WO2024029755A1 - Animal model of charcot-marie-tooth disease - Google Patents

Abstract

The present invention relates to an animal model of Charcot-Marie-Tooth disease caused by K141N or K141T mutations in HSPB8. In this animal model expressing the K141N or K141T mutant HSPB8 protein, symptoms of Charcot-Marie-Tooth disease, such as motor deficit, mitochondrial dysfunction, and reduced levels of mitophagy, were observed, confirming that Charcot-Marie-Tooth disease can be induced by K141N or K141T mutations in the HSPB8 protein. Therefore, the animal model expressing the K141N or K141T mutant HSPB8 protein can be used as an animal model of Charcot-Marie-Tooth disease. The symptoms of Charcot-Marie-Tooth disease were significantly reduced by the expression and activation of PINK1 or Parkin, so that the animal model can be used in therapeutic applications for Charcot-Marie-Tooth disease.

Description

Charcot-Marie-Tooth disease animal model

The present invention relates to an animal model of Charcot-Marie-Tooth disease caused by K141N or K141T mutation of HSPB8.

Hereditary peripheral neuropathy is largely divided into three types: hereditary motor and sensory neuropathy (HMSN), hereditary motor neuropathy (HMN), and hereditary sensory neuropathy (HSN). Among these, hereditary motor and sensory neuropathy accounts for the majority, which is better known as Charcot-MarieTooth disease (CMT). Charcot-Marie-Tooth disease was first known in 1886 by the Frenchmen Charcot and Marie, and the Englishman Tooth, and is commonly called CMT disease, named after the first letters of their names. Charcot-Marie-Tooth (CMT) disease, or hereditary motor and sensory neuropathy (HMSN), is also known as distal hereditary motor neuropathy (dHMN), hereditary sensory neuropathy (HMSN). ; HSN) and Hereditary Neuropathy with a liability to pressure palsy (HNPP), it is a clinically heterogeneous disease. Charcot-Marie-Tooth disease is a general term for all genetic diseases in which motor and sensory nerves are abnormal on nerve conduction tests. The incidence of CMT is one in 2,500 people, which is the highest among rare inherited diseases. In CMT patients, the muscles in the feet and hands gradually atrophy, resulting in weakened strength, deformed foot and hand shapes, facial paralysis, and hearing impairment. Onset usually occurs around the age of 10, but rarely occurs after the age of 30. Depending on the type of genetic mutation, patients' symptoms range from mild, almost normal, to very severe, requiring assistance with walking or relying on a wheelchair. In the past, it was recognized as a relatively simple disease with legs shaped like upside-down champagne bottles due to muscle atrophy in the distal lower extremities, but it is now recognized as a syndrome rather than a single disease. Recently, many new pathogenic mechanisms of CMT have been discovered, which has greatly helped in classifying complex clinical types and genotypes as well as pathophysiological research. During the last research period, more than 40 genetic loci for hereditary motor and sensory neuron diseases were discovered using gene cloning techniques, and more than 20 causative genes were identified. However, many patients with hereditary motor and sensory neuropathy still show no correlation with known loci, so it is reported that there will be more than 50 more causative genes in the future. Depending on the pattern of inheritance, CMT is divided into the myelin defect type (CMT1), which is inherited autosomal dominantly and recessively, the axonal type (CMT2), and the CMTX type, which is inherited via X-chromosome linkage. In the case of CMT type 1, it was named 1A, 1B, 1C, etc. in the order in which genetic mutations were reported. To date, more than 50 causative genes have been reported as the cause of CMT. It is caused by a single gene mutation in the causative gene, and although the severity of symptoms varies, it is inherited according to Mendel's laws. Therefore, the elements that make up various nervous tissues are gradually revealing themselves, and just as there are many types of hereditary muscle diseases, it is assumed that there will be quite a variety of types of hereditary neuropathy. In addition, since genetic defects and onset are directly related, the results of genetic diagnosis of the cause of CMT have the meaning of confirmation rather than a simple tendency or possibility of onset, making it very appropriate to utilize a genetic diagnosis system.

Genetic diagnosis of CMT relies on direct sequencing of the primary causative gene, but despite being a difficult process, it has low isolation efficiency. With the advent of Whole Exome Sequencing (WES) based on Next-generation Sequencing (NGS) technology, genetic research on various diseases, especially heterogeneous diseases such as CMT, is expanding and accelerating. In addition, onapriston, ascorbic acid, and NT-3 (neurotrophin-3) have been reported as reasonable and potential drug treatments for CMT1A, the most common type of hereditary motor sensory neuropathy. . Among the drugs for CMT treatment, there was a report that administration of onapristone, a progesterone receptor antagonist, reduced overexpression of Pmp22 mRNA in transgenic mice and improved the phenotype of hereditary motor-sensory neuropathy without side effects, and in peripheral nerves. Ascorbic acid, an essential substance for myelin formation, improved myelin remodeling and the phenotype of hereditary motor-sensory neuropathy in CMT1A transgenic mice, and neurotrophin-3 (NT-3) improved the phenotype in CMT1A patient groups. It was reported that it improved sensory symptoms by increasing myelinated nerve fibers. Therefore, accurate genetic diagnosis of CMT has made it possible to suppress its onset and provide customized treatment appropriate for the genetic cause, but the development of efficient diagnostic methods for hereditary neurological diseases is still insufficient. In order to provide customized treatment for motor-sensory neuropathy, which has genetically very heterogeneous characteristics, an accurate genetic diagnosis method is first required to be established.

