KR20200021145A - Methods for providing information on the diagnosis of sporadic Parkinson's disease and drug screening methods - Google Patents

Abstract

The present invention relates to a method for providing information capable of effectively diagnosing sporadic Parkinson′s disease, by utilizing a novel 3D marker gene TXNIP as an index factor, and measuring and comparing the expression level of the TXNIP gene in each sample of a patient with suspected sporadic Parkinson′s disease and a control group. In addition, the present invention relates to a method of screening a therapeutic drug for sporadic Parkinson′s disease by suppressing the expression of the TXNIP gene in patients with sporadic Parkinson′s disease.

Description

Method for providing information on the diagnosis of sporadic Parkinson's disease and drug screening methods

The present invention relates to a method for providing effective diagnostic information by using three-dimensional specific disease factors in diagnosing sporadic Parkinson's disease and a method for screening a therapeutic drug by suppressing the present disease factor.

Parkinson's Disease (Parkinson's Disease) is a chronic disease caused by the lack of neurotransmitters called dopamine in the brain, which are the main symptoms of tremor, stiffness, slow behavior, and postural dysfunction. Dopamine is produced from nerve cells called the black matter of the brain, and the nerve cells of the black matter are connected to a part called the basal nucleus of the brain, and the basal nucleus is connected to the motor cortex and many other parts of the brain in a complex way to smooth and harmonize the movement of the human body. It is also a very important part to ensure that it is done correctly. However, Parkinson's disease is caused by a lack of dopamine, a substance secreted to control the function of the basal ganglia in the black matter.

Parkinson's disease can be divided into primary and secondary symptoms. The primary symptoms are stiffness, tremor, slow or reduced movement of the body, imbalance, and impaired gait. Secondary symptoms refer to symptoms derived from primary symptoms or from involvement of the nervous system other than melanoma.

Therapies currently used for the treatment of Parkinson's disease include drug therapy, surgical therapy, and physical therapy. In the case of drug therapy, dopamine, which is generally lacking in the brain, is supplemented, and neurotransmitters are imbalanced due to the lack of dopamine. Drugs to control or delay the destruction of nerve cells and to control other symptoms such as depression, such as amantadine, anticholinergic drugs, el-dopa, cinemet, madopa, dopamine Agonists, eldepril and antidepressants. However, since these drugs cannot revive dead neurons, they are not intended to cure, but to control symptoms.

In the case of surgical treatment, stereotactic brain surgery and transplantation are mainly used. Stereotactic brain surgery is performed to remove the symptoms of Parkinson's disease on the opposite side of the thalamus by artificially creating lesions on the sagittal. The purpose of the method or symptom relief is that the therapeutic effect is weak. Transplantation is a surgical technique developed due to the recent development of cell culture and surgical techniques, in which dopamine-producing cells are directly implanted into the brain. Especially, fetal nerve cells or the patient's own adrenal medulla cells are transplanted. Is developed. However, this method is ideal in that it fundamentally corrects the cause of Parkinson's disease.

In addition, in the case of physiotherapy, most of the patient's symptoms are weak or not severe as a method of performing the exercise ability of the patient to help maximize, and prevents the joints to prevent.

On the other hand, the cause of this Parkinson's disease is still active research, but the exact cause is not yet known, only the infection theory of viral encephalitis, immune theory of immune system, innate innate substrate There are genetic theories, theories that free radicals destroy nerve cells, and that there is a problem with the production and metabolism of dopamine. However, there is not enough to clearly explain the entire Parkinson's disease. Sporadic and senile Parkinson's disease, developed by environmental factors, accounts for the majority of Parkinson's disease. For this reason, there is a need for more active research on the techniques for diagnosing sporadic and senile Parkinson's disease and for the treatment of sporadic Parkinson's disease.

In addition, according to the recent statistics, the elderly population is rapidly increasing, and the incidence of Parkinson's disease is increasing as the life expectancy is extended. This can be a very important problem.

Therefore, many researchers are using the animal model of Parkinson's disease to identify the cause of the Parkinson's disease and to develop a therapeutic agent. However, in many cases, the Parkinson's disease is not properly induced or the desired animal model is not produced due to the physiological changes of each animal. There are many problems with getting results. In addition, there is no direct way to prove that an animal model of Parkinson's disease has Parkinson's disease, and only by observing the difference in behavior between animals with Parkinson's disease and normal animals. Indirectly confirmed, there is a disadvantage that the measurement results are inaccurate and time-consuming problem. In addition, when developing a Parkinson's disease model using existing human cells, there have been great difficulties in identifying Parkinson's disease occurring in the three-dimensional brain because the cell culture itself is a two-dimensional culture.

In order to obtain more accurate experimental results for the development of the cause and treatment of sporadic Parkinson's disease in animal models of many Parkinson's diseases reported so far, a three-dimensional structure utilizing human cells is required. Therefore, various technologies have been developed so far. These techniques are the results obtained through observation of two-dimensional cells and tissues, and appropriate modeling in the method of identifying and treating Parkinson's disease with three-dimensional structure occurring in the human brain with three-dimensional structure. There is a big difficulty in doing this.

Korean Patent No. 1081358

An object of the present invention is to provide a method for providing information of sporadic Parkinson's disease diagnosis using a novel three-dimensional specific marker gene and a drug screening method through the same.

1. measuring the expression level of mRNA or protein of the TXNIP gene from a patient sample and a control sample; Comparing the measured expression levels with each other; And providing information for predicting or confirming the risk of developing sporadic Parkinson's disease-derived LRRK2-G2019S mutation when the expression level of the patient is higher than that of the control group.

2. treating the test substance with induced pluripotent stem cell-derived midbrain-like sporadic Parkinson's disease 3D organoid expressing TXNIP gene; Measuring the expression level of the gene of the organoid; And if the expression of the gene is reduced before the treatment of the test substance, selecting the test substance as a preventive or therapeutic agent for sporadic Parkinson's disease; and screening method for a candidate agent for preventing or treating sporadic Parkinson's disease-derived LRRK2-G2019S mutation .

3. A composition for preventing or treating sporadic Parkinson's disease-derived LRRK2-G2019S mutations comprising an oligonucleotide consisting of the sequence of SEQ ID NO: 4.

4. Spontaneous Parkinson's disease-derived LRRK2-G2019S mutations comprising a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence Diagnostic composition.

5. A sporadic Parkinson's disease diagnostic kit derived from LRRK2-G2019S mutation comprising the composition of claim 4.

The present invention provides a method for providing an information for effectively diagnosing sporadic Parkinson's disease using the novel three-dimensional specific marker gene TXNIP as an index factor, and a method for developing a treatment technique for sporadic Parkinson's disease using the gene.

