% PFA solution, and transferred to 30% sucrose for cryopreservation. The tissue was frozen in an optimal cutting temperature compound (Tissue-Tek OCT Compound, Sakura Finetek USA, Inc, Torrance, CA, USA) and cut to make 10 μm thick sections. All sections were infiltrated in 0.01% Triton X-100 for 15 minutes and incubated overnight with primary antibody at 4°C. After washing three times, the sections were incubated with a secondary antibody for 1 hour at room temperature. Tissue was captured using the Evos Fl Auto 2 Cell Imaging System (Thermo Fisher Scientific).

18. Analysis of the formation of neuromuscular junctions

In order to confirm the function of hiPSC-induced motor neurons, C2C12 cells were cultured on a Matrigel-coated confocal dish at a density of 880 cells/mm ² in maintenance medium. After 24 hours, the cells were transferred to differentiation medium to induce myoblast fusion. The medium was changed every other day. On day 4, HB9+ MN was dissociated and dispensed into myotubes at a density of 8800 cells/mm ² . Cells were neurotrophic factor (10 ng/ml insulin-like growth factor 1, Peptrotech, Rocky Hill, NJ, USA)), 10 ng/ml brain-derived neurotrophic factor (BDNF, Peptrotech), and 10 ng/ml ciliary nephrotrophic factor ( CNTF, R&D systems) was cultured for 7 days in a differentiation medium containing. For the evaluation of NMJ, quantification of the overlapped region of α-Bungarotoxin-488 and ChAT + was performed, and NMJ measurement was performed every 6 fields under co-culture conditions of iPSC-derived MN and C2C12 cells.

19. Electrophysiological recordings

Whole cell patch clamp recordings were performed to measure action potential (AP) and voltage gated sodium/potassium currents. Finally, the differentiated MN was put into the recording chamber and a glucose solution (3 mM/min) of 137 NaCl, 2.0 CaCl ₂ , 10 HEPES, and 20 glucose (pH 7.3 containing NaOH) was continuously perfused. Patch pipettes were made with borosilicate glass capillaries (Clark Electromedical Instruments, UK) using a pipette puller (PP-830, Narishige, Japan). Their resistance was 5-8 MΩ when filled with pipette solution (in mM): 140 K-gluconate, 5 NaCl, 1 MgCl ₂ , 0.5 EGTA, 10 HEPES (pH 7.25, KOH). Voltage or current clamp protocol generation and data acquisition was controlled by a computer equipped with an A/D converter, Digidata 1440 (Molecular Devices, Sunnyvale, CA USA) and pClamp 10.3 software (Molecular Devices). The signal was filtered at 5 kHz and sampled at 10 kHz using an Axopatch 200B amplifier (Molecular Devices). The induced AP was induced by injecting a step current from -0.1 to 0.2 nA in 0.02 nA increments for 500 ms. For the voltage gate current, a voltage step was used for 1 second in 10 mV increments from -70 to +90 mV. Clamfit (Molecular Devices), GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA) and Origin (Microsoft, Redmond, Washington, USA) were used for data analysis.

20. Statistical Analysis

The statistical significance of the difference between the two independent samples was analyzed using a non-parametric Mann-Whitney U-test. The experimental results were expressed as mean value ± standard error (SEM), and statistically significant differences were indicated by asterisks (ns: not significant, * P <0.05, ** P <0.01, *** P <0.001). All experiments were repeated at least 3 times.

Experiment result

1. Screening of SMN2 splicing modulators

Since SMN2 splicing defect is a major factor inducing disease phenotype in SMA patients, recovery of SMN2 splicing is a promising treatment for the treatment of SMA. Therefore, the present inventors have developed a new platform for SMA drug development that combines the Lucifer Raise Reporter assay for quantitative evaluation of SMN2 splicing and the validation of drug candidates using patient-specific iPSC-based assays (Fig. 1a). For the easy and quantitative measurement of SMN2 splicing, the present inventors have developed a robust SMN2 minigene in which the firefly luciferase (FLuc) gene is fused, exon 6 to 8 exons, and introns between them. A cell-based assay system (SMN2-luciferase splicing reporter cell line; SMN2-Luc stable cell line) was developed (Fig. 1B). In this assay system, since the mRNA containing exon 7 produces a larger protein fused with the FLuc protein, compounds that enhance the inclusion of exon 7 increase FLuc activity. In contrast, exon 7-deficient mRNA generates an in-frame translation stop codon in exon 8, resulting in the production of a pre-matured terminator protein without fusion of the FLuc protein. After treatment with 88 small-molecular compounds, the present inventors confirmed two compounds (No. 53 and No. 65) that significantly increased luciferase activity by more than three times that of the DMSO control group (Fig. 1c).