Meanwhile, in the development of new drugs and new treatment technologies, experiments using animal models are an essential step that must precede research on humans. However, compared to the numerous types of human diseases, the types of animal models that mimic them are extremely small, and this acts as a major constraint in the development of new drugs and new treatment technologies. There are various types of diseased animals with different characteristics depending on the method used, and each has different utility depending on the purpose of the experiment. The methods for producing disease model animals known to date can be roughly divided into three types. Traditionally, there has been a method of developing naturally occurring mutant species as disease models, and later, experimental disease-inducing methods such as chemical administration or artificial cell line transplantation have been developed and used. Recently, with the rapid development of molecular genetic methods, the transformation method by gene introduction has been in the spotlight as a powerful technology. In order to meet the diverse needs of research and development of new drugs and new treatments, it is very important to secure disease model production methods using various approaches and establish an effective model animal production system through cooperation between them.

The object of the present invention is to provide a composition for diagnosing Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a diagnostic kit for Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide an animal model for Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a method for providing information necessary to diagnose the possibility of developing Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a screening method for a candidate drug for the treatment of Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a method for diagnosing Charcot-Marie-Tooth disease.

Additionally, an object of the present invention is to provide a method of treating Charcot-Marie-Tooth.

Additionally, an object of the present invention is to provide a diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.

In addition, the object of the present invention is to provide a use for the prevention or treatment of Charcot-Marie-Tooth disease of a vector containing PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same.

In order to solve the above problems, the present invention provides a composition for diagnosing Charcot-Marie-Tooth disease, which includes an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.

Additionally, the present invention provides a kit for diagnosing Charcot-Marie-Tooth disease comprising the composition.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease, comprising PINK1 or Parkin protein, a nucleic acid molecule encoding it, or an activator thereof.

Additionally, the present invention provides a Charcot-Marie-Tooth disease animal model expressing HSPB8 protein containing a K141N or K141T mutation.

Additionally, the present invention provides a method for providing information necessary for diagnosing the possibility of developing Charcot-Marie-Tooth disease, which includes detecting the K141N or K141T mutation of the HSPB8 protein in a sample isolated from a subject.

Additionally, the present invention provides a screening method for a candidate drug for the treatment of Charcot-Marie-Tooth disease.

Additionally, the present invention provides a method for diagnosing Charcot-Marie-Tooth disease, comprising detecting a K141N or K141T mutation in the HSPB8 protein.

In addition, the present invention provides a method for treating Charcot-Marie-Tooth, comprising administering a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same to an individual suffering from Charcot-Marie-Tooth disease. provides.

Additionally, the present invention provides diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting K141N or K141T mutations in the HSPB8 protein.

In addition, the present invention provides the use of a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same for the prevention or treatment of Charcot-Marie-Tooth disease.

In an animal model expressing the K141N or K141T mutant HSPB8 protein of the present invention, symptoms of Charcot-Marie-Tooth disease, such as motor defects, mitochondrial dysfunction, and reduced mitophagy levels, were observed in the K141N or K141T mutation of the HSPB8 protein. Since it has been confirmed that Charcot-Marie-Tooth disease is caused by Since the expression of PINK1 or Parkin and PINK1 activation were significantly reduced, it can be used to treat Charcot-Marie-Tooth disease.

Figure 1 shows the results of HSPB8 expression and lifespan analysis of transgenic Drosophila:

Figure 1A: Results of immunoblot analysis of control Drosophila elav (elav-GAL4), wild-type HSPB8-expressing Drosophila ( elav HSPB8 ^WT ), HSPB8 ^K141T -expressing Drosophila ( elav HSPB8 ^K141T ) and HSPB8 ^K141E -expressing Drosophila ( elav HSPB8 ^K141E );

Figure 1B: Photomicrograph of a 5-day-old fruit fly;

Figure 1c: Lifespan curve of male Drosophila; and

Figure 1D: Lifespan curve of female Drosophila.

Figure 2 is a diagram confirming the occurrence of sensory defects due to HSPB8 mutation:

Figure 2A: Heat nociception assay results of L3 larvae ( ppk ) transgenic with the ppk-GAL4 driver; and

Figure 2b: Result of response delay analysis for thermal nociception in ppk- expressing larvae, HSPB8 ^WT -expressing larvae ( ppk HSPB8 ^WT ), HSPB8 ^K141T -expressing larvae ( ppk HSPB8 ^K141T ) and HSPB8 ^K141E- expressing larvae ( ppk HSPB8 ^K141E ) at 40°C.

Figure 3 is a diagram confirming changes in exercise capacity due to HSPB8 mutation:

elav : control Drosophila;

elav HSPB8 ^WT : Drosophila expressing wild type HSPB8;

elav HSPB8 ^K141T : Drosophila expressing HSPB8 ^K141T ;

elav HSPB8 ^K141E : Drosophila expressing HSPB8 ^K141E ;

Figure 3a and b: Average walking speed of 5-day-old fruit flies (A) and 15-day-old fruit flies (B); and

Figure 3c and d: Movement trajectories of 5-day-old fruit flies (C) and 15-day-old fruit flies (D).