1 shows (A) Bright-field microscopy images of the generation of 3D organoids for 2 months, (B) pluripotency markers (OCT4), neurogenic markers (SOX1) and dopaminergic nerve markers (TH and VRT2) qRT-PCR analysis results (data represent mean ± SEM. ANOVA, * p <0.05, ** p <0.01), (C) MAP2, MASH1 to confirm the presence of midbrain dopaminergic neurons at day 60 , Immunofluorescence (scale bar = 50 μm) for TUJ1, VMAT2, TH, DAT and GIRK2, (D) Schematic image of midbrain development (marginal zone (MZ) is distinguished from radial glia cells in ventricular zone (VZ)) Including midbrain dopaminergic neurons), (E) percent of MAP2 / MASH1 positive cells in MZ- and VZ-like regions on day 60, (F) gene expression profiling using qRT-PCR between 1 and 45 days Green indicates high and low gene expression levels, respectively, and synapsin-RFP-positive in midbrain-like 3D organoids (n = 3) and (G) per sample. KCL-induced dopamine levels in midbrain-like 3D organoids using fluorescence activated cell sorting analysis of cells, (H, I) liquid chromatography-mass spectrometry analysis (data show mean ± SEM. ANOVA, ** p < 0.01), (J) Microarray Gene set enrichment analysis data comparing midbrain-like 3D organoids and 2D organoids.
FIG. 2 shows (A) Immunofluorescence staining of midbrain-like 3D organoids in LRRK2-G2019S hiPSC (scale bar = 100 μm), (B) midbrain-like 3D organoid and LRRK2-G2019S for dopaminergic neuron markers TH, AADC and DAT QRT-PCR analysis of 3D organoids (data shows mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample), (C) midbrain-like 3D organoids and LRRK2-G2019S Immunostaining of TH- and cleaved caspase-3-positive cells of 3D organoids (scale bar = 20 μm), (D) midbrain-like 3D organoids of cleaved caspase-3 / TH-positive cells and LRRK2 − treated with 0.5 mM MPTP Proportions in G2019S 3D organoids (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample), (E, F) cleaved caspase-3 levels after MPTP treatment Western blot analysis showing an increase of (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01).
Figure 3 (A) Representative images showing colocalization analysis of pS129-α-synuclein-positive puncta and EEA1-positive endosome in wild-type and LRRK2-G2019S 3D organoids (scale bar = 20 μm), (B) pS129 among all EEA1-positive mucus Percentage of -α-synuclein- and EEA1-positive mucus (left), number of pS129-α-synuclein / EEA1-positive endosome per cell (right) in LRRK2-G2019S 3D organoids (data shows mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 10 per sample), (C) Thioflavin T (50 μm) staining of wild-type and LRRK2-G2019S 3D organoids (arrow indicates α-synuclein precipitate, scale bar = 20 μm), (D) Proportion of Thioflavin T positive cells among all TH-positive cells of wild-type and LRRK2-G2019S 3D organoids (data shows mean ± SEM, ANOVA, ** p <0.01, n = 3 per sample) ), (E) Quantifying the average area of Thioflavin T positive deposits in midbrain-like 3D organoids for 60 days Vector (data represent the mean ± SEM, ANOVA, ** p <0.01, n = 5 per sample). Data showing significantly reduced phosphorylated α-synuclein oligomer levels of LRRK2-G2019S 3D organoids when treated with (F, G) LRRK2 kinase inhibitor (GSK2578215A, 1 μm) (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample). (H) Real-time qPCR analysis data of dopaminergic neural markers (TH, AADC and DAT) in wild-type and LRRK2-G2019S 3D organoids treated with LRRK2 kinase inhibitor GSK2578215A (data show mean ± SEM, ANOVA, * p <0.05 , N = 3) per sample.
4 is a Venn diagram (A) comparing the wild type 2D organoid and LRRK2-G2019S knock-in 2D organoid, wild type 3D organoid and LRRK2-G2019S knock-in 3D organoid to show the overlap of differentially expressed genes. Venn diagrams show the number of genes regulated up or down by 1.5-fold), (B) heatmap and density color codes of 3965 genes that show differential expression in microarray analysis under wild-type and LRRK2-G2019S knock-in conditions ( C) Scatter plot of microarray data comparing LRRK2-G2019S knock-in 3D organoids and wild-type 3D organoids (top), LRRK0G2019S knock-in 2D organoids and wild-type 2D organoids (bottom), (D) LRRK2- Gene set enrichment analysis of microarray expression data comparing G2019S knock-in 3D organoid and wild-type 3D organoid, LRRK0G2019S knock-in 2D organoid and wild-type 2D organoid, (E) Graph of TXNIP protein interaction network related to Parkinson's disease risk factors (red indicates high expression and blue indicates low expression; square nodes represent proteins with genetic risk factors for Parkinson's disease) will be.
5 shows (A) Validation of microarray and real-time qPCR gene expression data, (B) Real-time qPCR analysis data of TXNIP expression in LRRK2-G2019S knock-in 3D organoids and midbrain-like 3D organoids (data is mean ± SEM , ANOVA, ** p <0.01, n = 3 per sample, (C) real-time qPCR analysis data comparing TXNIP expression to control and LRRK2-G2019S 3D organoids to control and LRRK-G2019S 2D organoids ( Data show mean ± SEM, ANOVA, ** p <0.01, n = 3 per sample), (D) TXNIP gene in LRRK2-G2019S knock-in 3D organoid treated with TXNIP-shRNA via real time qPCR Expression data (data represent mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample), (E) LRRK2-G2019S knock-in 3D organoids treated with TXNIP-shRNA Western blot analysis of pS129-α-synuclein in the data indicates a decrease in pS129-α-synuclein oligomer (data is mean ± SEM , ANOVA, * p <0.05, n = 3) per sample, (G) representative immunofluorescence images of LAMP1 and pS129-α-synuclein puncta of LRRK2-G2019S knock-in 3D organoids, (H, I) TXNIP- Quantitative data of LAMP1 + and pS129-α-synuclein + puncta showing a decrease in pS129-α-synuclein inclusion upon shRNA treatment (data shows mean ± SEM, ANOVA, * p <0.05, n = 5 per sample) .
6 (A) Schematic diagram of organizing midbrain-like 3D organoids in hiPSC, timeline of various differentiation protocols for generating effective and optimal 3D organoids (B) Representative immunofluorescence images of 3D organoid preparation for 2 months (Scale bar = 100 μm), (C) TUJ1, TH, VMAT2, DAT and GIRK2 positive midbrain dopamine neurons in midbrain-like 3D organoids on day 60, (D) AADC, PITX3, in midbrain-like 3D organoid immune precipitates Western blot analysis of VMAT2 and NURR1 shows representative images for the mature dopaminergic neuron markers PITX3 and AADC at 30 and 60 days.
7 shows (A) Fontana-masson staining showing neuromelanin-positive cells in midbrain-like 3D organoids on days 30 and 60; (B) Data quantifying the number of neuromelanin-positive cells in midbrain-like 3D organoids (data is average ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample), (C) age-related genes ANLN, GAL35ST1, FAH, MBP, ACSL1 and CLDN11 in midbrain-like 3D organoids Data showing increase at day 60, (D) Gene set enrichment analysis of microarray data showing significant enrichment of the brain of the elderly compared to the fetal brain (GSM2639572 and GSM175904, http://www.brain-map.org ), (E) immunostaining of histone H2AX phosphorylation (γH2AX) in midbrain-like 3D organoids on days 30 and 60 (scale bar = 50 μm), (F) of histone H2AX phosphorylation positive cells in midbrain-like 3D organoids Ratio (data represents mean ± SEM, ANOVA, * p <0.05, 1 N = 3 per plate, (G, H) Data showing increased expression of histone H2AX phosphorylated protein in midbrain-like organoids on day 60 (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01).
Figure 8 shows (A) representative immunofluorescence images of MAP2- and TH-positive phosphorylated dopaminergic neurons in 3D organoids and 2D organoids (scale bar = 100 μm), (B) TH among all DAPI positive cells of 3D and 2D organoids And data quantifying the number of MAP2 positive cells over 20 days (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample), (C) linked to 20 days Real-time qPCR analysis data of the expression of markers AADC, CHAT and MAPT and astrocyte markers GFAP, S100B and ALDH1L1 (data indicates mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample ), (D) scatter plot of microarray expression data comparing midbrain-like 3D organoids and 2D organoids, (E) gene ontology analysis of biological processes in midbrain-like 3D organoids and 2D organoids, (F) Human brain tissue, cerebral cortex of midbrain-like 3D organoids compared to 2D organoids Gene set of striatal microarray data showing a substantial enrichment of the concentration shows an analysis of data.
Figure 9 shows (A) the sequence of Cas9 that targets the locus in the human LRRK2 gene (single guide RNA target site in blue and adjacent 5'- NGG protospacer in red. Arrows indicate LRRK2-G2019S mutation sites) (B) SURVEYOR mutation detection assay gel showing modification of Cas9 target site by single guide RNA, (C) representative sequence of indels of human LRRK2 gene targeted by CRISPR-Cas9 (delete is indicated by dash), (D A) Schematic representation of a donor vector containing LRRK2-G2019S mutation site, (E) Sanger sequencing showing G2019S mutation site at exon 41 of LRRK2 gene after Cas9 LRRK2- targeting and donor plasmid introduction, (F) Amplifying puromycin resistant hiPSC colonies Representative images of PCR products using puromycin specific primers (hiPSC colonies transformed with CRE are appropriate primers shown in (D) (I) representative immunofluorescence images of colonies positive for pluripotency markers SOX2, NANOG, TRA1-60, OCT4 and SSEA1 (scale bar = 100 μm), (H) pluripotency in normal cells and LRRK2-G2019S target hiPSCs Quantitative RT-PCR analysis of normal hiPSCs and LRRK2-G2019S hiPSCs for markers OCT4, SOX2, NANOG and REX1 (data show mean ± SEM, ANOVA, n = 3 per sample). AP staining of (I) normal hiPSC and LRRK2-G2019S hiPSC, (J) quantification data of the number of alkaline phosphatase positive clones between normal hiPSC and LRRK2-G2019S hiPSC.
10 shows (A) Bright-field images of control and LRRK2-G2019S 3D organoids (scale bar = 1 mm), (B) Size measurement results of control and LRRK2-G2019S 3D organoids on day 45, (C) control on day 30 And real-time qPCR analysis data of pluripotency markers (OCT4 and NANOG) expression in LRRK2-G2019S 3D organoids (data shows mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample) . (D) Relative expression levels of dopaminergic linkage markers TH, AADC and DAT in control and LRRK2-G2019S 3D organoids using qRT-PCR, (E) Neural markers (VMAT2, synapsin1 and GAP43) and astrocytes on day 60 Quantitative RT-PCR analysis data of control and LRRK2-G2019S 3D organoids for marker (S100B) (data show mean ± SEM, ANOVA, * p <0.05, ** p <0.01, n = 3 per sample) It is shown.
FIG. 11 shows the relative expression levels of the mature linkage markers NURR1, PITX3, EN1, TH and MAPT of the control and LRRK2-G2019S 3D organoids versus the control and LRRK2-G2019S 2D organoids (data shows mean ± SEM, ANOVA, N = 3) per sample.
(A) Gene set enrichment analysis of microarray expression data for LRRK2-G2019S knock-in 2D organoid and wild type 2D organoid, (B) Graph of TXNIP protein interaction network of LRRK2-G2019S 3D organoid ( Red circles represent high representations and blue circles represent low representations.
Figure 13 shows (A) immunostaining of TH and α-synuclein in mutant synuclein expressing LRRK2-G2019S 3D organoid at day 40 (scale bar = 50 μm), (B) mutant synuclein expressing LRRK2-G2019S 3D organoid Quantitative data of Lewy bodies and Lewy neurites in (C, D) immunostaining and quantification data of TH and pS129-α-synuclein of mutant synuclein expressing LRRK2-G2019S organoids on day 40 (scale bar = 50 μm), ( E, F) Western blot analysis data showing an increase in pS129-α-synuclein oligomer in mutant synuclein expressing LRRK2-G2019S organoid on day 40.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of measuring the expression level of mRNA or protein of the TXNIP gene from the sample of the patient and the control sample; Comparing the measured expression levels with each other; And when the expression level of the patient is higher than the expression level of the control group provides an information providing method for predicting the risk or confirm the onset risk of sporadic Parkinson's disease derived from LRRK2-G2019S mutation.

The TXNIP gene (SEQ ID NO: 1) is a novel indicator that can determine the risk or predict the onset of sporadic Parkinson's disease (hereinafter, sporadic Parkinson's disease) derived from the LRRK2-G2019S mutation presented through the present invention, the indicator The higher the expression level of the factor, the higher the risk of developing sporadic Parkinson's disease can be predicted or confirmed.

The sporadic Parkinson's disease-derived LRRK2-G2019S mutation may be a form (G2019S) substituted with Serine instead of Glycine of the kinase protein, which is a final translation product due to a gene sequence mutation. In this case, the activity of LRRK2 kinase is upregulated to induce neuronal degeneration, which can lead to sporadic Parkinson's disease.

The sample of the patient and the sample of the control group refers to all samples obtained from an individual in which the expression of the indicator factor of the present invention can be detected as a biological sample, wherein the biological sample is a group consisting of biopsy, blood and skin tissue. It may be any one selected from, but is not particularly limited thereto, and may be prepared by treating by a method commonly used in the art.

As a method of measuring the expression level of the indicator factor, a method of measuring the concentration in the sample of the transcription factor TXNIP mRNA or the sample translation of the TXNIP protein as the final translation material of the indicator factor may be selected, but is not limited thereto. Instead, it may be carried out by taking the method commonly used in the art.

As a method of measuring the concentration in the sample of the TXNIP mRNA reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR) , RNase protection assay (RPA), Northern blotting, and DNA chips, but are not limited thereto.

As a method of measuring the concentration of the TXNIP protein in the sample, the amount of the protein can be confirmed using an antibody that specifically binds to the protein. Analytical methods for this include immunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectric Immobilohistochemistry, immunoprecipitation assays, complement fixation assays, fluorescence-activated cell sorting, and protein chips are not limited thereto. .

