CN115429796A - Liluzole targeted treatment of Wolfram syndrome - Google Patents

Abstract

The invention relates to the field of medicines, in particular to application of riluzole in preparation of medicines or kits. The present invention provides the use of riluzole in the manufacture of a medicament or kit for: 1) Treatment ofWFS1Diseases associated with genetic abnormalities; or, 2) treating Wolfram syndrome. The present invention is directed toWFS1The research and development of medicines are carried out on the influence of the deficiency on astrocytes and the influence on neurons, and the treatment effect of riluzole on neuron damage caused by abnormal glutamate metabolism of astrocytes in Wolfram syndrome is unexpectedly found, so that the new application of riluzole in preparing medicines or kits is provided.

Description

Liluzole targeted treatment of Wolfram syndrome

Technical Field

The invention relates to the field of medicines, in particular to application of riluzole in preparation of medicines or kits.

Background

Wolfram syndrome is an autosomal recessive genetic disease caused by a loss-of-function mutation in the WFS1 gene. Wolfram syndrome is characterized by juvenile onset diabetes, diabetes insipidus, bilateral optic atrophy, deafness, and a wide range of neurological and psychiatric manifestations including brainstem and cerebellar atrophy, peripheral neuropathy, epilepsy, cognitive decline, depression, and anxiety, with the majority of patients dying from brainstem atrophy and neuropathy-induced respiratory failure. Molecular genetic studies have shown that 90% of WS patients have a WFS1 gene loss of function mutation, WFS1 encodes the transmembrane protein Wolframin (WFS 1) located on the ER membrane, with higher expression in brain, heart and pancreas. The morbidity and mortality of Wolfram syndrome increases with the age of the patient. The average life expectancy of patients with WS is 30 years, and no specific and effective treatment is currently available, and effective treatment of WS, particularly neuropathy, is urgently needed.

According to the existing research reports, the pathogenic mechanism of Wolfram syndrome is mainly the following problems caused by WFS1 gene mutation: high levels of endoplasmic reticulum sustained stress cause unfolded protein responses, imbalances in endoplasmic reticulum calcium ion homeostasis, mitochondrial dysfunction, and the like. In view of the various pathogenic mechanisms, researchers have also sought drug targets from different aspects in the drug development of Wolfram syndrome. A common molecular feature of Wolfram syndrome is structural mutation of the WFS1 protein resulting from mutations in the WFS1 allele, thereby causing endoplasmic reticulum stress in the cell. Researchers have tested chemical chaperones associated with optimized mutant WFS1 protein structures, such as 4-phenylbutyric acid (4-phenylbutyric acid) and taurodeoxycholic acid (Tauroursodeoxycholic acid), for this property, to restore or stabilize the native conformation of the mutant WFS1 protein to relieve endoplasmic reticulum stress and thereby reduce protein aggregation. Aiming at high-level endoplasmic reticulum stress in Wolfram syndrome, researchers find that Valproic acid (Valproic acid) can induce WFS1 expression and regulate endoplasmic reticulum stress response and provide protection effect on the occurrence of apoptosis, and the medicine is subjected to a2 nd-stage double-blind test; glucagon-like peptide receptor agonists (GLP-1 receptor agonists) have been found to inhibit apoptosis in rodent and cellular models. With respect to the imbalance in the homeostasis of the endoplasmic reticulum calcium ions, researchers have found that the FDA approved drug Dantrolene sodium (Dantrolene sodium) for the treatment of malignant hyperthermia and muscle spasm can act as an endoplasmic reticulum calcium ion stabilizer by targeting the endoplasmic reticulum calcium ion transporter. Dantrolene sodium was found to inhibit apoptosis and dysfunction in neurons and beta cells in the Wolfram syndrome mouse model and in the Induced Pluripotent Stem Cell (iPSCs) model, and this drug is currently undergoing phase 2 clinical trials to evaluate its efficacy, safety and tolerability in children and adults. There is no relevant drug research for mitochondrial dysfunction in Wolfram syndrome, and some mitochondrial modulators that restore mitochondrial function can be further studied. Meanwhile, from the viewpoint of gene therapy and cell replacement therapy, studies have been conducted to explore the possibility of performing replacement therapy for repairing mutant cells or regenerating normal cells of patients in the future. In summary, the existing drug research mainly aims at the defects caused by the endoplasmic reticulum stress of cells and the steady state unbalance of calcium ions of the endoplasmic reticulum caused by WFS1 gene mutation.