Figure 4 shows the occurrence of mitochondrial dysfunction caused by mutations and the expression of PINK1 and parkin . This is a diagram confirming the recovery of mitochondrial dysfunction by introducing:

elav HSPB8 ^WT : Drosophila expressing wild type HSPB8

elav HSPB8 ^WT : Drosophila expressing wild type HSPB8;

elav HSPB8 ^K141T : Drosophila expressing HSPB8 ^K141T ;

elav HSPB8 ^K141E : Drosophila expressing HSPB8 ^K141E ;

elav HSPB8 ^K141T PINK1 : Drosophila expressing HSPB8 ^K141T and PINK1 ;

elav HSPB8 ^K141E PINK1 : Drosophila expressing HSPB8 ^K141E and PINK1 ;

elav HSPB8 ^K141T Parkin : Drosophila expressing HSPB8 ^K141T and Parkin ;

elav HSPB8 ^K141E Parkin : Drosophila expressing HSPB8 ^K141E and Parkin ;

Figure 4A: Mitochondrial membrane potential (ΔΨ) measured in Drosophila larva VNC;

Figure 4b: mt-Keima fluorescence image of Drosophila larva VNC;

Figure 4C: Quantitative analysis of mitophagy in Drosophila larvae VNC;

Figure 4d: Climbing ability analysis of 15-day-old fruit flies;

Figure 4e: Average walking speed of 15-day-old fruit flies; and

Figure 4f: Movement trajectory of female fruit fly.

Figure 5 is a diagram confirming motor defects caused by motor neuron-specific HSPB8 mutant expression:

D42 : Drosophila expressing motor neuron-specific D42 -GAL4;

D42 HSPB8 ^WT : Drosophila expressing motor neuron-specific HSPB8 ^WT ;

D42 HSPB8 ^K141T : Drosophila expressing motor neuron-specific HSPB8 ^K141T ;

D42 HSPB8 ^K141E : Drosophila expressing motor neuron-specific HSPB8 ^K141E ;

Figure 5A: Average walking speed of 5-day-old fruit flies;

Figure 5b: Average walking speed of 15-day-old fruit flies;

Figure 5c: Movement trajectory of 5-day-old fruit flies; and

Figure 5d: Movement trajectory of 15-day-old fruit flies.

Figure 6 shows 15-day-old male transgenic Drosophila ( elav HSPB8 ^WT , elav HSPB8 ^K141T ) following PINK1 activation by KR administration (1 and 5 mM). and elav HSPB8 ^K141E ), which confirmed the recovery of motor skills:

Figure 6A: Average walking speed of fruit flies;

Figure 6b: Movement trajectory; and

Figure 6c: Climbing ability analysis.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

As used herein, the terms “step of” or “step of” do not mean “step for.”

In this specification, the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more components selected from the group consisting of the components described in the Markushi format expression, It means including one or more selected from the group consisting of.

In this specification, the description of “A and/or B” means “A, B, or A and B.”

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.

In one aspect, the present invention provides a composition for diagnosing Charcot-Marie-Tooth disease (CMT) disease, comprising an agent capable of detecting the K141N or K141T mutation of the small heat shock protein B8 (HSPB8) protein. It's about.

In one embodiment, detecting the K141N or K141T mutation of the HSPB8 protein may be detecting a nucleic acid molecule encoding the HSPB8 protein comprising the K141N or K141T mutation or the HSPB8 protein comprising the K141N or K141T mutation.

In one embodiment, the agent capable of detecting a mutation is an antibody, antibody fragment, aptamer, Affilin, Affibody, Affimer, Affitin, Alphabody, Anticlin, DARpin, Fynomoer, Kunitz domain peptide, avidity multimer, peptidomimetic It may include peptidomimetics, receptors, ligands, or cofactors, and detection of protein mutations can be performed using Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, or immunosorbent assay. It may be performed by one or more methods selected from the group consisting of electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

In one embodiment, the antibody is a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimetic, chimeric antibody, antibody conjugate, human antibody, or humanized antibody. Fragments with immunological activity include Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, and Fv of the antibody. It may be a dimer, a complementarity determining region fragment, or a diabody.

In one embodiment, an agent capable of detecting a mutation may include a primer pair, or probe, or a primer pair and probe that specifically recognizes a nucleic acid molecule encoding the HSPB8 protein, Detection of nucleic acid molecules can be performed using polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, and nuclease protection assay (RNase, S1 nuclease assay). ), in situ hybridization, nucleic acid microarray, Northern blot, DNA chip, multiplex PCR, or ddPCR.

As used herein, the term “detection” or “measurement” means confirming the presence or absence of a detected or measured object.

As used herein, the terms “gene,” “nucleic acid,” “polynucleotide,” or “oligonucleotide” refer to DNA molecules, RNA molecules, or analogs thereof. As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” include, but are not limited to, DNA molecules, such as cDNA, genomic DNA, or synthetic DNA and RNA molecules, such as guide RNA, messenger RNA, or Contains synthetic RNA. Moreover, as used herein, the terms “nucleic acid” and “polynucleotide” include single-stranded and double-stranded forms.