In the information providing method of the present invention, the expression level of the TXNIP indicator factor is measured from a sample of a suspected sporadic Parkinson's disease and a sample of a control group (unsuspected patient) according to the above method, and the results are compared with each other. When the expression level of the indicator factor is measured higher than that of the control group in the suspected patient's sample, the risk of developing sporadic Parkinson's disease may be predicted or the occurrence of sporadic Parkinson's disease may be confirmed, and the expression factor of the indicator factor may be If the sample is measured to be similar or lower than the control sample, it may provide information to predict that sporadic Parkinson's disease is less likely to be invented or to confirm that sporadic Parkinson's disease does not develop.

The present invention comprises the steps of treating a test substance to induced pluripotent stem cell-derived midbrain-like sporadic Parkinson's disease 3D organoid expressing TXNIP gene; Measuring the expression level of the gene of the organoid; And if the expression of the gene is reduced before the treatment of the test substance, selecting the test substance as a preventive or therapeutic agent for sporadic Parkinson's disease; and screening method for a candidate agent for preventing or treating sporadic Parkinson's disease-derived LRRK2-G2019S mutation To provide.

The sporadic Parkinson's disease-derived LRRK2-G2019S mutation may be a form (G2019S) substituted with Serine instead of Glycine of the kinase protein, which is a final translation product due to a gene sequence mutation. In this case, the activity of LRRK2 kinase is upregulated to induce neuronal degeneration, which can lead to sporadic Parkinson's disease.

The form of the LRRK2 mutation may be the introduction of a G2019S mutation through substitution or deletion of some nucleotide sequences, which may be introduced using CRISPR / Cas9 gene scissors, but are not limited thereto, and are commonly used in the art. This can be done by any method chosen.

The organoid may be an organoid prepared by culturing induced pluripotent stem cells into which a gene mutation of LRRK2 (SEQ ID NO: 2) is introduced by a three-dimensional culture method, and the organoid may be two-dimensional or three-dimensional ( It may be a three-dimensional organoid, but may be a three-dimensional organoid (3D organoid) cultured in a three-dimensional culture method more similar to the actual organs having better alignment and functionality of cells and cells.

The test substance may be a newly synthesized or known compound and may include, without limitation, a substance which is expected to be effective in preventing or treating Parkinson's disease, for example, nucleic acid, nucleotides, proteins, peptides, amino acids, sugars, lipids. And at least one selected from the group consisting of compounds, but is not particularly limited thereto.

In the screening method of the present invention, after the test substance is administered to the sample, a candidate drug for the treatment of sporadic Parkinson's disease, the expression level of TXNIP, which is an index factor of the present invention, is measured, and before the treatment of the test substance, When the expression decreases, the test substance is selected as a prophylactic or therapeutic agent for sporadic Parkinson's disease, and when the expression of the gene is unchanged or the expression is increased compared to before the treatment of the test substance, the test substance is selected as an agent for preventing or treating sporadic Parkinson's disease. Otherwise, candidate candidates for preventing or treating sporadic Parkinson's disease can be screened.

The present invention provides a composition for preventing or treating sporadic Parkinson's disease-derived LRRK2-G2019S mutations comprising an oligonucleotide consisting of the sequence of SEQ ID NO: 4.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders.

"Prevention" means any action that inhibits or delays the development of a related disease. It will be apparent to those skilled in the art that the compositions herein can prevent the initial symptoms, or related diseases, if administered before they appear.

Substances that inhibit the expression of the TXNIP gene include low molecular weight drugs, genetic drugs, protein drugs, extracts, nucleic acids, nucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, aptamers, chemicals, enzymes, amino acids, sugars, It may be at least one selected from the group consisting of lipids, compounds (natural compounds and / or synthetic compounds) and constituent elements thereof, but is not particularly limited as long as it has a substance capable of inhibiting the expression of the TXNIP gene.

The sporadic Parkinson's disease-derived LRRK2-G2019S mutation may be a form (G2019S) substituted with Serine instead of Glycine of the kinase protein, which is a final translation product due to a gene sequence mutation. In this case, the activity of LRRK2 kinase is upregulated to induce neuronal degeneration, which can lead to sporadic Parkinson's disease.

The oligonucleotide may be composed of the sequence of SEQ ID NO: 4, which inhibits the expression of the TXNIP gene by RNAi that destroys the mRNA by binding complementarily to TXNIP mRNA, which is a transcript of the TXNIP gene, to form a RISC complex.

The oligonucleotide is functionally equivalent to the oligonucleotide although the functional equivalent of the nucleic acid molecule constituting it, eg, some nucleotide sequence of the oligonucleotide, has been modified by deletion, substitution or insertion. The idea is to include variants that can do the same thing.

The oligonucleotides may be isolated or prepared using standard molecular biology techniques such as chemical synthesis or recombinant methods, or may be commercially available. In addition, the composition of the present invention may include not only the oligonucleotide itself, but also other substances capable of increasing the expression rate of the oligonucleotide in cells, such as compounds, natural products, novel proteins, and the like.

On the other hand, the oligonucleotide may be provided included in a vector for intracellular expression.

The oligonucleotides can be introduced into cells using a variety of transformation techniques, such as complexes of DNA and DEAE-dextran, complexes of DNA and nuclear proteins, complexes of DNA and lipids, and for this purpose the oligonucleotides are introduced into cells. It may be in a form contained within the carrier to enable efficient introduction. The carrier is preferably a vector, and both viral and non-viral vectors can be used. As a viral vector, for example, lentiviruses, retroviruses, adenoviruses, adenoviruses, herpesviruses, and abipoxvirus vectors may be used. Preferably is a lentiviral vector, but is not limited thereto. Lentiviruses are a type of retrovirus that infects dividing as well as dividing cells due to the nucleophilicity of a pre-integrated complex (virus "shell") that enables active introduction into the nucleopore or the complete nuclear membrane. There are features that can be made.

In addition, the vector containing the oligonucleotide preferably further comprises a selection marker. In the present invention, the term "selection marker" is intended to facilitate selection of transformed cells in which the oligonucleotide is introduced. The selectable markers that can be used in the vector are not particularly limited as long as they are genes capable of easily detecting or measuring the introduction of the vector, but typically, drug resistance, nutritional requirements, resistance to cytotoxic agents, or surface proteins. Markers that confer a selectable phenotype, such as expression, for example GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol dihydro Genase (histidinol dehydrogenase gene: hisD) and guanine phosphosribosyltransferase (Gpt), and the like, and preferably GFP (green fluorescent protein) and puromycin markers can be used.

On the other hand, the composition may further comprise a pharmaceutically acceptable carrier, it may be formulated with the carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition of the present invention is applicable to any formulation containing the oligonucleotide as an active ingredient, it can be prepared in oral or parenteral formulations. Pharmaceutical formulations of the present invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

The composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels depend on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other factors well known in the medical field. Can be determined. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the composition of the present invention varies widely depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and the appropriate dosage is, for example, Depending on the amount of drug accumulated in the body and / or the specific efficacy of the oligonucleotide used. It can be calculated on the basis of EC50, which is generally determined to be effective in in vivo animal models and in vitro, for example from 0.01 μg to 1 g per kg of body weight, in unit periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition can be administered for relapse.

The composition of the present invention may further contain a compound which maintains / increases the solubility and / or absorption of one or more of the active ingredients or the active ingredients exhibiting the same or similar functions in connection with the treatment of sporadic Parkinson's disease. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The present invention relates to sporadic Parkinson's disease derived from LRRK2-G2019S mutation comprising a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence. It provides a diagnostic composition of.

The sporadic Parkinson's disease-derived LRRK2-G2019S mutation may be a form (G2019S) substituted with Serine instead of Glycine of the kinase protein, which is a final translation product due to a gene sequence mutation. In this case, the activity of LRRK2 kinase is upregulated to induce neuronal degeneration, which can lead to sporadic Parkinson's disease.

The substance that specifically binds to the protein may specifically be an antibody, and the antibody refers to a specific immunoglobulin directed toward an antigenic site, and the antibody may refer to an antibody that specifically binds to the TXNIP protein. Means, the TXNIP gene can be cloned into an expression vector to obtain the TXNIP protein encoded by the TXNIP gene, and antibodies can be prepared according to conventional methods in the art from the obtained TXNIP protein. The forms of the antibody include polyclonal antibodies or monoclonal antibodies, and include all immunoglobulin antibodies. Said antibody does not have the structure of a full form intact antibody having two light chains and two full length heavy chains, as well as a full form intact antibody having two light chains and two heavy chains, but is directed against antigenic sites Also included are functional fragments of 'antibody' molecules that possess specific antigen binding sites (binding domains) and retain antigen binding function. In diagnosing sporadic Parkinson's disease using the composition of the present invention, sporadic Parkinson's disease can be diagnosed by hybridization using the TXNIP protein and the antibody and measuring the expression level of TXNIP through the degree of hybridization. Selection of the appropriate antibody and hybridization conditions may be appropriately selected according to techniques known in the art.

The nucleotide sequence, the sequence complementary to the nucleotide sequence, and the substance specifically binding to the fragment of the nucleotide may be specifically a probe or a primer.

The probe refers to a nucleotide fragment such as RNA or DNA, which is capable of specifically binding to nucleotides such as mRNA, which is short to several bases to hundreds of bases, and is labeled with radioactive elements, and thus the presence or absence of a specific mRNA. , The content (expression) can be confirmed. The probe may be prepared in the form of oligonucleotide (probe), single strand DNA (probe), double strand DNA (double strand DNA) probe, RNA "probe", etc., the probe is complementary to the mRNA of TXNIP gene Sporadic Parkinson's disease can be diagnosed by performing hybridization by measuring the expression level of mRNA through the degree of hybridization. The selection of appropriate probes and the hybridization conditions can be appropriately selected according to techniques known in the art.

The primer refers to a short nucleotide sequence that can form a base pair with a complementary template with a nucleotide sequence having a short free 3-terminal hydroxyl group and serves as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase / polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures, PCR amplification using mRNA primers can be used to diagnose sporadic Parkinson's disease by measuring the expression level of the desired TXNIP protein. PCR conditions and the length of the primer set may be appropriately selected according to techniques known in the art.

Since the nucleotide sequence encoding the TXNIP gene, the sequence complementary to the nucleotide sequence, or the primer or primer that specifically binds to the fragment of the nucleotide is known, the nucleotide sequence of the TXNIP gene is known. Probes can be designed according to conventional methods in the art.

The probe or primer may be chemically synthesized using phosphoramidite solid support synthesis or other well known methods, and may be 10 to 100 nucleotides in length (hereinafter referred to as 'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 20 to 30 nt, or 20 to 25 nt.