Astrocytes have long been considered as an important cell type, accounting for nearly half of the brain cells, providing the necessary support for neurons. In recent years, studies have found that astrocytes are closely linked to the pathogenic mechanisms associated with various neurodevelopmental and neurodegenerative diseases, and that dysfunctional astrocytes lead to neuronal apoptosis, reduced axon and dendrite growth, and impaired synapse formation. Among them, an imbalance in astrocytes regulating glutamate homeostasis is a central feature of various diseases. In Alzheimer's disease, amyloid beta induces glutamate release from neurons and astrocytes in vitro, inhibiting GLT-1 and GLAST levels in astrocytes, resulting in decreased glutamate uptake by astrocytes. Down syndrome patient-specific iPSCs-derived astrocytes induce neuronal apoptosis and fail to promote neuronal maturation and synapse formation. Knocking down wfs1 in drosophila neurons and glial cells can lead to premature death of drosophila and significantly exacerbate behavioral deficits and neurodegenerative diseases of drosophila, which indicates that wfs1 has an important function in both cell types. At present, the role and function of WFS1 in astrocytes is not clear, and the lack of influence on astrocytes and its influence on neurons on WFS1 is unknown. Therefore, we will use human embryonic stem cells to study the role of WFS1 in astrocytes and target the effects of astrocytes on neurons, for the first time conducting related drug development.

Currently, there is no specific and effective treatment for the neuropathy of Wolfram syndrome, and the redevelopment of drugs that have been approved by regulatory agencies has great advantages in terms of safety, tolerability, and adverse effects, most often the most rapid and effective way to develop Wolfram syndrome drugs.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of riluzole in the manufacture of a medicament or a kit for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides the use of riluzole for the preparation of a medicament or a kit for:

1) Treating diseases related to WFS1 gene abnormality; or the like, or, alternatively,

2) Treating Wolfram syndrome.

In some embodiments of the invention, the riluzole is a single active ingredient.

In some embodiments of the invention, the WFS1 gene abnormality is low expression of WFS 1.

In some embodiments of the invention, the medicament or kit is used for treating diseases related to abnormal glutamate uptake associated with WFS1 gene abnormality.

In some embodiments of the invention, the medicament or kit is for treating Wolfram syndrome associated with abnormal glutamate uptake.

In some embodiments of the invention, the glutamate uptake abnormality is an increase in glutamate levels.

In some embodiments of the invention, the glutamate uptake abnormality is a glutamate uptake abnormality caused by an abnormality in the GLT-1 gene.

In some embodiments of the invention, the GLT-1 gene abnormality is low expression of GLT-1.

In some embodiments of the invention, the medicament or the kit is used for treating a WFS1 gene abnormality related disease related to neuronal damage, wherein the neuronal damage is a morphological structure defect and/or neuronal apoptosis of neurons.

In some embodiments of the invention, the medicament or kit is for treating Wolfram syndrome associated with neuronal damage that is a morphological structural defect of a neuron and/or neuronal apoptosis.

Drawings

FIG. 1 is a graph showing the comparison between the length of dendrites and the number of branches of neurons in the WFS1 knock-out group and the control group in example 3 of the present invention.

FIG. 2 is a graph showing the comparison of the number of dendritic spines of neurons between the WFS1 knock-out group and the control group in example 3 of the present invention.

FIG. 3 is a graph showing the comparison of the neuron numbers between the WFS1 knock-out group and the control group in example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

For the sake of brevity, only some numerical ranges are explicitly disclosed herein. However, any lower limit may be combined with any upper limit to form a range not explicitly recited, and any lower limit may be combined with other lower limits to form a range not explicitly recited, as well as any upper limit may be combined with any other upper limit to form a range not explicitly recited. Also, although not explicitly recited, each point or individual value between endpoints of a range is encompassed within the range. Thus, each point or individual value can form a range not explicitly recited as its own lower or upper limit in combination with any other point or individual value or in combination with other lower or upper limits.