As used in the present invention, the term "primer" is a nucleic acid sequence with a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.

As used in the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. You can check. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to the AR or AR-V7 gene, and the level of gene expression can be diagnosed based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art, so the present invention is not particularly limited thereto.

Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoronucleotide). amidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).

As used in the present invention, the term “antibody” is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the amino acid substitution mutation of HSPB8, which is a marker of the present invention, and the antibody can be produced using a well-known method. This also includes partial peptides that can be made from the above proteins. The form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

In one aspect, the present invention relates to a diagnostic kit for Charcot-Marie-Tooth disease comprising the composition of the present invention.

In one embodiment, the kit may be an RT-PCR kit, a microarray chip kit, or a protein chip kit.

In one aspect, the present invention provides a pharmaceutical for the prevention or treatment of Charcot-Marie-Tooth disease, comprising a vector containing a PINK1 (PTEN-induced putative kinase 1) or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same. It relates to composition.

In one embodiment, the Charcot-Marie-Tooth disease may be Charcot-Marie-Tooth disease caused by a K141N or K141T mutation in the HSPB8 protein.

In one embodiment, the activator of PINK1 may be kinetin riboside (KR) (N6-furfuryl adenine riboside).

In one embodiment, the composition can improve symptoms of motor defects, mitochondrial dysfunction, or reduced mitophagy levels caused by the K141N or K141T mutation of the HSPB8 protein.

As used herein, the term “vector” refers to any nucleic acid containing a competent nucleotide sequence that is inserted into a host cell and recombines with and integrates into the host cell genome, or replicates spontaneously as an episome. These vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.

In the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the above, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., and carriers for parenteral administration, such as water, suitable oils, saline solutions, aqueous glucose and glycols, etc. and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As for other pharmaceutically acceptable carriers, those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention can be formulated in a suitable form according to methods known in the art along with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods, and a representative example of the formulation for parenteral administration is an isotonic aqueous solution or suspension as an injectable formulation. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline solution or buffer solution and formulated for injection. Additionally, dosage forms for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules. The pharmaceutical composition formulated in the manner described above can be administered in an effective amount through various routes including orally, transdermally, subcutaneously, intravenously, or intramuscularly. The term “administration” refers to introducing a predetermined substance into a patient by any appropriate method. This means that the substance can be administered through any general route as long as it can reach the target tissue. In the above, “effective amount” refers to the amount that exhibits a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, gender, weight, sensitivity to the drug, type of current treatment, administration method, target cells, etc., and may vary depending on the field of the art. can be easily determined by experts. Additionally, the pharmaceutical composition of the present invention can be administered in combination with conventional therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. Preferably, considering all of the above factors, the amount that can obtain the maximum effect with the minimum amount without side effects can be administered, more preferably 1 to 10,000 ㎍/kg of body weight/day, and even more preferably 10 to 1,000. The effective dose is ㎎/kg body weight/day and can be administered repeatedly several times a day.

In one aspect, the invention relates to an animal model of Charcot-Marie-Tooth disease expressing HSPB8 protein containing a K141N or K141T mutation.

In one embodiment, the animal model is capable of expressing K141N or K141T mutant HSPB8 protein in neurons, motor neurons, or multi-dendritic sensory neurons.

In one embodiment, the animal model may be Drosophila.

In one embodiment, in the animal model, K141N or K141T mutant HSPB8 in the neuron-specific promoter elav , motor neuron-specific promoter D42 , or multi-dendritic sensory neurons-specific promoter ppk. Nucleic acid molecules encoding the protein can be operably linked to express the HSPB8 protein containing the K141N or K141T mutation specifically in neurons, motor cells, or multi-dendritic sensory neurons.

As used herein, the term “operably linked” means that one nucleic acid fragment is linked to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.

Promoters used in the present invention can be of various natures and origins and have various properties. This is because the choice of promoter used depends particularly on the gene in question. Accordingly, the promoter can be driven, for example, by a promoter that is strong or weak, ubiquitous or tissue/cell specific, or specific to a physiological or pathophysiological state (the activity may depend on the state of cell differentiation or on a specific stage of the cell cycle). being). Promoters may be of eukaryotic, prokaryotic, viral, animal, plant, artificial, human, etc. origin.

In one aspect, the present invention relates to a vector for constructing a Charcot-Marie-Tooth disease animal model comprising a nucleic acid encoding a K141N or K141T mutant HSPB8 protein and a neuron-specific promoter operably linked thereto.

In one embodiment, the neuron-specific promoter may be the neuron-specific promoter elav , the motor neuron-specific promoter D42 , or the multi-dendritic sensory neuron-specific promoter ppk .

In one aspect, the present invention relates to a method of providing information necessary for diagnosing the possibility of developing Charcot-Marie-Tooth disease, including detecting the K141N or K141T mutation of the HSPB8 protein in a sample isolated from a subject.

In one embodiment, the method may further include determining that the subject is likely to develop Charcot-Marie-Tooth disease when the K141N or K141T mutation of the HSPB8 protein is detected in a sample isolated from the subject. You can.