The present invention provides a LRRK2-G2019S mutation comprising a composition comprising a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence. Provided is a kit for diagnosing sporadic Parkinson's disease derived.

The sporadic Parkinson's disease-derived LRRK2-G2019S mutation may be a form (G2019S) substituted with Serine instead of Glycine of the kinase protein, which is a final translation product due to a gene sequence mutation. In this case, the activity of LRRK2 kinase is upregulated to induce neuronal degeneration, which can lead to sporadic Parkinson's disease.

The kit can diagnose sporadic Parkinson's disease by measuring the expression level of TXNIP protein by measuring the expression level of TXNIP mRNA or TXNIP protein. The kit not only includes a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence, but also the TXNIP used by the kit. It may include one or more other component compositions, solutions or devices suitable for assays that measure the amount of protein expression.

When the kit is a kit for measuring the expression level of TXNIP mRNA, it may be a kit including the necessary elements necessary to perform RT-PCR. RT-PCR kits include test tubes or other appropriate containers, reaction buffers, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerases and reverse transcriptases, as well as individual “primer” pairs specific for mRNA of the marker gene. Inhibitors, DEPC-water, sterile water, and the like. In addition, it may include a "primer" pair specific to the gene used as a quantitative control.

The kit comprises a substrate, a suitable buffer for immunological detection of a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence. It may include a secondary antibody, a coloring substrate, labeled with a coloring enzyme or a fluorescent material. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a glass slide glass, and the like. The chromophore may be a peroxidase, an alkaline phosphatase ( alkaline phosphatase) may be used, the fluorescent material may be FITC, RITC, etc., and the chromogenic substrate may be 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB) and the like can be used.

The kit may be a sporadic Parkinson's disease microarray for measuring the amount of TXNIP protein or TXNIP mRNA expression. The microarray can be easily prepared by those skilled in the art using the TXNIP indicator, according to a method known in the art. According to one embodiment, the cDNA is a probe of the sequence corresponding to the mRNA or fragment thereof of the TXNIP gene. It may be a microarray attached to the substrate as a.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example  1. Experimental Materials and Methods

1. Human iPSC Culture and Analysis

Human fibroblasts include human cells comprising Dulbecco's modified Eagle's medium, 10% calf serum, 1% non-essential amino acids (Gibco), 0.1% β-mercaptoethanol and 1% penicillin / streptomycin (Gibco). Cultured in the culture medium. To generate hiPSCs (human induced pluripotent cells) from fibroblasts, cells in passage 6 were infected twice a day with lentiviral constructs containing Oct4, Sox2, C-myc and Klf4. hiPSCs were cultured in human embryonic stem cell growth medium (CEFO Co., Ltd. Seoul, Korea) on Matrigel at 37 ° C. and 5% CO ₂ . For transduction into donor and CRISPR / Cas9 vectors, hiPSCs were treated with Accutase (A6964, Sigma) and transformed with the Gene Pulser Xcell Electroporation System (Bio-Rad). These cells were sprinkled on Matrigel coated plates and human embryonic stem cell growth medium was replaced daily. hiPSCs were immunized using primary and appropriate fluorescent secondary antibodies (Invitrogen) against OCT4 (Santa Cruz), SOX2 (R & D), NANOG (Bethyl Laboratories), SSEA1 (Santa Cruz) and TRA 1-60 (Millipore) Stained.

2. Midbrain-like 3D organoid culture

hiPSCs were treated with Accutase to induce dissociation into single cells, and embryoid bodies were maintained for 3 days in human embryonic stem cell growth medium without ROCK inhibitor. To generate 3D organoids and their structural construction, embryonic bodies (EBs) were inserted into Matrigel matrix (Corning) containing 60% laminin, 30% collagen IV and 8% enactin. It was. EB embedded in Matrigel was maintained in 37 ° C and 5% CO ₂ incubators. Human embryonic stem cell growth medium was replaced with neural stem cell medium (Advanced DMEM / F12 and neurobasal medium, 1x N2, 1x B27, 5% Albumax-I, 2 mM GlutaMAX, 0.1 mM beta-mercaptoethanol, 3 μM CHIR99021, 0.5 μM A83-01, and 10 ng / ml LIF). In addition, fibroblast growth factor-2 (20 ng / ml) and epidermal growth factor (20 ng / ml) were added to neural stem cell medium for neural induction. To promote patterning and maturation of the midbrain, ascorbic acid (200 nM), brain-derived neurotrophic factor (20 ng / ml), sonic hedgehog (SHH, 100 ng) / ml) and glia cell-derived neurotrophic factor (20 ng / ml) aggregated cells were cultured in neural stem cell medium. The culture medium was changed every 3 days and the organisms were maintained in an orbital shaker (PSU-10i, Biosan) at 37 ° C. and 5% CO ₂ incubator.

3. Flow cytometry

Midbrain-like 3D organoids were dissociated using a homogenizer and treated with 0.125% trypsin-EDTA for 5 minutes. Single cells were resuspended in 4% paraformaldehyde-dissolved phosphate buffered saline and incubated at room temperature for 10 minutes. Fixed cells were washed twice with 1% bovine serum albumin and filtered to remove aggregated cells. After filtration, the cells were resuspended in fluorescence-activated cell sorting buffer. Flow cytometry was performed using Accuri instrument (Becton-Dickinson) and data analysis was performed by FlowJo vX software (TreeStar).

4. Immunofluorescence Staining Assay

Organoids were washed with 1 × phosphate buffered saline before being fixed in phosphate buffered saline with 4% paraformaldehyde dissolved and cryoprotected in 30% sucrose overnight. Frozen organoids were cut to 15-20 μm using Thermo Shandon cryotome and collected on glass microscope slides. OTX2 (Abcam), FOXA2 (Abcam), NGN2 (Millipore), MASH1 (BD Biosciences), TUJ1 (Sigma), MAP2 (Cell Signaling), TH (Pel-Freez) (Abcam), AATC (Abcam), NURR1 (Santa Cruz), GIRK2 (Abcam), cleaved-caspase-3 (Cell Signaling), α-synuclein (BD Biosciences), LAMP1 (Development Research Hybridoma Bank), γH2AX (cell signal) and EEA1 (cell signal) primary antibodies Sections were immunostained according to standard protocols. Appropriate fluorescent secondary antibodies were obtained from Invitrogen. Next, the sections were treated with 6-diamino-2-phenylindole (6-diamidino-2-phenylindole, DAPI, Invitrogen) and mounted on Fluoromount-G mounting medium. Representative images were captured using a Nikon Eclipse Ti microscope and a confocal laser scanning microscope (ZEISS, LSM800).

5. Western blot analysis

Organoids were dissociated by homogenizer and washed with lx phosphate buffered saline. Homogenized organoids were dissolved in lysis buffer (1% NP-40, 0.5% DOC, 0.1% SDS, 150 mmol / L NaCl in 50 mmol / L Tris, pH 8.0, Sigma-Aldrich; and 1x proteinase inhibitor mixture, Roche) Extracted. The extracted protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. These membranes include NURR1 (1: 200, Santa Cruz), VMAT2 (1: 1000, Abcam), PITX3 (1: 1000, Invitrogen), pS129-alpha-synuclein (1: 500, Abcam), cleaved caspase-3 (1: 500, Cell Signaling) and beta-actin (1: 1000, AbFrontier). Representative images of Western blots were displayed by Chemidoc TRS + with Image Lab software (Bio-Rad).

6. qRT-PCR Analysis

Total RNA was isolated from organism samples using the RNeasy RNA isolation kit (QIAGEN). Complementary DNA was then synthesized using AccuPower RT-PCR PreMix (Bioneer). QRT-PCR was performed using SYBR Green Real-time PCR Master Mix (Invitrogen). qRT-PCR analysis was performed in a Rotor-Gene Q RT-PCR cycler (QIAGEN) after 1/50 dilution of reverse transcription reaction. Gene expression of all target genes was normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase in each sample.

7. Gene expression profiling using microarray

Affymetrix GeneChip Human Gene 1.0 ST array was performed according to manufacturer's protocol. Robust Multiarray Averaging (RMA) method using affy R package was used for normalization and summarization. Comparative analysis of test and control samples was performed using the LPE test and the fold change of the null hypothesis that there was no difference between the two groups. FDR (False Discovery Rate) was controlled by p value using Benjamini-Hochberg algorithm.

8. Network Analysis

Protein interactions of homo sapiens were obtained from STRING (https://string-db.org/, v10.5). To identify the network between Parkinson's disease and the TXNIP gene, Cytoscape (http://www.Cytoscape.org, v3.6.1) included the DEG and CTD gene sets from 3D_LRRK2 / 3d_Control.

9. Statistical Analysis

All data are expressed as mean ± standard deviation of three independent experiments. n represents the number of independent experiments performed or the number of individual experiments or mice. For independent in vitro experiments, at least three technical replicates were used, and at least three independent experiments were performed to ensure adequate statistical capability. In all analyzes, group differences were considered statistically significant at p <0.05 (* p <0.05, ** p <0.01). ANOVA was used for the multidimensional comparison, and after the normal distribution was confirmed, the student's t-test was used for the two-component comparison.