2. Candidate compound Ligosertip promotes splicing correction of SMN2 exon 7

Next, the present inventors investigated whether the two selected compounds increase the amount of SMN protein in SMA fibroblasts in SMA type I patients. The present inventors performed immunoassays to specifically detect SMN proteins using specific antibodies that provide a quantitative measurement of SMN proteins in cells. Of the two compounds, only compound number 65 significantly increased the SMN protein (1.62 times that of the DMSO control) (FIG. 2A ). Therefore, subsequent studies were conducted only with compound 65, which is a well-known PLK (Polo-like kinase) inhibitor, igosertip (ON 01910.Na). To further define whether the effect of igosertip on SMN2 splicing is related to PLK inhibition, four other PLK inhibitors (BI2536, GSK461364, HMN214 and BI6727) were also tested in SMN2-Luc cells. Of the five PLK inhibitors tested, Rigosertip showed the strongest effect of increasing the luciferase activity of SMN2-Luc cells by about 4 times at 100 and 1,000 nM, but other inhibitors had little or no effect on the luciferase activity. (Fig. 2b). Ligosertip had little effect on luciferase activity in SMN1-Luc cells (Fig. 2b). Collectively, these results indicate that Ligosertip exhibits a strong splicing improving activity against SMN2 and that its activity is not related to PLK inhibition.

The present inventors investigated whether igosertip can increase the expression of SMN protein by other mechanisms. In order to analyze the effect of Rigosertip on the activation of SMN2 transcription, the present inventors constructed a highly reactive luciferase reporter vector containing the SMN2 promoter by fusing the firefly luciferase protein with the PEST sequence. As a result of the experiment, Rigosertip did not induce a significant change in transcriptional activation, but it was confirmed that positive controls including the HDAC inhibitors SAHA and SB significantly induce transcriptional activation without affecting cell viability (Fig. ). The possible effect of igosertip on SMN protein stability was also investigated. Considering that the degradation of SMNΔ7 occurs very sensitively by the proteasome, 293T cells (FLuc-SMNΔ7 stable cell line) stably expressing the FLuc-SMNΔ7 protein were first established. Luciferase activity indicates the instability of SMNΔ7, which was rapidly reduced by treatment with the protein synthesis inhibitor cycloheximide (CHX) alone. Further treatment with MG132 significantly increased the luciferase activity as a result of inhibition of the proteasome. In contrast, Ligosertip had little effect on the degradation of SMNΔ7 without significantly affecting the cell viability (FIG. 2D ). These results clearly exclude the association between transcription and protein stability in the effect of igosertip on SMN expression.

3. Generation of iPSCs from type 1 SMA patients

In order to establish an in vitro human SMA model, the present inventors differentiated type 1 SMA patient fibroblasts and SMA iPSCs into disease-related cell types to generate SMA iPSCs for experimental validation of Rigosertip. The present inventors reprogrammed SMA fibroblasts into iPSCs using a non-integrating oriP/EBNA-1 based episomal vector. The properties of SMA iPSCs were confirmed by the expression of hESC-like forms and pluripotent markers including AP, OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4. The in vitro and in vivo differentiation potential of SMA iPSCs was confirmed by the presence of three embryonic layers: EB and teratomas derived from SMA iPSCs, respectively. Short tandem repeat (STR) analysis confirmed that SMA iPSCs were derived from fibroblasts of SMA patients and that cultured SMA iPSCs maintained a normal karyotype (results not shown). This result indicates that SMA fibroblasts were successfully reprogrammed with SMA iPSCs.

4. Development of an in vitro SMA model using SMA iPSC-derived pMN (SMA iPSC-pMN)

SMA is due to the low levels of SMN protein due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene, which primarily affects α-MNs in the lower spinal cord. Therefore, in order to observe disease-related phenotypes, WT iPSCs and SMA iPSCs were differentiated into MNs through high-purity pMN (FIG. 3A). IPSCs derived from WT human fibroblasts and SMA iPSCs are neuroepithelial progenitors (NEPs) in the presence of SB431542 (inhibitor of activin-nodal signaling), DMH1 (inhibitor of bone morphogenetic protein signaling) and CHIR99021 (Wnt agonist) Differentiated into Next, it was treated with RA for caudalization and Pur (SHH signaling agonist) for ventralization. A robust population of OLIG2+ pMN was isolated and discontinued 12 days after differentiation with iPSCs without intraneuronal precursors labeled NKX2.2. For terminal differentiation, OLIG2+ pMNs were treated with RA and Pur suspensions to induce differentiation into HB9+ and ISL1+ mature MNs, and then further differentiated into mature ChAT+ and SMI32+ MNs for 10 days in the presence of compound E (FIG. 3b ). In addition, the ratio of OLIG2+ and ChAT+ cells of differentiated pMNs and MNs maintained at least 75.6% of OLIG2+ and 75% of ChAT+ in WT and SMA groups, respectively (Fig. 3c). These data imply that there is no significant difference in the likelihood of differentiation between WT and SMA groups. Importantly, most pMNs derived from WT iPSCs and SMA iPSCs had high proliferative properties as indicated by Ki67 expression, and pMNs were expanded to at least 5 passages with strong OLIG2 expression (Fig. 3d). In addition, OLIG2+ pMNs can be frozen and thawed and finally differentiated into mature ChAT+ MNs as needed.