In the description herein, it is to be noted that, unless otherwise specified, "above" and "below" are inclusive, and "one or more" of "plural" means two or more.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. At various points throughout this application, guidance is provided through a series of embodiments that can be used in various combinations. In various embodiments, the list is provided as a representative group only and should not be construed as an exhaustive list.

The invention provides in a first aspect the use of riluzole in the manufacture of a medicament or kit for the treatment of a disease associated with WFS1 gene abnormalities and/or Wolfram syndrome. After the WFS1 gene was knocked out in human embryonic stem cells, WFS1 gene-knocked out embryonic stem cells were induced to differentiate into WFS 1-knocked out Neural Precursor Cells (NPCs), neurons (neurones) and astrocytes (astrocytes) by in vitro induced differentiation. The present inventors found that astrocytes (WFS 1) were knocked out when neurons (WT neurons) and WFS1 were present in the control group ^-/- astrocytes) and causing obvious defects on the structure of the neuron, and the WFS1 knockout astrocyte can generate impaired glutamate homeostasis, but when the cells exist in riluzole, the situation that the apoptosis level of the astrocyte is reversed and the neuron damage is also improved, thereby proving that the riluzole can be used for treating WFS1 gene abnormality related diseases and/or treating Wolfram syndrome.

Riluzole (CAS number: 1744-22-5, molecular formula: C) ₈ H ₅ F ₃ N ₂ OS, 2-amino-6-trifluoromethoxybenzothiazole), the chemical structure of which is shown below:

riluzole is an approved drug that can be used to treat ALS.

In the medicine or the kit provided by the invention, riluzole can be used as a single effective component, and can also be combined with other active components to be jointly used for treating WFS1 gene abnormality related diseases and/or Wolfram syndrome.

In the medicine or the kit provided by the invention, WFS1 gene abnormality is usually expressed as low expression of WFS 1. WFS1 low expression may refer to the loss of WFS1 expression, or the expression level of WFS1 is below a certain level, either at the molecular level or at the protein level. For example, the WFS1 under-expression may be an expression of mRNA of WFS1 that is not clearly detectable in neural precursor cells, neurons, or astrocytes, the WFS1 under-expression may be an expression of protein of WFS1 that is not clearly detectable in the above-mentioned cells, the WFS1 under-expression may be an expression level of mRNA of WFS1 that is significantly lower than that of wild-type-related cells, and the WFS1 under-expression may be an expression level of protein of WFS1 that is significantly lower than that of wild-type-related cells.

In the medicament or the kit provided by the invention, the WFS1 gene abnormality related diseases and/or Wolfram syndrome are generally related to glutamate uptake abnormality. The results show that the glutamic acid homeostasis of the WFS1 knockout astrocyte is damaged due to the influence of glutamate transport, so that the WFS1 gene abnormality related diseases and/or Wolfram syndrome are generally related to glutamate uptake abnormality.

In the medicine or the kit provided by the invention, the WFS1 gene abnormality related diseases and/or Wolfram syndrome are generally related to neuron damage. When the neurons of the control group and the WFS1 knockout astrocyte are co-cultured, the survival rate of the neurons is obviously reduced, which indicates that the WFS1 knockout astrocyte can cause the apoptosis of the neurons. Meanwhile, the inventor also finds that after the neurons of the control group and the WFS1 knockout astrocyte are co-cultured, the total dendritic length and the dendritic branch number of the neurons of the control group are obviously reduced, the density of dendritic spines is obviously increased, and the results show that the morphological structure of the neurons can be obviously deficient by the WFS1 knockout astrocyte. However, the inventor finds that riluzole can reverse the reduction of the length and branch of the neuron dendrites and the damage of synapse formation caused by the WFS1 knockout astrocyte, and can improve the survival rate of the neuron, thereby proving that riluzole can be used for treating diseases related to the WFS1 gene abnormality and/or Wolfram syndrome related to the neuron damage.