Example  2. from hiPSC  Midbrain Similar 3D Organoids  Comparison of Manufacturing and Similarity with Human Midbrain

To prepare midbrain organoids, the self-organizing principle was applied to hiPSC culture. First, hiPSCs were dissociated into embryoid bodies (EBs) and organized to form 3D structures using Matrigel. These EBs were treated with GSK-3 inhibitors (CHIR99021) and TGF-β inhibitors (Noggin and SB431542) to promote neuroectoderm differentiation. Addition of FGF8, EGF and Sonic Hedgehog (SHH) triggers patterning to fate of the midbrain (FIGS. 1A and 6A). While mRNA expression of the neuroectoderm marker SOX1 increased significantly on day 5 in EB, expression of the pluripotency marker OCT4 was markedly decreased immediately after organoid production. From the 15th day, at the onset of further differentiation into the midbrain-like stage in three-dimensional conditions, the expression of the dopaminergic nerve markers VMAT2 and TH increased rapidly (FIG. 1B). Consistently, expression of midbrain markers NURR1 and DAT were detected in midbrain organoids at 6 and 8 weeks, respectively (FIG. 6B). Dopaminergic neurons expressing mature neuronal markers TH, VMAT2, GIRK2 and DAT were analyzed by immunostaining to confirm the production of mature dopaminergic neurons in midbrain organoids on day 60 (FIGS. 1C and 6C). In addition, the expression of PITX3 and AADC significantly increased from day 30 (Figs. 6D and 6E). In addition, we found that most of the MASH1 positive cells (50%) stayed in the ventricular zone (VZ), through which radical glia cells matured into MAP2 positive cells as they migrated to the marginal zone (MZ) (FIG. 1C, 1D and 1E). qPCR (FIG. 1F) was used to observe potent expression of additional midbrain markers in the midbrain organoids, suggesting that on day 45 the organoids more closely resemble mature dopaminergic organoids. In addition, flow cytometry was performed to analyze red fluorescent protein positive cells derived from organoids carrying a synapsin1-red fluorescent protein reporter. FACS analysis showed that red fluorescent protein positive cells increased from 2.3% on day 15 to 21.5% on day 45 (FIG. 1G). Liquid chromatography-mass spectrometry analysis of dopamine release showed that dopamine levels increased significantly in midbrain-like organoids on day 45 (FIGS. 1H and 1I). In addition, the gene database derived from the human midbrain revealed a large number of genes differentially expressed in organoids on day 45 (Figure 1J). In addition, to demonstrate whether the organoids reproduce the 3D brain environment, the expression of neuromelanin in the midbrain organoids was examined and found that consistently significant amounts of neuromelanin were detected in the organoids on day 60 (FIG. 7A and 7B). In addition, genes such as ANLN, GLA35ST1, FAH, MBP, ACSL1 and CLDN11, which are known to be highly expressed in elderly human midbrain, were highly expressed in midbrain organoids on day 60 (FIG. 7C). In addition, genes differentially expressed in organoids on day 60 were expressed with many genes differentially expressed from aged human brain (derived from the database) (FIG. 7D). In addition, phosphorylation of histone H2AX, a marker of aging, significantly increased in midbrain organoids on day 60 (FIGS. 7E and 7F). Taken together, this data suggests that at about 60 days the midbrain organoids are very similar to the midbrain of the elderly.

Midbrain-like 3D organoids were also compared to 2D organoids. At 45 days after differentiation, the 3D organisms significantly increased the proportions with the tissue structures of MAP2 + and TH + cells (FIGS. 8A and 8B). In addition, the expression of AADC, CHAT, MAPT, and astrocyte glial marker genes GFAP, S100B, ALDH1L1 were significantly increased in the midbrain-like 3D organoids, while the 2D organoids were slowly increased (FIGS. In addition, gene ontological analysis of biological processes in 3D organoids has been shown to be associated with neuronal development, neuronal differentiation, and cell development, and 3D midbrain organoids are more efficient at dopamine neurons (FIGS. 8D and 8E). Implies that Moreover, gene set enrichment analysis showed that genes upregulated in 3D organoids were enriched in the elderly midbrain compared to 2D organoids (FIG. 1J and FIG. 8F).

Example  3. LRRK2 - G2019S  Mutant-derived Parkinson's Disease Model Midbrain-like 3D Organoids  Preparation and Comparison of Similarities with the Human Brain in Parkinson's Disease

For modeling Parkinson's disease, a heterozygous LRRK2-G2019S point mutation was introduced into hiPSC by generating an isogenic hiPSC using the CRISPR / Cas9 nuclease system. A guide RNA target site 4 bp away from the G2019S mutation site at exon 41 of the human LRRK2 locus (FIG. 9A) was designed. Surveyor nuclease assay and Sanger sequencing analysis performed to confirm that the CRISPR / Cas9 nuclease system can efficiently cleave the LRRK2 locus of hiPSC showed LRRK2 − / − cells with deletions of 3 to 26 bp (FIG. 9B). And 9C). Donor plasmids containing G2019S mutations were transfected simultaneously with the guide RNA / Cas9 vector and then puromycin and CRE-selected cells were identified by PCR based assays (FIGS. 9D-9F). The control and LRRK2-G2019S mutants introduced hiPSCs expressed endogenous pluripotent genes such as TRA 1-60, SOX2, OCT4, NANOG and SSEA1 with no difference in pluripotency and self-renewal assays (FIG. 9G To 9J).

Next, mutant midbrain organoids were generated from hiPSCs (FIGS. 10A-10D) into which the LRRK2-G2019S mutation was introduced using the 3D organoid protocol. Neuronal identity of the LRRK2-G2019S midbrain organoids was confirmed by immunohistochemistry of the neuronal markers TUJ1, MAP2, NURR1 and DAT (FIG. 2A). Expression of the dopaminergic nerve markers TH, AADC, VMAT2 and DAT was significantly reduced in organoids into which the LRRK2-G2019S mutation was introduced at 60 days compared to the control (FIGS. 2B and 10E). Mature neuronal genes such as NURR1, PITX3, EN1, TH, and MAPT were reduced in organoids into which the LRRK2-G2019S mutation was introduced, but not in 2D organoids, and the 3D organoid model showed stronger disease pathology (Figure 11A). ).

Example  4. LRRK2 - G2019S  Mutant-derived Parkinson's Disease Model Midbrain-like 3D Organoids  Possible diagnostic or drug screening analysis

Next, the susceptibility of organoids to which the LRRK2-G2019S mutation was introduced for neurotoxic damage caused by MPTP was examined. A significant increase in caspase-3 + apoptotic DA neurons cleaved from organoids to which LRRK2-G2019S mutants treated with all concentrations of MPTP was introduced (FIGS. 2C-2F). In addition, to assess the effect of the LRRK2-G2019S mutation on Parkinson's disease-related etiology, it was determined whether the organoids with the LRRK2-G2019S mutation exhibit abnormal localization of phosphorylated α-synuclein in the endosomal compartment of the vesicle. Checked. It was observed that pS129-α-synuclein-positive vesicles were localized to endosome positive for the early endosome marker EEA1 in 3D organoids (FIG. 3A) introduced with LRRK2-G2019S mutation. The LRRK2-G2019S mutation effectively increased the number of pS129-α-synuclein and EEA1 double positive endosome (FIG. 3B). Surprisingly, thioflavin T positive deposition in 3D organoids at day 60 was significantly increased by LRRK2-G2019S mutations (FIGS. 3C-3E), which were introduced into the LRRK2-G2019S mutations in which pS129-α-synuclein was aged. Suggests that it is abnormally localized. To further demonstrate whether this 3D model system can be used to test treatment strategies for Parkinson's disease, pharmacological inhibition of LRRK2 kinase activity was evaluated. Treatment of LRRK2 inhibitors in organoids into which the LRRK2-G2019S mutation was introduced substantially reduced the accumulation of phosphorylated α-synuclein (FIGS. 3F and 3G). Consistently, expression of the dopaminergic neuronal marker genes TH, AADC and DAT was partially recovered in organoids introduced with LRRK2-G2019S mutations treated with LRRK2 kinase inhibitors (FIG. 3H). Thus, this 3D organoid model system can test treatment strategies for Parkinson's disease.

Example  5. LRRK2 - G2019S  Mutation introduced Organoid  Analysis of gene expression patterns and discovery of indicator factor TXNIP

To gain insight into the molecular mechanism of Parkinson's disease pathology associated with LRRK2-G2019S in midbrain 3D organoids, we compared the overall gene expression patterns of the LRRK2 control and the 3D organoids introduced with the LRRK2-G2019S mutation. Midbrain 3D organoids expressing LRRK2-G2019S showed a dynamic change in overall gene expression (FIG. 4A). Genes differentially expressed in the presence of the LRRK2-G2019S mutation were significantly different in 3D organoids compared to 2D organoids (FIGS. 4A and 4B). The differentially expressed genes also overlap between LRRK2-G2019S 3D organoids and LRRK2-G2019S 2D organoids, indicating that they are specific genes affected by LRRK2-G2019S mutations (FIGS. 4A and 4C). In particular, one of the most differentially expressed genes in LRRK2-G2019S 3D organoids is the thioredoxin-interacting protein (TXNIP), which has been reported to be associated with lysosomal dysfunction in α-synuclein overexpressed cultures. It was.

Next, to investigate the extent to which the LRRK2-G2019S mutant Parkinson's disease organoids are molecularly similar to the sporadic Parkinson's disease tissue, a differential genetic pattern between the LRRK2-G2019S mutant organoids and the brain tissue of the sporadic Parkinson's disease patient (MeSH: D010300) is examined. Compared. Gene sets differentially expressed in LRRK2-G2019S 3D organoids compared to LRRK2-G2019S 2D organoids were found to express a large number of genes isolated from sporadic Parkinson's disease brain tissue (FIG. 4D), which is a LRRK2-G2019S mutation Is a suitable 3D organoid for the study of the molecular pathology of LRRK2-G2019S-related sporadic Parkinson's disease. The co-expression network was constructed by further analyzing the protein-protein interaction HTRldb database of genes differentially expressed in organoids into which the LRRK2-G2019S mutation was introduced. Interestingly, the TXNIP protein interaction network has been defined as a major subnetwork that includes Parkinson's disease hereditary risk factors (FIGS. 4E and 12A).

Example  6. LRRK2 - G2019S  Mutation introduced At organoids  Analysis of TXNIP Gene Expression as an Indicator of Sporadic Parkinson's Disease

TXNIP mRNA levels of 3D organoids into which wild-type 3D organoids and LRRK2-G2019S mutations were introduced were analyzed by real time qPCR (FIG. 5A). At the basal level, a significant increase in TXNIP mRNA levels induced by the LRRK2-G2019S mutation in 3D organoids was found in comparison to 2D organoids (FIGS. 5B and 5C). To analyze whether TXNIP functionally contributes to the Parkinson's disease phenotype derived from the LRRK2-G2019S mutation, α-synuclein aggregates were examined in 3D organoids into which the LRRK2-G2019S mutation was introduced upon TXNIP knockdown. Interestingly, α-synuclein aggregation of 3D organoids with LRRK2-G2019S mutant introduced significantly decreased in pS129-α-synuclein analysis by Western blot when TXNIP inhibition compared to wild type 3D organoids (Figures 5D-5F). . In addition, the lysosomal effect of LRRK2-G2019S-related Parkinson's disease was investigated. TXNIP knockdown inhibited aggregation of phosphorylated α-synuclein with LAMP1 (lysosomal marker) -positive puncta (FIGS. 5G and 5H). Consistent with these data, the area of pS129-α-synuclein inclusion was significantly reduced in TXNIP knockdown (Figure 5I). Overall, these results show that human TXNIP is functionally linked to the LRRK2-G2019S mutation in the 3D environment and can exacerbate LRRK2-G2019S related sporadic Parkinson's disease in the 3D environment compared to the 2D environment.