During pMN and MN differentiation, cells differentiated from SMA iPSCs showed different and equal defect properties compared to WT cells. First, as a result of RT-PCR analysis, SMA iPSC-pMNs and SMA iPSC-MNs had lower levels of SMN-FL transcripts, including SMN1 and SMN2, and SMN-FL band intensity was compared with WT iPSC-pMNs. It was 21.54% in 1-pMNs and 26.55% in SMA#2-pMN, and 33.98% in SMA#1-MN and 24.44% in SMA#2-MN, respectively, compared to WT iPSC-MN, respectively. It showed a higher level of SMNΔ7 transcription (FIG. 4A ).

Second, at the protein level, the expression of SMN-FL protein in SMA iPSC-pMNs and SMA iPSC-MNs was significantly reduced. That is, when compared with the WT pMN control group, SMA#1-pMNs decreased by 16.82% and SMA#2-pMNs by 35.19%, and when compared with the WT MN control group, SMA#1-MN decreased by 27.73% and SMA#2-MN. It was confirmed through Western blotting (Fig. 4b) and immunocytochemistry (Fig. 4c) results that each decreased by 30.27%. Nuclear gem body was hardly present in SMA iPSC-pMNs and SMA iPSC-MNs (Fig. 4c). These results indicate a reduced level of functional SMN-FL protein in SMA iPSC-pMNs and SMA iPSC-MNs.

Third, as reported by other groups than before, increased apoptosis was observed in SMA cells compared to WT cells (1.59 times in SMA#1-pMNs, 1.77 times in SMA#2-pMNs, SMA# respectively. 1.71 fold in 1-MNs, 1.76 fold in SMA#2-MNs) (Figure 4d).

Fourth, it was confirmed that the neurites in SMA iPSC-MN were significantly shorter compared to WT#1 iPSC-MNs, similar to the previously reported results (Fig. 4e).

In addition, when WT and SMA iPSC-MNs differentiated for NMJ formation were cultured with muscle cells differentiated from mouse C2C12 cells, aggregated α-bungarotoxin (α-BTX) acetylcholine receptors (AChR) on myotubes were derived from WT iPSC. It was found to overlap with the neurite within the MNs, suggesting the formation of NMJ. However, AChR clustering in myotubes cultured with SMA iPSC-derived MN was markedly impaired (results not shown).

In addition, the present inventors analyzed whether the final differentiated MNs in WT and SMA iPSCs have functional membrane properties using electrophysiological analysis. When -0.1 nA to 0.2 nA was injected into the MN with a current of 0.02 nA, AP was generated from MN to 0.2 nA induced in WT iPSC (Vm> 0 mV). However, multiple APs were induced in SMA iPSC-derived MNs (results not shown). Mean Na+ currents in control MNs were 54.8 ± 6.4 pA/pF for WT#1 iPSC-MNs and -53.7 ± 8.3 pA/pF for WT#2 iPSC-MNs, and 74.3 ± 14.7 pA in SMA#1 iPSC-MNs. /pF and SMA#2 iPSC-MNs were 85.2 ± 10.8 pA/pF. Thus, these data demonstrate that SMA MNs are more easily excited than WT MNs with higher AP generation and INa current density, as reported previously. These data imply that both SMA iPSC-pMN and SMA iPSC-MN in our culture system are useful for analyzing SMA pathology and can be applied to in vitro disease models. SMA iPSC-pMNs were chosen for later experiments due to advantages such as a more marked reduction in SMN-FL transcripts and proteins and the ability to quickly yield sufficient amounts to perform cell culture based assays.

5. Cell damage in SMA iPSC-pMNs caused by SMN2 splicing defect was improved by Ligosertip treatment

Candidate Ligosertips selected in the primary screening using the SMN2-Luc stable cell line showed significant splicing corrective activity against SMN2. Therefore, the present inventors tested whether igosertip can alleviate the defective phenotype by inhibiting splicing of SMN2 exon 7 in the SMA iPSC-pMNs, which can mimic the disease phenotype in vitro.