In a second aspect, the present invention provides a composition comprising riluzole for: 1) Treating diseases related to WFS1 gene abnormality; or, 2) treating Wolfram syndrome.

In a third aspect, the invention provides a method of modulation that can be used to modulate (e.g., ameliorate, reverse, etc.) neuronal damage (e.g., a defect in the morphological structure of a neuron, apoptosis of a neuron, etc.) in an individual or tissue. The regulation method may include, for example: administering to the individual or cell an effective amount of riluzole, or a composition as provided by the second aspect of the invention.

In a fourth aspect, the invention provides a method of treatment comprising: administering to the subject a therapeutically effective amount of riluzole, or a composition provided by the second aspect of the invention.

In the present invention, "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the formulation, kit or combined formulation.

In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is capable of achieving the effect of treating the diseases as listed above.

The invention carries out drug research and development aiming at the influence of WFS1 deletion on astrocytes and the influence of the astrocytes on neurons, and unexpectedly discovers the treatment effect of riluzole on neuron damage caused by abnormal glutamate metabolism of astrocytes in Wolfram syndrome, thereby providing a new application of riluzole in preparing drugs or kits.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Unless otherwise indicated, the methods of testing, methods of preparation, and methods of preparation disclosed herein employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature and are described in particular in Sambrook et al, molecular CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHOD IN ENZYMOLOGY, vol.304, chromatin (P.M. Wassarman and A.P. Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.

Example 1

Construction of WFS1 Gene knockout human embryonic stem cell line

1. Plasmid construction

a. Plasmid design and sgRNA screening

Using tool web site (http://crispr.mit.edu/) 2 sgrnas are designed for the first coding exon near the 5' end of the human WFS1 gene, and are inserted into a vector carrying a Cas9 protein gene and a puromycin resistance gene (P2U 6) in a PCR and enzyme digestion connection manner. According to the scoring height of the sgRNA targeting position and the off-target rate, the following sgRNAs are screened out:

sgRNA1：ATTGAGTCGGGAACGCGCC(SEQ ID NO.1)

sgRNA2：GCGGAGCAGTGTTGGAGTC(SEQ ID NO.2)

the plasmid sequence is as follows:

aattgaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgATTGAGTCGGGAACGCGCCGTTTTtctagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgtttaaacacacggttcctagtatcggtccgaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgGACTCCAACACTGCTCCGCgtgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggcccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcgtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcgggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccatctccagcctcggggctgtccgcagggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagtgcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattctctagagatatcgctagcaccatggactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagatggcccccaagaagaagaggaaggtgggcattcaccgcggggtacccatggacaagaagtactccattgggctcgatatcggcacaaacagcgtcggctgggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagaccatcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaagactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcggctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagtcttcacgagcacatcgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctcaaagatgattctattgataataaagtgttgacaagatccgataaaaatagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaagtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctggtctcagatttcagaaaggactttcagttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaaggaactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatgagcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagacagcagggctgaccccaagaagaagaggaaggtgaccggtgcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggaccgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgatgtacaagtaactgcagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctagggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatcgatctcccgatcccctagggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgac(SEQ ID NO.3)

b. plasmid transformation and sequencing

The chemically competent cell DH 5. Alpha. For cloning was thawed on ice in advance, 10. Mu.L of the ligation product was added to 100. Mu.L of the competent cell, and the tube wall was flicked and mixed well, and left on ice for 25min. After heat shock in a 42 ℃ water bath for 45s, the mixture was cooled for 2min on ice. 900. Mu.L of LB liquid medium without antibiotics (operating in a clean bench) was added and shaken at 37 ℃ for 1h (rotation speed 200-250 rpm). The LB solid medium plate with the corresponding resistance is preheated in an incubator at 37 ℃, centrifuged at 5000rpm for 1min, 900. Mu.L of supernatant is discarded, about 100. Mu.L of cell is resuspended and then added to the plate, the plate is coated with coating beads, and inverted culture is carried out in the incubator at 37 ℃ for 12 to 16h. Then, the single clone is selected and sent to a sequencing company for sequencing, and plasmids which are successfully connected are selected for large-scale plasmid extraction.