<110> DONGGUK UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Methods for providing information on the diagnosis of sporadic          Parkinson's disease and drug screening methods <130> 18P05030 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1176 <212> DNA <213> Artificial Sequence <220> <223> TXNIP gene sequence <400> 1 atggtgatgt tcaagaagat caagtctttt gaggtggtct ttaacgaccc tgaaaaggtg 60 tacggcagtg gcgagaaggt ggctggccgg gtgatagtgg aggtgtgtga agttactcgt 120 gtcaaagccg ttaggatcct ggcttgcgga gtggctaaag tgctttggat gcagggatcc 180 cagcagtgca aacagacttc ggagtacctg cgctatgaag acacgcttct tctggaagac 240 cagccaacag gtgagaatga gatggtgatc atgagacctg gaaacaaata tgagtacaag 300 ttcggctttg agcttcctca ggggcctctg ggaacatcct tcaaaggaaa atatgggtgt 360 gtagactact gggtgaaggc ttttcttgac cgcccgagcc agccaactca agagacaaag 420 aaaaactttg aagtagtgga tctggtggat gtcaataccc ctgatttaat ggcacctgtg 480 tctgctaaaa aagaaaagaa agtttcctgc atgttcattc ctgatgggcg ggtgtctgtc 540 tctgctcgaa ttgacagaaa aggattctgt gaaggtgatg agatttccat ccatgctgac 600 tttgagaata catgttcccg aattgtggtc cccaaagctg ccattgtggc ccgccacact 660 taccttgcca atggccagac caaggtgctg actcagaagt tgtcatcagt cagaggcaat 720 catattatct cagggacatg cgcatcatgg cgtggcaaga gccttcgggt tcagaagatc 780 aggccttcta tcctgggctg caacatcctt cgagttgaat attccttact gatctatgtt 840 agcgttcctg gatccaagaa ggtcatcctt gacctgcccc tggtaattgg cagcagatca 900 ggtctaagca gcagaacatc cagcatggcc agccgaacca gctctgagat gagttgggta 960 gatctgaaca tccctgatac cccagaagct cctccctgct atatggatgt cattcctgaa 1020 gatcaccgat tggagagccc aaccactcct ctgctagatg acatggatgg ctctcaagac 1080 agccctatct ttatgtatgc ccctgagttc aagttcatgc caccaccgac ttatactgag 1140 gtggatccct gcatcctcaa caacaatgtg cagtga 1176 <210> 2 <211> 7584 <212> DNA <213> Artificial Sequence <220> <223> LRRK2 gene sequence <400> 2 atggctagtg gcagctgtca ggggtgcgaa gaggacgagg aaactctgaa gaagttgata 60 gtcaggctga acaatgtcca ggaaggaaaa cagatagaaa cgctggtcca aatcctggag 120 gatctgctgg tgttcacgta ctccgagcac gcctccaagt tatttcaagg caaaaatatc 180 catgtgcctc tgttgatcgt cttggactcc tatatgagag tcgcgagtgt gcagcaggtg 240 ggttggtcac ttctgtgcaa attaatagaa gtctgtccag gtacaatgca aagcttaatg 300 ggaccccagg atgttggaaa tgattgggaa gtccttggtg ttcaccaatt gattcttaaa 360 atgctaacag ttcataatgc cagtgtaaac ttgtcagtga ttggactgaa gaccttagat 420 ctcctcctaa cttcaggtaa aatcaccttg ctgatattgg atgaagaaag tgatattttc 480 atgttaattt ttgatgccat gcactcattt ccagccaatg atgaagtcca gaaacttgga 540 tgcaaagctt tacatgtgct gtttgagaga gtctcagagg agcaactgac tgaatttgtt 600 gagaacaaag attatatgat attgttaagt gcgttaacaa attttaaaga tgaagaggaa 660 attgtgcttc atgtgctgca ttgtttacat tccctagcga ttccttgcaa taatgtggaa 720 gtcctcatga gtggcaatgt caggtgttat aatattgtgg tggaagctat gaaagcattc 780 cctatgagtg aaagaattca agaagtgagt tgctgtttgc tccataggct tacattaggt 840 aattttttca atatcctggt attaaacgaa gtccatgagt ttgtggtgaa agctgtgcag 900 cagtacccag agaatgcagc attgcagatc tcagcgctca gctgtttggc cctcctcact 960 gagactattt tcttaaatca agatttagag gaaaagaatg agaatcaaga gaatgatgat 1020 gagggggaag aagataaatt gttttggctg gaagcctgtt acaaagcatt aacgtggcat 1080 agaaagaaca agcacgtgca ggaggccgca tgctgggcac taaataatct ccttatgtac 1140 caaaacagtt tacatgagaa gattggagat gaagatggcc atttcccagc tcatagggaa 1200 gtgatgctct ccatgctgat gcattcttca tcaaaggaag ttttccaggc atctgcgaat 1260 gcattgtcaa ctctcttaga acaaaatgtt aatttcagaa aaatactgtt atcaaaagga 1320 atacacctga atgttttgga gttaatgcag aagcatatac attctcctga agtggctgaa 1380 agtggctgta aaatgctaaa tcatcttttt gaaggaagca acacttccct ggatataatg 1440 gcagcagtgg tccccaaaat actaacagtt atgaaacgtc atgagacatc attaccagtg 1500 cagctggagg cgcttcgagc tattttacat tttatagtgc ctggcatgcc agaagaatcc 1560 agggaggata cagaatttca tcataagcta aatatggtta aaaaacagtg tttcaagaat 1620 gatattcaca aactggtcct agcagctttg aacaggttca ttggaaatcc tgggattcag 1680 aaatgtggat taaaagtaat ttcttctatt gtacattttc ctgatgcatt agagatgtta 1740 tccctggaag gtgctatgga ttcagtgctt cacacactgc agatgtatcc agatgaccaa 1800 gaaattcagt gtctgggttt aagtcttata ggatacttga ttacaaagaa gaatgtgttc 1860 ataggaactg gacatctgct ggcaaaaatt ctggtttcca gcttataccg atttaaggat 1920 gttgctgaaa tacagactaa aggatttcag acaatcttag caatcctcaa attgtcagca 1980 tctttttcta agctgctggt gcatcattca tttgacttag taatattcca tcaaatgtct 2040 tccaatatca tggaacaaaa ggatcaacag tttctaaacc tctgttgcaa gtgttttgca 2100 aaagtagcta tggatgatta cttaaaaaat gtgatgctag agagagcgtg tgatcagaat 2160 aacagcatca tggttgaatg cttgcttcta ttgggagcag atgccaatca agcaaaggag 2220 ggatcttctt taatttgtca ggtatgtgag aaagagagca gtcccaaatt ggtggaactc 2280 ttactgaata gtggatctcg tgaacaagat gtacgaaaag cgttgacgat aagcattggg 2340 aaaggtgaca gccagatcat cagcttgctc ttaaggaggc tggccctgga tgtggccaac 2400 aatagcattt gccttggagg attttgtata ggaaaagttg aaccttcttg gcttggtcct 2460 ttatttccag ataagacttc taatttaagg aaacaaacaa atatagcatc tacactagca 2520 agaatggtga tcagatatca gatgaaaagt gctgtggaag aaggaacagc ctcaggcagc 2580 gatggaaatt tttctgaaga tgtgctgtct aaatttgatg aatggacctt tattcctgac 2640 tcttctatgg acagtgtgtt tgctcaaagt gatgacctgg atagtgaagg aagtgaaggc 2700 tcatttcttg tgaaaaagaa atctaattca attagtgtag gagaatttta ccgagatgcc 2760 gtattacagc gttgctcacc aaatttgcaa agacattcca attccttggg gcccattttt 2820 gatcatgaag atttactgaa gcgaaaaaga aaaatattat cttcagatga ttcactcagg 2880 tcatcaaaac ttcaatccca tatgaggcat tcagacagca tttcttctct ggcttctgag 2940 agagaatata ttacatcact agacctttca gcaaatgaac taagagatat tgatgcccta 3000 agccagaaat gctgtataag tgttcatttg gagcatcttg aaaagctgga gcttcaccag 3060 aatgcactca cgagctttcc acaacagcta tgtgaaactc tgaagagttt gacacatttg 3120 gacttgcaca gtaataaatt tacatcattt ccttcttatt tgttgaaaat gagttgtatt 3180 gctaatcttg atgtctctcg aaatgacatt ggaccctcag tggttttaga tcctacagtg 3240 aaatgtccaa ctctgaaaca gtttaacctg tcatataacc agctgtcttt tgtacctgag 3300 aacctcactg atgtggtaga gaaactggag cagctcattt tagaaggaaa taaaatatca 3360 gggatatgct cccccttgag actgaaggaa ctgaagattt taaaccttag taagaaccac 3420 atttcatccc tatcagagaa ctttcttgag gcttgtccta aagtggagag tttcagtgcc 3480 agaatgaatt ttcttgctgc tatgcctttc ttgcctcctt ctatgacaat cctaaaatta 3540 tctcagaaca aattttcctg tattccagaa gcaattttaa atcttccaca cttgcggtct 3600 ttagatatga gcagcaatga tattcagtac ctaccaggtc ccgcacactg gaaatctttg 3660 aacttaaggg aactcttatt tagccataat cagatcagca tcttggactt gagtgaaaaa 3720 gcatatttat ggtctagagt agagaaactg catctttctc acaataaact gaaagagatt 3780 cctcctgaga ttggctgtct tgaaaatctg acatctctgg atgtcagtta caacttggaa 3840 ctaagatcct ttcccaatga aatggggaaa ttaagcaaaa tatgggatct tcctttggat 3900 gaactgcatc ttaactttga ttttaaacat ataggatgta aagccaaaga catcataagg 3960 tttcttcaac agcgattaaa aaaggctgtg ccttataacc gaatgaaact tatgattgtg 4020 ggaaatactg ggagtggtaa aaccacctta ttgcagcaat taatgaaaac caagaaatca 4080 gatcttggaa tgcaaagtgc cacagttggc atagatgtga aagactggcc tatccaaata 4140 agagacaaaa gaaagagaga tctcgtccta aatgtgtggg attttgcagg tcgtgaggaa 4200 ttctatagta ctcatcccca ttttatgacg cagcgagcat tgtaccttgc tgtctatgac 4260 ctcagcaagg gacaggctga agttgatgcc atgaagcctt ggctcttcaa tataaaggct 4320 cgcgcttctt cttcccctgt gattctcgtt ggcacacatt tggatgtttc tgatgagaag 4380 caacgcaaag cctgcatgag taaaatcacc aaggaactcc tgaataagcg agggttccct 4440 gccatacgag attaccactt tgtgaatgcc accgaggaat ctgatgcttt ggcaaaactt 4500 cggaaaacca tcataaacga gagccttaat ttcaagatcc gagatcagct tgttgttgga 4560 cagctgattc cagactgcta tgtagaactt gaaaaaatca ttttatcgga gcgtaaaaat 4620 gtgccaattg aatttcccgt aattgaccgg aaacgattat tacaactagt gagagaaaat 4680 cagctgcagt tagatgaaaa tgagcttcct cacgcagttc actttctaaa tgaatcagga 4740 gtccttcttc attttcaaga cccagcactg cagttaagtg acttgtactt tgtggaaccc 4800 aagtggcttt gtaaaatcat ggcacagatt ttgacagtga aagtggaagg ttgtccaaaa 4860 caccctaagg gcattatttc gcgtagagat gtggaaaaat ttctttcaaa aaaaaggaaa 4920 tttccaaaga actacatgtc acagtatttt aagctcctag aaaaattcca gattgctttg 4980 ccaataggag aagaatattt gctggttcca agcagtttgt ctgaccacag gcctgtgata 5040 gagcttcccc attgtgagaa ctctgaaatt atcatccgac tatatgaaat gccttatttt 5100 ccaatgggat tttggtcaag attaatcaat cgattacttg agatttcacc ttacatgctt 5160 tcagggagag aacgagcact tcgcccaaac agaatgtatt ggcgacaagg catttactta 5220 aattggtctc ctgaagctta ttgtctggta ggatctgaag tcttagacaa tcatccagag 5280 agtttcttaa aaattacagt tccttcttgt agaaaaggct gtattctttt gggccaagtt 5340 gtggaccaca ttgattctct catggaagaa tggtttcctg ggttgctgga gattgatatt 5400 tgtggtgaag gagaaactct gttgaagaaa tgggcattat atagttttaa tgatggtgaa 5460 gaacatcaaa aaatcttact tgatgacttg atgaagaaag cagaggaagg agatctctta 5520 gtaaatccag atcaaccaag gctcaccatt ccaatatctc agattgcccc tgacttgatt 5580 ttggctgacc tgcctagaaa tattatgttg aataatgatg agttggaatt tgaacaagct 5640 ccagagtttc tcctaggtga tggcagtttt ggatcagttt accgagcagc ctatgaagga 5700 gaagaagtgg ctgtgaagat ttttaataaa catacatcac tcaggctgtt aagacaagag 5760 cttgtggtgc tttgccacct ccaccacccc agtttgatat ctttgctggc agctgggatt 5820 cgtccccgga tgttggtgat ggagttagcc tccaagggtt ccttggatcg cctgcttcag 5880 caggacaaag ccagcctcac tagaacccta cagcacagga ttgcactcca cgtagctgat 5940 ggtttgagat acctccactc agccatgatt atataccgag acctgaaacc ccacaatgtg 6000 ctgcttttca cactgtatcc caatgctgcc atcattgcaa agattgctga ctacggcatt 6060 gctcagtact gctgtagaat ggggataaaa acatcagagg gcacaccagg gtttcgtgca 6120 cctgaagttg ccagaggaaa tgtcatttat aaccaacagg ctgatgttta ttcatttggt 6180 ttactactct atgacatttt gacaactgga ggtagaatag tagagggttt gaagtttcca 6240 aatgagtttg atgaattaga aatacaagga aaattacctg atccagttaa agaatatggt 6300 tgtgccccat ggcctatggt tgagaaatta attaaacagt gtttgaaaga aaatcctcaa 6360 gaaaggccta cttctgccca ggtctttgac attttgaatt cagctgaatt agtctgtctg 6420 acgagacgca ttttattacc taaaaacgta attgttgaat gcatggttgc tacacatcac 6480 aacagcagga atgcaagcat ttggctgggc tgtgggcaca ccgacagagg acagctctca 6540 tttcttgact taaatactga aggatacact tctgaggaag ttgctgatag tagaatattg 6600 tgcttagcct tggtgcatct tcctgttgaa aaggaaagct ggattgtgtc tgggacacag 6660 tctggtactc tcctggtcat caataccgaa gatgggaaaa agagacatac cctagaaaag 6720 atgactgatt ctgtcacttg tttgtattgc aattcctttt ccaagcaaag caaacaaaaa 6780 aattttcttt tggttggaac cgctgatggc aagttagcaa tttttgaaga taagactgtt 6840 aagcttaaag gagctgctcc tttgaagata ctaaatatag gaaatgtcag tactccattg 6900 atgtgtttga gtgaatccac aaattcaacg