In one aspect, the present invention provides treatment of a test compound or composition to a cell line or animal model expressing HSPB8 protein containing a K141N or K141T mutation; Determining the degree of mitochondrial dysfunction in cell lines or animal models treated with the test compound or composition; and a method for screening a candidate for the treatment of Charcot-Marie-Tooth disease, comprising selecting a test compound or composition in which mitochondrial dysfunction is improved compared to a control cell line untreated with the test compound or composition.

In one aspect, the present invention provides treatment of a test compound or composition to a cell line or animal model expressing HSPB8 protein containing a K141N or K141T mutation; Confirming the expression of PINK1 or Parkin, or its activity, in cell lines or animal models treated with the test compound or composition; And a method for screening a candidate for the treatment of Charcot-Marie-Tooth disease, comprising selecting a test compound or composition with increased expression or activity of PINK1 or Parkin compared to a control cell line untreated with the test compound or composition. .

In one aspect, the present invention relates to a method for diagnosing Charcot-Marie-Tooth disease, comprising detecting a K141N or K141T mutation in the HSPB8 protein.

In one aspect, the present invention provides treatment for Charcot-Marie-Tooth disease, comprising administering a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same to an individual suffering from Charcot-Marie-Tooth disease. It's about treatment methods.

In one aspect, the invention relates to the diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.

In one aspect, the present invention relates to the use of a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same for the prevention or treatment of Charcot-Marie-Tooth disease.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. HSPB8 Creation and validation of mutant animal models

1-1. Construction of HSPB8 K141N/K141T mutant model

To create a K141N mutant animal model and a K141T mutant animal model of the HSPB8 (small heat shock protein B8) (also called HSP22) gene, elav -GAL4, a nerve (neuron)-specific GAL4 driver, and motor neuron-specific GAL4 driver Wild-type human HSPB8 ( HSPB8 ^WT ), K141T mutant HSPB8 ( HSPB8 ^K141T ), and K141E mutant HSPB8 in Drosophila neurons using D42 -GAL4 and ppk -GAL4, a multi-dendritic sensory neurons-specific GAL4 driver, respectively. Drosophila models that specifically expressed the UAS transgene ( HSPB8 ^K141E ) were created. Specifically, K141T and K141E mutations were induced in the cDNA of human HSPB8, respectively, using the QuikChange™ site-directed mutagenesis kit (Agilent Technologies) using the following primer pairs: HSPB8 K141T forward primer: 5'-ctg cag gaa gct gga ttt tcg ttg tga agt tct tag aaa ca-3' and K141T reverse primer: 5'-tgt ttc taa gaa ctt cac aac gaa aat cca gct tcc tgc ag), and HSPB8 K141E forward primer: 5'-gaa gct gga ttt tct ctg tga agt tct tag aaa caa tgc cac c-3' and K141E reverse primer: 5'-ggt ggc att gtt tct aag aac ttc aca gag aaa atc cag ctt c-3'. Afterwards, the wild-type HSPB8 cDNAs and the mutant HSPB8 cDNAs were each inserted into the pACU2 vector and microinjected into y w;PBac y[+]-attP-3B VK00001 embryos. elav -GAL4, ppk -GAL4, and D42 -GAL4 lines were purchased from Bloomington Stock Center. In addition, Drosophila lines expressing UAS-PINK1 and UAS-Parkin were also created using the method described in Park et al., 2006. The genotypes of the constructed Drosophila lines are as follows: elav (elav-GAL4/+); elav HSPB8 ^WT (elav-GAL4/+; UAS-HSPB8 ^WT /+); elav HSPB ^K141T (elav-GAL4/+; UAS-HSPB8 ^K141T /+); elav HSPB ^K141E (elav-GAL4/+;; UAS-HSPB8 ^K141E /+); ppk (ppk-GAL4/+); ppk HSPB8 ^WT (ppk-GAL4/UAS-HSPB8 ^WT ); ppk HSPB ^K141T (ppk-GAL4/UAS-HSPB8 ^K141T ); ppk HSPB ^K141E (ppk-GAL4/UAS-HSPB8 ^K141E ); D42 (D42-GAL4/+); D42 HSPB8 ^WT (UAS-HSPB8 ^WT /+; D42-GAL4/+); D42 HSPB ^K141T (UAS- HSPB ^K141T /+; D42-GAL4/+); D42 HSPB ^K141E (UAS-HSPB8 ^K141E /+; D42-GAL4/+); elav HSPB ^K141T PINK1 (elav-GAL4/+; UAS-HSPB8 ^K141T /UAS-PINK1); elav HSPB ^K141T Parkin (elav-GAL4/+; UAS-HSPB8 ^K141T /UAS-Parkin); elav HSPB ^K141E PINK1 (elav-GAL4/+; UAS-HSPB8 ^K141E /UAS-PINK1); elav HSPB ^K141E Parkin (elav-GAL4/+; and UAS-HSPB8 ^K141E /UAS-Parkin) .