2. The culture conditions of the embryonic stem cells are as follows:

a. 1; washed twice with DMEM/F12 (Invitrogen, # 11330-082) medium.

b. Human embryonic Stem cells (HuES 8) were seeded, cultured using commercial mTeSR medium (Stem Cell, # 85850), and 1000X diluted Y27632 (Stem Cell, # 72304) was added on the first day of culture.

c. The cells were cultured in an incubator containing 5% CO2 at 37 ℃ and the medium was removed Y27632 after the next day of complete exchange. Cells were then passaged every 3-4 days.

3. Plasmid electrotransfer and cell line screening

a. Culturing embryonic stem cells to about 80% density, digesting with TrypLE, and filtering the cells with a 40 μm cell filter screen to disperse the cells into single cells;

b. taking cells with the total amount of about 0.5M to ensure that plasmids carrying sgRNA and Cas9 protein genes enter the cells in an electrotransfection mode (see SEQ ID NO. 3);

c. after electroporation, embryonic stem cells were resuspended in mTeSR medium and plated on 3.5cm cell culture dishes for two days, after which cell selection was performed by adding 500ng/mL puromycin. After 48-60h of culture, removing puromycin, and changing culture medium every day until the clone grows to 30-50 μ M;

d. and (3) selecting the monoclonal cells, transferring the monoclonal cells into a 96-well plate for amplification culture, cracking a part of the cells by using RIPA lysate, then extracting cell genomes, and verifying whether the WFS1 gene is knocked out or not by sequencing. Sequencing results show that 49bp (19-67 del) of the first coding exon of WFS1 is knocked out, so that a stop codon is advanced, and the gene is stopped to express.

4. In vitro induced differentiation of embryonic stem cells into Neural Precursor Cells (NPCs), neurons (Neurons) and Astrocytes (Astrocytes)

After the WFS1 gene is knocked out in a human embryonic stem cell line, an NPCs-rich area containing a rose-like structure is manually picked for amplification culture by an induction method of an Embryoid Body (EB), the characteristics of NPCs are identified by immunofluorescence staining stage-specific markers SOX2, nestin and Musashi of the NPCs, and corresponding NPCs are established for subsequent experiments. Further withdrawal of bFGF from N2/B27 medium allowed NPCs to differentiate towards Neurons. Based on the natural process of differentiation and maturation of astrocytes in vivo, NPCs are induced to generate Neurospheres (Neurospheres) firstly, and the Neurospheres can migrate and grow to generate GFAP positive astrocytes along with the extension of differentiation time. Experimental results show that the experimental method disclosed above can be used for obtaining WFS1 knockout neural precursor cells, neurons and astrocytes.

Example 2

Effect of WFS1 knock-out on astrocytes and its Effect on neurons

To investigate the effect of WFS1 knockout astrocytes on Neurons, experimental validation was performed using a neuron and astrocyte co-culture system (Neurons-astrocytes co-culture). The specific scheme is as follows: firstly, NPCs express GFP started by a neuron specific promoter (Synapsin 1) through infecting lentiviruses, and then the NPCs infected by the lentiviruses are differentiated to neurons for 3 weeks to obtain Syn:: GFP marks positive neurons. And simultaneously, according to a well established differentiation system, differentiating the NPCs of the WFS1 knockout group and the NPCs of the control group into the astrocytes. Then, the WFS1 knockout group and the control group of astrocytes (cell number 3X 10) formed by induced differentiation were used ⁵ ) Syn with differentiation for 3 weeks: GFP-labeled-Positive neurons (cell count 2X 10) ⁵ ) Co-culture was performed for 3 days, and on the third day, the cells were fixed with 4% PFA in preparation for the subsequent experiments. Fixed cells are subjected to immunofluorescence staining of a neuron marker MAP2 and a presynaptic marker Syn1, then the number and the length of dendritic branches of Syn:ofneurons positive to GFP and MAP2 are counted, meanwhile, the density of Syn1 on the dendritic branches of the neurons positive to Syn:: GFP and MAP2 is counted, and the neurons are compared with a WFS1 knockout group and a control group respectivelyDendritic growth and synapse formation differences following group astrocyte co-culture. Meanwhile, we counted the number of neurons positive for GFP and MAP2 in Syn, and compared the survival rate of neurons after astrocyte co-culture with WFS1 knock-out and control groups.