gaaagaaatg taatgtgggg aggatgtggc 6960 acaaagattt tctccttttc taatgatttc accattcaga aactcattga gacaagaaca 7020 agccaactgt tttcttatgc agctttcagt gattccaaca tcataacagt ggtggtagac 7080 actgctctct atattgctaa gcaaaatagc cctgttgtgg aagtgtggga taagaaaact 7140 gaaaaactct gtggactaat agactgcgtg cactttttaa gggaggtaat ggtaaaagaa 7200 aacaaggaat caaaacacaa aatgtcttat tctgggagag tgaaaaccct ctgccttcag 7260 aagaacactg ctctttggat aggaactgga ggaggccata ttttactcct ggatctttca 7320 actcgtcgac ttatacgtgt aatttacaac ttttgtaatt cggtcagagt catgatgaca 7380 gcacagctag gaagccttaa aaatgtcatg ctggtattgg gctacaaccg gaaaaatact 7440 gaaggtacac aaaagcagaa agagatacaa tcttgcttga ccgtttggga catcaatctt 7500 ccacatgaag tgcaaaattt agaaaaacac attgaagtga gaaaagaatt agctgaaaaa 7560 atgagacgaa catctgttga gtaa 7584 <210> 3 <211> 7584 <212> DNA <213> Artificial Sequence <220> <223> LRRK2-G2019S mutated gene sequence <400> 3 atggctagtg gcagctgtca ggggtgcgaa gaggacgagg aaactctgaa gaagttgata 60 gtcaggctga acaatgtcca ggaaggaaaa cagatagaaa cgctggtcca aatcctggag 120 gatctgctgg tgttcacgta ctccgagcac gcctccaagt tatttcaagg caaaaatatc 180 catgtgcctc tgttgatcgt cttggactcc tatatgagag tcgcgagtgt gcagcaggtg 240 ggttggtcac ttctgtgcaa attaatagaa gtctgtccag gtacaatgca aagcttaatg 300 ggaccccagg atgttggaaa tgattgggaa gtccttggtg ttcaccaatt gattcttaaa 360 atgctaacag ttcataatgc cagtgtaaac ttgtcagtga ttggactgaa gaccttagat 420 ctcctcctaa cttcaggtaa aatcaccttg ctgatattgg atgaagaaag tgatattttc 480 atgttaattt ttgatgccat gcactcattt ccagccaatg atgaagtcca gaaacttgga 540 tgcaaagctt tacatgtgct gtttgagaga gtctcagagg agcaactgac tgaatttgtt 600 gagaacaaag attatatgat attgttaagt gcgttaacaa attttaaaga tgaagaggaa 660 attgtgcttc atgtgctgca ttgtttacat tccctagcga ttccttgcaa taatgtggaa 720 gtcctcatga gtggcaatgt caggtgttat aatattgtgg tggaagctat gaaagcattc 780 cctatgagtg aaagaattca agaagtgagt tgctgtttgc tccataggct tacattaggt 840 aattttttca atatcctggt attaaacgaa gtccatgagt ttgtggtgaa agctgtgcag 900 cagtacccag agaatgcagc attgcagatc tcagcgctca gctgtttggc cctcctcact 960 gagactattt tcttaaatca agatttagag gaaaagaatg agaatcaaga gaatgatgat 1020 gagggggaag aagataaatt gttttggctg gaagcctgtt acaaagcatt aacgtggcat 1080 agaaagaaca agcacgtgca ggaggccgca tgctgggcac taaataatct ccttatgtac 1140 caaaacagtt tacatgagaa gattggagat gaagatggcc atttcccagc tcatagggaa 1200 gtgatgctct ccatgctgat gcattcttca tcaaaggaag ttttccaggc atctgcgaat 1260 gcattgtcaa ctctcttaga acaaaatgtt aatttcagaa aaatactgtt atcaaaagga 1320 atacacctga atgttttgga gttaatgcag aagcatatac attctcctga agtggctgaa 1380 agtggctgta aaatgctaaa tcatcttttt gaaggaagca acacttccct ggatataatg 1440 gcagcagtgg tccccaaaat actaacagtt atgaaacgtc atgagacatc attaccagtg 1500 cagctggagg cgcttcgagc tattttacat tttatagtgc ctggcatgcc agaagaatcc 1560 agggaggata cagaatttca tcataagcta aatatggtta aaaaacagtg tttcaagaat 1620 gatattcaca aactggtcct agcagctttg aacaggttca ttggaaatcc tgggattcag 1680 aaatgtggat taaaagtaat ttcttctatt gtacattttc ctgatgcatt agagatgtta 1740 tccctggaag gtgctatgga ttcagtgctt cacacactgc agatgtatcc agatgaccaa 1800 gaaattcagt gtctgggttt aagtcttata ggatacttga ttacaaagaa gaatgtgttc 1860 ataggaactg gacatctgct ggcaaaaatt ctggtttcca gcttataccg atttaaggat 1920 gttgctgaaa tacagactaa aggatttcag acaatcttag caatcctcaa attgtcagca 1980 tctttttcta agctgctggt gcatcattca tttgacttag taatattcca tcaaatgtct 2040 tccaatatca tggaacaaaa ggatcaacag tttctaaacc tctgttgcaa gtgttttgca 2100 aaagtagcta tggatgatta cttaaaaaat gtgatgctag agagagcgtg tgatcagaat 2160 aacagcatca tggttgaatg cttgcttcta ttgggagcag atgccaatca agcaaaggag 2220 ggatcttctt taatttgtca ggtatgtgag aaagagagca gtcccaaatt ggtggaactc 2280 ttactgaata gtggatctcg tgaacaagat gtacgaaaag cgttgacgat aagcattggg 2340 aaaggtgaca gccagatcat cagcttgctc ttaaggaggc tggccctgga tgtggccaac 2400 aatagcattt gccttggagg attttgtata ggaaaagttg aaccttcttg gcttggtcct 2460 ttatttccag ataagacttc taatttaagg aaacaaacaa atatagcatc tacactagca 2520 agaatggtga tcagatatca gatgaaaagt gctgtggaag aaggaacagc ctcaggcagc 2580 gatggaaatt tttctgaaga tgtgctgtct aaatttgatg aatggacctt tattcctgac 2640 tcttctatgg acagtgtgtt tgctcaaagt gatgacctgg atagtgaagg aagtgaaggc 2700 tcatttcttg tgaaaaagaa atctaattca attagtgtag gagaatttta ccgagatgcc 2760 gtattacagc gttgctcacc aaatttgcaa agacattcca attccttggg gcccattttt 2820 gatcatgaag atttactgaa gcgaaaaaga aaaatattat cttcagatga ttcactcagg 2880 tcatcaaaac ttcaatccca tatgaggcat tcagacagca tttcttctct ggcttctgag 2940 agagaatata ttacatcact agacctttca gcaaatgaac taagagatat tgatgcccta 3000 agccagaaat gctgtataag tgttcatttg gagcatcttg aaaagctgga gcttcaccag 3060 aatgcactca cgagctttcc acaacagcta tgtgaaactc tgaagagttt gacacatttg 3120 gacttgcaca gtaataaatt tacatcattt ccttcttatt tgttgaaaat gagttgtatt 3180 gctaatcttg atgtctctcg aaatgacatt ggaccctcag tggttttaga tcctacagtg 3240 aaatgtccaa ctctgaaaca gtttaacctg tcatataacc agctgtcttt tgtacctgag 3300 aacctcactg atgtggtaga gaaactggag cagctcattt tagaaggaaa taaaatatca 3360 gggatatgct cccccttgag actgaaggaa ctgaagattt taaaccttag taagaaccac 3420 atttcatccc tatcagagaa ctttcttgag gcttgtccta aagtggagag tttcagtgcc 3480 agaatgaatt ttcttgctgc tatgcctttc ttgcctcctt ctatgacaat cctaaaatta 3540 tctcagaaca aattttcctg tattccagaa gcaattttaa atcttccaca cttgcggtct 3600 ttagatatga gcagcaatga tattcagtac ctaccaggtc ccgcacactg gaaatctttg 3660 aacttaaggg aactcttatt tagccataat cagatcagca tcttggactt gagtgaaaaa 3720 gcatatttat ggtctagagt agagaaactg catctttctc acaataaact gaaagagatt 3780 cctcctgaga ttggctgtct tgaaaatctg acatctctgg atgtcagtta caacttggaa 3840 ctaagatcct ttcccaatga aatggggaaa ttaagcaaaa tatgggatct tcctttggat 3900 gaactgcatc ttaactttga ttttaaacat ataggatgta aagccaaaga catcataagg 3960 tttcttcaac agcgattaaa aaaggctgtg ccttataacc gaatgaaact tatgattgtg 4020 ggaaatactg ggagtggtaa aaccacctta ttgcagcaat taatgaaaac caagaaatca 4080 gatcttggaa tgcaaagtgc cacagttggc atagatgtga aagactggcc tatccaaata 4140 agagacaaaa gaaagagaga tctcgtccta aatgtgtggg attttgcagg tcgtgaggaa 4200 ttctatagta ctcatcccca ttttatgacg cagcgagcat tgtaccttgc tgtctatgac 4260 ctcagcaagg gacaggctga agttgatgcc atgaagcctt ggctcttcaa tataaaggct 4320 cgcgcttctt cttcccctgt gattctcgtt ggcacacatt tggatgtttc tgatgagaag 4380 caacgcaaag cctgcatgag taaaatcacc aaggaactcc tgaataagcg agggttccct 4440 gccatacgag attaccactt tgtgaatgcc accgaggaat ctgatgcttt ggcaaaactt 4500 cggaaaacca tcataaacga gagccttaat ttcaagatcc gagatcagct tgttgttgga 4560 cagctgattc cagactgcta tgtagaactt gaaaaaatca ttttatcgga gcgtaaaaat 4620 gtgccaattg aatttcccgt aattgaccgg aaacgattat tacaactagt gagagaaaat 4680 cagctgcagt tagatgaaaa tgagcttcct cacgcagttc actttctaaa tgaatcagga 4740 gtccttcttc attttcaaga cccagcactg cagttaagtg acttgtactt tgtggaaccc 4800 aagtggcttt gtaaaatcat ggcacagatt ttgacagtga aagtggaagg ttgtccaaaa 4860 caccctaagg gcattatttc gcgtagagat gtggaaaaat ttctttcaaa aaaaaggaaa 4920 tttccaaaga actacatgtc acagtatttt aagctcctag aaaaattcca gattgctttg 4980 ccaataggag aagaatattt gctggttcca agcagtttgt ctgaccacag gcctgtgata 5040 gagcttcccc attgtgagaa ctctgaaatt atcatccgac tatatgaaat gccttatttt 5100 ccaatgggat tttggtcaag attaatcaat cgattacttg agatttcacc ttacatgctt 5160 tcagggagag aacgagcact tcgcccaaac agaatgtatt ggcgacaagg catttactta 5220 aattggtctc ctgaagctta ttgtctggta ggatctgaag tcttagacaa tcatccagag 5280 agtttcttaa aaattacagt tccttcttgt agaaaaggct gtattctttt gggccaagtt 5340 gtggaccaca ttgattctct catggaagaa tggtttcctg ggttgctgga gattgatatt 5400 tgtggtgaag gagaaactct gttgaagaaa tgggcattat atagttttaa tgatggtgaa 5460 gaacatcaaa aaatcttact tgatgacttg atgaagaaag cagaggaagg agatctctta 5520 gtaaatccag atcaaccaag gctcaccatt ccaatatctc agattgcccc tgacttgatt 5580 ttggctgacc tgcctagaaa tattatgttg aataatgatg agttggaatt tgaacaagct 5640 ccagagtttc tcctaggtga tggcagtttt ggatcagttt accgagcagc ctatgaagga 5700 gaagaagtgg ctgtgaagat ttttaataaa catacatcac tcaggctgtt aagacaagag 5760 cttgtggtgc tttgccacct ccaccacccc agtttgatat ctttgctggc agctgggatt 5820 cgtccccgga tgttggtgat ggagttagcc tccaagggtt ccttggatcg cctgcttcag 5880 caggacaaag ccagcctcac tagaacccta cagcacagga ttgcactcca cgtagctgat 5940 ggtttgagat acctccactc agccatgatt atataccgag acctgaaacc ccacaatgtg 6000 ctgcttttca cactgtatcc caatgctgcc atcattgcaa agattgctga ctacagcatt 6060 gctcagtact gctgtagaat ggggataaaa acatcagagg gcacaccagg gtttcgtgca 6120 cctgaagttg ccagaggaaa tgtcatttat aaccaacagg ctgatgttta ttcatttggt 6180 ttactactct atgacatttt gacaactgga ggtagaatag tagagggttt gaagtttcca 6240 aatgagtttg atgaattaga aatacaagga aaattacctg atccagttaa agaatatggt 6300 tgtgccccat ggcctatggt tgagaaatta attaaacagt gtttgaaaga aaatcctcaa 6360 gaaaggccta cttctgccca ggtctttgac attttgaatt cagctgaatt agtctgtctg 6420 acgagacgca ttttattacc taaaaacgta attgttgaat gcatggttgc tacacatcac 6480 aacagcagga atgcaagcat ttggctgggc tgtgggcaca ccgacagagg acagctctca 6540 tttcttgact taaatactga aggatacact tctgaggaag ttgctgatag tagaatattg 6600 tgcttagcct tggtgcatct tcctgttgaa aaggaaagct ggattgtgtc tgggacacag 6660 tctggtactc tcctggtcat caataccgaa gatgggaaaa agagacatac cctagaaaag 6720 atgactgatt ctgtcacttg tttgtattgc aattcctttt ccaagcaaag caaacaaaaa 6780 aattttcttt tggttggaac cgctgatggc aagttagcaa tttttgaaga taagactgtt 6840 aagcttaaag gagctgctcc tttgaagata ctaaatatag gaaatgtcag tactccattg 6900 atgtgtttga gtgaatccac aaattcaacg gaaagaaatg taatgtgggg aggatgtggc 6960 acaaagattt tctccttttc taatgatttc accattcaga aactcattga gacaagaaca 7020 agccaactgt tttcttatgc agctttcagt gattccaaca tcataacagt ggtggtagac 7080 actgctctct atattgctaa gcaaaatagc cctgttgtgg aagtgtggga taagaaaact 7140 gaaaaactct gtggactaat agactgcgtg cactttttaa gggaggtaat ggtaaaagaa 7200 aacaaggaat caaaacacaa aatgtcttat tctgggagag tgaaaaccct ctgccttcag 7260 aagaacactg ctctttggat aggaactgga ggaggccata ttttactcct ggatctttca 7320 actcgtcgac ttatacgtgt aatttacaac ttttgtaatt cggtcagagt catgatgaca 7380 gcacagctag gaagccttaa aaatgtcatg ctggtattgg gctacaaccg gaaaaatact 7440 gaaggtacac aaaagcagaa agagatacaa tcttgcttga ccgtttggga catcaatctt 7500 ccacatgaag tgcaaaattt agaaaaacac attgaagtga gaaaagaatt agctgaaaaa 7560 atgagacgaa catctgttga gtaa 7584 <210> 4 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> TXNIP-shRNA sequence <400> 4 agtggaggtg tgtgaagtta ctcgtgtca 29