1-2. Mutation model validation

HSPB8 expression in the transgenic fruit flies prepared above was confirmed by Western blot analysis, and their lifespan analysis was performed. Specifically, to confirm the expression of HSPB8, 20 10-day-old male fruit fly heads were disrupted with disruption buffer, then centrifuged, and SDS sample buffer was added and heated. Afterwards, the samples were electrophoresed on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution for 30 minutes and then incubated with anti-HSPB8 antibody (1:1,000, Cell Signaling Technology, # 95357 ) or anti-beta-actin antibody (1:1,000, Santa Cruz Biotechnology, SC-1616) as the primary antibody. The reaction was performed as described in Kang et al., 2020, and antibodies bound to the membrane were detected using the ImageQuant LAS 4000 system (GE Healthcare Life Sciences). Additionally, for lifespan analysis, 90 fruit flies were divided into three groups (n = 30 in each group), transferred to each vial, and survival scores were scored every 3 or 4 days at 25°C. Afterwards, Kaplan-Meier estimation was performed on the cumulative survival data to determine the effect of each genotype on the individual's lifespan using Online Application Survival Analysis 2 Lifespan Assays ( http://sbi.postech.ac.kr/oasis2 ). And the log-rank test was performed.

As a result, the transgenic fruit flies successfully developed into adults, and there was no significant difference in the protein expression level of HSPB8 between them (Figures 1a and b). Additionally, lifespan analysis showed that expression of wild type and HSPB8 mutants caused a partial reduction in lifespan, but no significant decrease in survival was observed within 15 days (Figures 1c and d).

Example 2. Confirmation of sensory symptoms due to mutation

To confirm whether sensory defects occur due to HSPB8 mutation, a thermal nociception assay was performed using Drosophila larvae. Specifically, each L3 larvae (hatched) of HSPB8 WT fruit flies, HSPB8 K141N mutant fruit flies, and HSPB8 K141T mutant fruit flies that expressed the HSPB8 transgene in multi-dendritic sensory neurons using ppk-GAL4 After [AEL] 120 h) (minimum of 50 larvae) were rinsed with distilled water, placed in Petri dishes, allowed to acclimatize for 10 s, and observed under a microscope. Abdominal A4-A5 segments of each larva were placed in a custom-made 0.6-microprocessor temperature-controlled larvae. It was touched with a mm-wide thermal probe. The time taken to induce an evasive corkscrewlike rolling response was measured as pain assessment (withdrawal latency), and larvae that did not show a rolling response within the cutoff value of 20 seconds were considered unresponsive. As a result, all larvae showed no response to the heat probe at 36°C, whereas they showed a rapid rolling response to the heat probe at 46°C (Figure 2a). Accordingly, after selecting the temperature of the heat probe at 40°C, heat nociception was observed in ppk- expressing larvae, HSPB8 ^WT -expressing larvae ( ppk HSPB8 ^WT ), HSPB8 ^K141T -expressing larvae ( ppk HSPB8 ^K141T ), and HSPB8 ^K141E -expressing larvae ( ppk HSPB8 ^K141E ), respectively. Analyzes were performed and response latencies were analyzed. As a result, no significant differences were found in each group of larvae, confirming that the HSPB8 transgene was unable to induce sensory abnormalities in the Drosophila heat nociception model (Figure 2b).

Example 3. Confirmation of decreased exercise capacity due to mutation

3-1. behavioral analysis

In order to confirm changes in locomotor activity due to mutations in HSPB8, the behavior of the transgenic Drosophila lines HSPB8 ^WT , HSPB8 ^K141T , and HSPB8 ^K141E ( elav, elav HSPB8 ^WT , elav HSPB8 ^K141T , and elav HSPB8 ^K141E ) produced in Example 1, respectively. It was recorded with a digital video camera, and walking trajectory and speed were calculated with Ctrax and Matlab. Specifically, 5-day-old adult male transgenic fruit flies expressing HSPB8 ^WT ( elav HSPB8 ^WT ), HSPB8 ^K141T ( elav HSPB8 ^K141T ), or HSPB8 ^K141E ( elav HSPB8 ^K141E ) were each spaced at 675 mm × 440 mm × 410 mm equipped with a camera. They were placed in an insulated box and acclimatized for 30 minutes at 25°C, and their free movement in a transparent, round space with a height of 2 mm and a diameter of 60 mm was recorded at 30.06 fps (frames per second). The recorded video was converted to a MATLAB file using Ctrax and the behavioral microarray toolbox was used to obtain average velocity and trajectory graphs.

As a result, the group of fruit flies expressing HSPB8 ^WT showed no significant difference in movement from the control fruit flies expressing only the elav-GAL4 driver, but the group of fruit flies expressing HSPB8 ^K141T or HSPB8 ^K141E showed obvious defects in walking speed and trajectory. (Figure 3A and B). This mutation-specific movement defect was also observed in 5-day-old female fruit flies. Additionally, when measuring the exercise capacity of 15-day-old fruit flies, the HSPB8 ^WT fruit fly group consistently showed no significant change in exercise ability, but the HSPB8 ^K141T or HSPB8 ^K141E fruit fly group showed a serious decrease in exercise ability compared to 5-day-old fruit flies. (Figures 3c and d).

3-2. Climbing Analysis

A climbing assay was also used to determine climbing ability, which has proven useful in identifying motor deficits in fruit fly models of various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Specifically, the fruit fly group (n=15) expressing HSPB8 ^WT , HSPB8 ^K141T or HSPB8 ^K141E of Example 3-1 was transferred to climbing ability test vials, acclimatized to the environment for 1 h at room temperature, and the flies were knocked by tapping the vials. After being dropped to the floor, the number of climbing flies was counted for 10 seconds. Climbing scores (percentage of number of climbing flies relative to total number of flies) were calculated after 10 attempts for each group.