The experimental result shows that compared with the control group, after the neurons are co-cultured with astrocytes of the WFS1 knockout group, the dendritic length and the branch number of the neurons are obviously reduced, the dendritic spine number of each neuron is obviously increased, and the dendritic growth and synapse formation of the neurons are influenced. Meanwhile, after the cells are co-cultured with the WFS1 knockout astrocytes, the number of GFP and MAP2 positive neurons is obviously reduced, which indicates that the survival rate of the neurons is also influenced.

Further to investigate how WFS1 knockout astrocytes have an effect on neurons, a series of experiments were performed on WFS1 knockout astrocytes. First, we planted astrocytes of WFS1 knockout group and control group induced by differentiation with the same number of cells (3X 10) ⁵ ) After two days of culture, fresh medium was changed and 100. Mu.l of the medium was collected at 48 hours and 96 hours, respectively, followed by measurement of glutamic acid level. Meanwhile, we collected cell samples of astrocytes of the WFS1 knock-out group and the control group, and analyzed the expression of mRNA level of transporter associated with glutamate uptake of astrocytes by extracting RNA. Experimental results show that compared with a control group, the glutamic acid level in the astrocytes of the WFS1 knockout group is obviously increased, which indicates that the glutamic acid metabolism is abnormal, and meanwhile, the mRNA level of a glutamic acid transporter GLT-1 is obviously reduced in the astrocytes of the WFS1 knockout group, which indicates that the uptake of glutamic acid is problematic.

In conclusion, the WFS1 knockout astrocyte has an effect on dendritic growth, synapse formation and survival of neurons, and the action mechanism of the astrocyte is probably glutamate metabolism abnormality caused by the decreased expression level of GLT-1, a glutamate transporter in the WFS1 knockout astrocyte.

Example 3

Therapeutic effect of riluzole on the effects of WFS1 knockout astrocytes on neurons

To investigate the therapeutic effect of riluzole on the neuronal effect of WFS1 knockout astrocytes, astrocytes (cell number 3X 10) were first induced to differentiate between WFS1 knockout and control groups ⁵ ) Syn with differentiation for 3 weeks: GFP-labeled-Positive neurons (cell count 2X 10) ⁵ ) In the co-culture system, drug gradient experiments are carried out by adding riluzole with the concentration of 5-100 mu M for 48h, and the situation that the cells have severe apoptosis at the drug concentration of more than 10 mu M is found, and then the concentration is reduced to 5 mu M and 10 mu M. After comparing the two concentrations of 5 μ M and 10 μ M, the cell state of 5 μ M was found to be good, and the experimental index of treatment was better, so that the drug concentration in the subsequent experiments was 5 μ M, and the culture time was 48h. Through MAP2 and Syn1 immunofluorescence staining on cells, statistics is carried out on the number and length of dendritic branches of neurons with GFP and MAP2 positive and the density of Syn1 on the dendrites of the neurons with GFP and MAP2 positive, and whether difference exists between dendritic growth and synapse formation of the neurons co-cultured with the WFS1 knockout group astrocytes after the addition of riluzole is compared.

Meanwhile, statistics is carried out on the number of neurons positive for GFP and MAP2 in Syn, and the survival rate of neurons co-cultured with WFS1 knockout astrocytes after adding riluzole is compared.

As shown in fig. 1 and fig. 2, the experimental results show that after riluzole is added, the dendritic length and the number of branches of the neurons co-cultured with the WFS1 knockout astrocytes are significantly increased, the number of dendritic spines of each neuron is significantly reduced, and the morphological structure of the neurons co-cultured with the control astrocytes is restored, which indicates that riluzole has a significant therapeutic effect on the defect of the neuronal morphological structure caused by the WFS1 knockout astrocytes.