Claims (5)

Measuring the expression level of mRNA or protein of the TXNIP gene from the sample of the patient and the sample of the control group;
Comparing the measured expression levels with each other; And
When the expression level of the patient is higher than the expression level of the control method for providing information for predicting or confirming the risk of developing sporadic Parkinson's disease-derived LRRK2-G2019S mutation.
Treating the test material with induced pluripotent stem cell-derived midbrain-like sporadic Parkinson's disease 3D organoid expressing TXNIP gene;
Measuring the expression level of the gene of the organoid; And
When the expression of the gene is reduced compared to before the treatment of the test material, selecting the test material as a sporadic Parkinson's disease prevention or treatment agent; Screening method for a candidate substance for preventing or treating sporadic Parkinson's disease derived from LRRK2-G2019S mutation.
A composition for preventing or treating sporadic Parkinson's disease-derived LRRK2-G2019S mutations comprising an oligonucleotide consisting of the sequence of SEQ ID NO: 4.
Diagnostic composition for sporadic Parkinson's disease derived from LRRK2-G2019S mutation comprising a nucleotide sequence encoding a TXNIP gene, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance specifically binding to a protein encoded by the nucleotide sequence .
Sporadic Parkinson's disease diagnostic kit derived from LRRK2-G2019S mutation comprising the composition of claim 4.

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