As a result, expression of HSPB8 ^WT did not induce a significant difference in climbing ability, but expression of HSPB8 ^K141T or HSPB8 ^K141E induced a significant decrease in locomotion in both 5-day-old male and female flies (Figures 4a and b). This loss of climbing ability was also consistently observed in 15-day-old fruit flies expressing HSPB8 ^K141T or HSPB8 ^K141E (Figures 4a and b).

3-3. Check for motor nerve abnormalities

Since motor neuron abnormalities are a major symptom in HSPB8-related human pathology, the D42-GAL4 driver was used to express the HSPB8 ^WT , HSPB8 ^K141T or HSPB8 ^K141E transgene in the motor neurons of Drosophila, and then perform transformation. Motor ability as a phenotype of Drosophila was confirmed by the method described in Examples 3-1 and 3-2 above.

As a result, no significant change in climbing ability was observed in the fruit fly group expressing HSPB8 ^WT compared to the control group, whereas in the fruit fly group expressing HSPB8 ^K141T or HSPB8 ^K141E , locomotor ability was significantly reduced compared to the control or HSPB8 ^WT expression group. was found to be (Figure 4c and d). Additionally, video tracking analysis also showed that the walking speed and trajectory of fruit flies expressing the HSPB8 transgene using the D42-GAL4 driver were significantly reduced (Figure 5), suggesting that expression in motor neurons of human HSPB8 mutants consistently impaired locomotor performance. It was confirmed that it decreased. This shows that expression of human HSPB8 ^K141T or HSPB8 ^K141E causes clear movement defects in Drosophila.

Example 4. Confirmation of mitochondrial dysfunction caused by mutation

4-1. mitochondrial membrane potential

Since mitochondrial dysfunction has been reported to play an essential role in the pathophysiology of multiple neurodegenerative diseases, to determine whether mutations in HSPB8 affect mitochondrial membrane potential in Drosophila neurons, Membrane potential was measured using TMRM in the ventral nerve cord (VNC) (functionally equivalent to the spinal cord of mammals containing motor neurons) of larvae from each HSPB8 transgenic Drosophila group. Specifically, to measure mitochondrial membrane potential, the VNC of larvae of each transgenic Drosophila group was dissected in PBS and incubated with 2.5 nM tetramethylrhodamine methyl ester (TMRM, Molecular Probes, MA) (in PBS+0.1% Triton X-100). was stained for 30 minutes. TMRM fluorescence was analyzed using a Zeiss LSM 800 confocal microscope, TMRM signal intensity was measured using Zeiss Zen software, and the average TMRM fluorescence intensity (±SD) was calculated for 10 independent samples.

As a result, mitochondrial membrane potential (transmembrane potential) was decreased throughout both HSPB8 ^K141T mutant larvae and HSPB8 ^K141E mutant larvae compared to wild-type transgenic larvae (Figure 4a), indicating that HSPB8 mutant Drosophila was more susceptible to patients with locomotor loss and mitochondrial dysfunction. It was confirmed that their phenotypes were successfully expressed.

4-2. Mitophage level

To further investigate the mitochondrial defects induced by HSPB8 mutations, in the VNC of HSPB8 transgenic Drosophila larvae in which the elav-GAL4 driver was coexpressed with the mitochondria-targeted fluorescent protein Keima (mt-Keima), mt -Mitochondrial QC ( The level of mitophagy, which is essential for the mitochondria quality control mechanism, was confirmed. Specifically, the VNC of each group of larvae expressing mt-Keima was dissected in PBS, and the fluorescence of mt-Keima was measured using a Zeiss LSM 800 confocal microscope in two sequential waves using an excitation bandwidth of 595-700 nm. Images were imaged with excitation lasers (488 nm and 555 nm), and the images were analyzed pixel by pixel using Zeiss Zen. The level of mitophagy (% of mitophagy) was defined as the number of pixels with a high red/green ratio divided by the total number of pixels. Mean mitophagy levels (±SD) were calculated for 10 independent samples.

As a result, mitophagy levels were also found to be reduced in both HSPB8 ^K141T mutant larvae and HSPB8 ^K141E mutant larvae compared to the wild-type control (Figure 4b).

Through this, it was confirmed that mutations in the HSPB8 protein interfere with mitochondrial QC and consequently impair mitochondrial function.

Example 5. PINK1 and parkin of Restoration of mitochondrial dysfunction by introduction

To determine whether the reduction in mitochondrial activity and QC in Drosophila caused by HSPB8 mutation is restored by PINK1 or Parkin, transgenes of PINK1 or Parkin were introduced into HSPB8 mutant Drosophila ( elav HSPB8 ^K141T PINK1, elav HSPB8 ^K141E PINK1, elav HSPB8 ^K141T Parkin and elav HSPB8 ^K141E Parkin ) Changes in mitochondrial membrane potential, mitophagy level, and exercise capacity were confirmed. As a result, loss of mitochondrial membrane potential was restored by expression of PINK1 or Parkin in both HSPB8 ^K141T or HSPB8 ^K141E transgenic Drosophila (Figure 4a), as well as reduced mitophagy level, reduced climbing ability, walking speed, and locomotor trajectory. appeared to recover (Figures 4c to f).