Meanwhile, as shown in fig. 3, after riluzole is added, the number of neurons co-cultured with WFS1 knockout astrocytes is also significantly increased, which indicates that riluzole has a significant inhibitory effect on neuronal apoptosis caused by WFS1 knockout astrocytes.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.

Sequence listing

<110> university of Tongji

<120> Linuzole Targeted treatment for Wolfram syndrome

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

attgagtcgg gaacgcgcc 19

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcggagcagt gttggagtc 19

<210> 3

<211> 9994

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aattgaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac 60

gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag 120

tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 180

gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt 240

tatatatctt gtggaaagga cgaaacaccg attgagtcgg gaacgcgccg tttttctagt 300

tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 360

caccgagtcg gtgctttttt tgtttaaaca cacggttcct agtatcggtc cgaaggtcgg 420

gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 480

tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 540

acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 600

ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 660

gaaaggacga aacaccggac tccaacactg ctccgcgtgt tttagagcta gaaatagcaa 720

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 780

tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 840

tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 900

accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 960

aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc 1020

agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 1080

gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 1140

ctacgtatta gtcatcgcta ttaccatggg tcgaggtgag ccccacgttc tgcttcactc 1200

tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt 1260

gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag 1320

gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 1380

aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 1440

cgggcgggag tcgctgcgtt gccttcgccc cgtgccccgc tccgcgccgc ctcgcgccgc 1500

ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct 1560

cctccgggct gtaattagcg cttggtttaa tgacggctcg tttcttttct gtggctgcgt 1620

gaaagcctta aagggctccg ggagggccct ttgtgcgggg gggagcggct cggggggtgc 1680

gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggcccg cgctgcccgg cggctgtgag 1740