As a result, Rigosertip increased SMN-FL mRNA levels in SMA iPSC-pMNs 72 hours after treatment (FIG. 5A). As a result of qRT-PCR analysis using forward primers spanning exons 7 and 8 and reverse primers for exon 8, SMA#1 iPSC-pMNs and SMA#2 iPSC-pMNs had the amount of SMN-FL transcript WT#1, respectively. Compared to iPSC-pMNs, it was lower at 74.21% and 50.42% (Figure 5b). There were no significant differences between the WT controls.

Rigosertip increased the mRNA expression of SMN-FL by including exon 7 by 2.78 and 1.46 times in SMA#1 iPSC-pMNs and SMA#2 iPSC-pMNs, respectively. Ligosertip treatment increased the amount of SMN protein in patient-induced pMN, as determined by Western blotting analysis (Fig. 5c) and immunocytochemistry (Fig. 5d), and restores the formation of gem body in SMA iPSC-pMNs. (Fig. 5d).

To confirm the protective role of Rigosertip in SMA, the effect of Rigosertip on the apoptosis of MN derived from SMA iPSC was investigated. The ratio of dead cells in SMA iPSC-pMN was significantly reduced by the treatment with igosertip (Fig. 5e). Therefore, in order to understand the mechanism of cell death by SMN reduction, MitoSOX Red, an indicator of mitochondrial superoxide, was used to compare mitochondrial superoxide, which is known to exhibit functional defects of spinal cord MN in SMA iPSC-pMNs. As a result, significantly more MitoSOX+ cells were observed in WT iPSC-pMNs than in SMA iPSC-pMNs (Fig. 5f). In the SMA iPSC-pMN-treated group, the production of MitoSOX+ cells was reduced to a level similar to that of the WT control group (FIG. 5F). Moreover, Rigosertip treatment prevented the reduction of neurite growth due to SMN deficiency in terminally differentiated SMA iPSC-MNs, which showed a phenotype closer to differentiated WT iPSC-MNs (FIG. 5G ). These results suggest that the SMN2 splicing regulator Rigosertip regulates SMN2 splicing in favor of exon 7 inclusion, thereby increasing the production of SMN protein in the in vitro human SMA model and further enhancing the apoptosis induced by mitochondrial oxidative stress. It suggests that it can be improved.

6. Intraperitoneal administration of Rigosertip increases the expression level of SMN protein in the spinal cord of SMA mice.

To investigate whether Rigosertip increases the amount of SMN protein in vivo, Rigosertip was administered intraperitoneally to SMNΔ7 transgenic mice on the 3rd day after birth for 8 days. SMNΔ7 transgenic mice are the most widely used SMA mouse model that exhibits a severe type of SMA and survives only for 2 weeks. 5 hours after the final administration of Rigosertip, a spinal cord extract was prepared and quantitative Western blotting analysis was performed with SMN antibody. It was found that the amount of SMN protein in the spinal cord of SMA mice treated with igosertip increased 2.13 times as compared to that of SMA mice (FIGS. 6A and 6B ). In addition, the spinal cord tissue sections of the same mouse were evaluated histologically. The SMN protein was highly observed in the spinal cord tissue of heterozygous mice, but the SMN protein was very weakly expressed in the spinal cord of SMA mice (Fig. 6c). As with the Western blotting analysis, the SMN+ cell population in the spinal cord increased by 3.18 times compared to the SMA mice in the group of mice treated with Rigosertip, indicating the in vivo efficacy of the Rigosertip (FIGS. 6C and 6D ).

According to the present invention, Rigosertip represented by Chemical Formula 1 exhibits an effect of correcting splicing defects of SMN2 pre-mRNA, thereby increasing the expression of SMN protein, and exhibiting the effect of improving abnormal lesions related to disease It can be very useful in the development of therapeutic agents for neuromuscular diseases including muscular dystrophy, so it has excellent industrial applicability.

Claims (6)

Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS), Duchenne Muscular Dystrophy, or muscle tone containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient A pharmaceutical composition for preventing or treating sexual dystrophy (Mytonic Dystrophy).
[Formula 1]

delete delete The pharmaceutical composition of claim 1, wherein the compound increases a survival motor neuron (SMN) protein.
The pharmaceutical composition according to claim 4, wherein the SMN protein increase is due to the correction of splicing defects of SMN2 pre-mRNA.
Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS), Duchenne Muscular Dystrophy, or muscle tone containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient Food composition for preventing or improving sexual dystrophy (Mytonic Dystrophy).
[Formula 1]

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