Through the results of recovery of motor defects accompanied by recovery of mitochondrial activity and mitophagy, it was confirmed that mitochondrial dysfunction plays an important role in the development of neuropathies induced by HSPB8 mutations.

Example 6. Recovery of mitochondrial dysfunction by PINK1 activation

To determine whether the movement defects of HSPB8 mutant Drosophila are restored by activation of PINK1, kinetin riboside (KR) (N6-furfuryl adenine riboside), which activates PINK1 independently of mitochondrial depolarization, was administered to mice at 1 day of age. After oral administration of 1 mM or 5 mM to HSPB8 WT fruit flies, HSPB8 K141N mutant fruit flies, and HSPB8 K141T mutant fruit flies, respectively, for 14 days, locomotor activity was confirmed using video tracking analysis and climbing analysis.

As a result, the locomotor activity of the wild-type transgenic fruit flies appeared to be unchanged compared to the control group (vehicle administered group) due to KR administration (FIGS. 6a to 6c), confirming that there were no side effects caused by KR. In addition, in HSPB8 mutant transgenic fruit flies, locomotor activity was shown to be significantly restored depending on the administered concentration of KR (Figures 6a to c).

Through this, it was confirmed that PINK1 activation can restore the phenotype induced by HSPB8 mutation and that KR can suppress HSPB8-related pathogenesis.

Claims (20)

1. A composition for diagnosing Charcot-Marie-Tooth disease (CMT) disease, comprising an agent capable of detecting the K141N or K141T mutation of the small heat shock protein B8 (HSPB8) protein.
2. The method of claim 1, wherein the agent capable of detecting a mutation is an antibody, antibody fragment, aptamer, Affilin, Affibody, or Affimer that specifically binds to the HSPB8 protein. , Affitin, Alphabody, Anticlin, DARpin, Fynomoer, Kunitz domain peptide, avidity multimer, peptido A composition for diagnosing Charcot-Marie-Tooth disease, comprising peptidomimetics, receptors, ligands or cofactors.
3. The method of claim 1, wherein the agent capable of detecting a mutation comprises a primer pair, or a probe, or a primer pair and a probe that specifically recognizes a nucleic acid molecule encoding the HSPB8 protein, for Charcot-Marie-Tooth disease. Diagnostic composition.
4. A diagnostic kit for Charcot-Marie-Tooth disease comprising the composition of any one of claims 1 to 3.
5. The diagnostic kit for Charcot-Marie-Tooth disease according to claim 4, which is an RT-PCR kit, a microarray chip kit, or a protein chip kit.
6. A pharmaceutical composition for the prevention or treatment of Charcot-Marie-Tooth disease, comprising a vector containing PINK1 (PTEN-induced putative kinase 1) or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same.
7. The pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease according to claim 6, which is Charcot-Marie-Tooth disease caused by K141N or K141T mutation of HSPB8 protein.
8. The pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease according to claim 6, comprising kinetin riboside (KR) (N6-furfuryl adenine riboside).
9. The pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease according to claim 6, which improves motor defects or mitochondrial dysfunction.
10. Charcot-Marie-Tooth disease animal model expressing HSPB8 protein containing K141N or K141T mutations.
11. 11. The Charcot-Marie-Tooth disease animal model according to claim 10, which expresses the K141N or K141T mutant HSPB8 protein in neurons, motor neurons, or multi-dendritic sensory neurons.
12. The Charcot-Marie-Tooth disease animal model according to claim 10, which is Drosophila.
13. The method of claim 10, wherein the nucleic acid molecule encoding the K141N or K141T mutant HSPB8 protein is selected from the neuron-specific promoter elav , the motor neuron-specific promoter D42 , or the multi-dendritic sensory neurons-specific promoter. A Charcot-Marie-Tooth disease animal model operably linked to the promoter ppk .
14. A method for providing information necessary for diagnosing the possibility of developing Charcot-Marie-Tooth disease, comprising detecting the K141N or K141T mutation of the HSPB8 protein in a sample isolated from a subject.
15. The method of claim 14, further comprising the step of determining that the subject is likely to develop Charcot-Marie-Tooth disease when the K141N or K141T mutation of the HSPB8 protein is detected in the sample isolated from the subject. -A method of providing the information necessary to diagnose the possibility of developing Marie-Tooth disease.
16. 1) Treating the test compound or composition to a cell line or animal model expressing HSPB8 protein containing K141N or K141T mutation;

    2) measuring the degree of mitochondrial dysfunction in cell lines or animal models treated with the test compound or composition; and

    3) A screening method for a candidate for the treatment of Charcot-Marie-Tooth disease, comprising selecting a test compound or composition with improved mitochondrial dysfunction compared to a control cell line untreated with the test compound or composition.
17. A method for diagnosing Charcot-Marie-Tooth disease, comprising detecting a K141N or K141T mutation in the HSPB8 protein.
18. A method of treating Charcot-Marie-Tooth, comprising administering a vector containing PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same to an individual suffering from Charcot-Marie-Tooth disease.
19. Diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.
20. Use of a vector containing PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same for the prevention or treatment of Charcot-Marie-Tooth disease.

Images