cgctgcgggc gcggcgcggg gctttgtgcg ctccgcgtgt gcgcgagggg agcgcggccg 1800

ggggcggtgc cccgcggtgc gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1860

gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc 1920

acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc 1980

gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg 2040

cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg 2100

gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg 2160

cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca 2220

ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg 2280

agggccttcg tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgtc 2340

cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 2400

tgaccggcgg ctctagtgcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 2460

tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttctctagag 2520

atatcgctag caccatggac tacaaagacc atgacggtga ttataaagat catgacatcg 2580

attacaagga tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggcattcacc 2640

gcggggtacc catggacaag aagtactcca ttgggctcga tatcggcaca aacagcgtcg 2700

gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa gttctgggca 2760

ataccgatcg ccacagcata aagaagaacc tcattggcgc cctcctgttc gactccgggg 2820

agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga 2880

atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaaggtg gatgactctt 2940

tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac gagcgccacc 3000

caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca accatatatc 3060

atctgaggaa gaagcttgta gacagtactg ataaggctga cttgcggttg atctatctcg 3120

cgctggcgca tatgatcaaa tttcggggac acttcctcat cgagggggac ctgaacccag 3180

acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat cagcttttcg 3240

aagagaaccc gatcaacgca tccggagttg acgccaaagc aatcctgagc gctaggctgt 3300

ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag aagaacggcc 3360

tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa tctaacttcg 3420

acctggccga agatgccaag cttcaactga gcaaagacac ctacgatgat gatctcgaca 3480

atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca aagaacctgt 3540

cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc aaagctccgc 3600

tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact ttgctgaagg 3660

cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat cagtctaaaa 3720

atggctacgc cggatacatt gacggcggag caagccagga ggaattttac aaatttatta 3780

agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag 3840

atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag attcacctgg 3900

gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg aaagataaca 3960

gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc cccctcgccc 4020

ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc actccctgga 4080

acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa aggatgacta 4140

actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagt 4200

acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc 4260

cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc aagacgaacc 4320

ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa tgtttcgact 4380

ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg tatcacgatc 4440

tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag gacattcttg 4500

aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa gaacgcttga 4560

aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagagg cgccgatata 4620

caggatgggg gcggctgtca agaaaactga tcaatgggat ccgagacaag cagagtggaa 4680

agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc atgcagttga 4740

tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt tctggccagg 4800

gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc aaaaagggaa 4860

tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg cataagcccg 4920

agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga cagaagaaca 4980

gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc caaatcctta 5040

aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg tactacctgc 5100

agaacggcag ggacatgtac gtggatcagg aactggacat caatcggctc tccgactacg 5160

acgtggatca tatcgtgccc cagtcttttc tcaaagatga ttctattgat aataaagtgt 5220

tgacaagatc cgataaaaat agagggaaga gtgataacgt cccctcagaa gaagttgtca 5280

agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca caacggaagt 5340

tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa gccggcttca 5400

tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgatt 5460

cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta 5520

ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat aaggtgagag 5580

agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta ggcactgcac 5640

ttatcaaaaa atatcccaag cttgaatctg aatttgttta cggagactat aaagtgtacg 5700

atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc gctaagtact 5760

tcttttacag caatattatg aattttttca agaccgagat tacactggcc aatggagaga 5820

ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg 5880

gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa 5940

agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa aggaacagcg 6000

acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga ttcgattctc 6060

ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac 6120

tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa 6180

accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagac ctcatcatta 6240

agcttcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg 6300

cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt 6360

atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc 6420

tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata agcgaattct 6480

ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct tacaataagc 6540

acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt actctgacca 6600

acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga aagcggtaca 6660

cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatg 6720

aaacaagaat cgacctctct cagctcggtg gagacagcag ggctgacccc aagaagaaga 6780

ggaaggtgac cggtgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga 6840

atcctggacc gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca 6900

gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 6960

atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 7020

ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 7080

cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 7140

tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 7200

ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg 7260

gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 7320

ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 7380

tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 7440

gtgcctgatg tacaagtaac tgcagcgcgg ggatctcatg ctggagttct tcgcccaccc 7500

caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 7560

aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 7620

ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct 7680

gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 7740

aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 7800

actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 7860

cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 7920

gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 7980

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 8040

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 8100

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 8160

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 8220

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 8280

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 8340

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 8400

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 8460

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 8520

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 8580

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 8640

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 8700

gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 8760

ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 8820

ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 8880

tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 8940

accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 9000

atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 9060

cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 9120

tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 9180

tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 9240

gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 9300

agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 9360

aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 9420

gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 9480

tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 9540

gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 9600

tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 9660

aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 9720

catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 9780

acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg 9840

agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata 9900

gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa 9960

aatttaagct acaacaaggc aaggcttgac cgac 9994

Claims (10)

1. Use of riluzole in the manufacture of a medicament or kit for:

1) Treating diseases related to WFS1 gene abnormality; or the like, or a combination thereof,

2) Treating Wolfram syndrome.

2. The use of claim 1, wherein riluzole is a single active ingredient.

3. The use of claim 1 wherein the WFS1 gene abnormality is low expression of WFS 1.

4. The use according to claim 1 wherein the medicament or kit is for the treatment of a disease associated with an abnormal glutamate uptake associated with an abnormal WFS1 gene.

5. The use of claim 1, wherein the medicament or kit is for the treatment of Wolfram syndrome associated with abnormal glutamate uptake.

6. The use of claim 4 or 5, wherein the abnormal glutamate uptake is an elevated glutamate level.

7. The use according to claim 4 or 5, wherein the glutamate uptake abnormality is glutamate uptake abnormality caused by an abnormality of the GLT-1 gene.

8. The use according to claim 7, wherein the GLT-1 gene abnormality is low expression of GLT-1.

9. The use of claim 1, wherein the medicament or kit is for treating a disease associated with WFS1 gene abnormality associated with neuronal damage, said neuronal damage being a morphological structural defect of a neuron and/or neuronal apoptosis.

10. The use of claim 1, wherein the medicament or kit is for the treatment of Wolfram syndrome associated with neuronal damage that is a morphological structural defect of a neuron and/or neuronal apoptosis.

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